Univesity of Cape Town Genetic studies on the region downstream of the unc operon of Thiobacillus ferrooxidans. by Joseph emick-Shank Oppon. Submitted in partial fulfilment of the requirements for the degree of Master of Science in the department of Microbiology, University of Cape Town. April, 1996.
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Unives
ity of
Cap
e Tow
n
Genetic studies on the region downstream of the unc operon of
Thiobacillus ferrooxidans
by
Joseph emick-Shank Oppon
Submitted in partial fulfilment of the requirements for the degree of
Master of Science in the department of Microbiology University of Cape Town
April 1996
The copyright of this thesis vests in the author No quotation from it or information derived from it is to be published without full acknowledgement of the source The thesis is to be used for private study or non-commercial research purposes only
Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author
Univers
ity of
Cap
e Tow
n
TABLE OF CONTENTS
ACKNOWLEOOEMENTS ii
ABBREVIATIONS iii
LIST OF TABLES iv
ABSTRACT v
Chapter 1 General Introduction 1
Chapter 2 Cosmid p8181 and its subclones 37
Chapter 3 T ferrooxidans glucosamine synthetase gene 53
Chapter 4 Tn7-like transposon of T ferrooxidans 81
Chapter 5 General Conclusions 124
Appendix 1 Media 130
Appendix 2 Buffers and Solutions 131
Appendix 3 Bacterial strains 138
Appendix 4 Standard Techniques 139
References 143
ii
ACKNOWLEDGEMENTS
I am most grateful to my supervisor Prof Doug Rawlings for his excellent supervision
guidance and encouragement throughout the research Without him this work would not
have been accomplished I am also indebted to my colleagues in the Thiobacillus Research
Unit Ant Smith Ros Powles Cliff Dominy Shelly Deane and the rest of my colleagues
in the department who in one way or the other helped me throughout the period of study
I am grateful to all the members of the Department of Microbiology for their assistance
and support during the course of this research Special thanks to Di James Anne Marie
who is always there when you need her and Di DeVillers for her amazing concern to let it
be there when you need it
My friends Kojo Joyce Pee Chateaux Vic Bob Felix Mawanda Moses the list of
which I cannot exhaust you have not been forgotten Thank you for your encouragement
which became the pillar of my strength Last but not the least I acknowledge the financial
support from Foundation for Research and Development of CSIR and the General Mining
Corp of South Africa
iii
A Ala amp Arg Asn Asp ATCC ATP
bp
C CGSC Cys
DNA dNTP(s) DSM
EDTA
9 G-6 P GFAT GIn Glu Gly
hr His
Ile IPTG
kb kD
I LA LB Leu Lys
M Met min ml NAcG-6-P
ORF (s) oriC
ABBREVIATIONS
adenine L alanine ampicillin L-Arginine L-Asparagine L-aspartic acid American Type Culture Collection adenos triphosphate
base pair(s)
cytosine Coli Genetic Stock Centre L-cysteine
deoxyribonucleic acid deoxyribonucleotide triphosphates Deutsche Sammlungvon Mikroorganismen (The German Collection) ethylenediamine tetraacetic acid
pSIS1 construct was further subcloned to plasm ids IS30 IS40 pSlS50
pS183S p81841 and p81 (Fig Some of subclones were extensively mapped
although not all sites are shown in 22 exact positions of the ends of
T jerrooxidans plasmids pTfatpl and pTfatp2 on the cosmid p8I81 were identified
21)
Tfeooxidans
that the cosmid was
unrearranged DNA digests of cosmid pSISI and T jerrooxidans chromosomal DNA were
with pSI The of the bands gave a positive hybridization signal were
identical each of the three different restriction enzyme digests of p8I8l and chromosomal
In order to confirm of pSI81 and to
49
kb kb ~
(A)
11g 50Sts5
middot 2S4
17 bull tU (probe)
- tosect 0S1
(e)
kb
+- 10 kb
- 46 kb
28---+ 26-+
17---
Fig 23 Hybrdization of cosmid pSISI and T jerrooxitians (A TCC 33020) chromosomal
DNA by the KpnI-SalI fragment of pSIS52 (probe) (a) Autoradiographic image of the
restriction digests Lane x contains the probe lane 13 and 5 contain pSISI restricted with BamHl HindIII and Bgffi respectively Lanes 2 4 and 6 contain T errooxitians chromosomal DNA also restricted with same enzymes in similar order (b) The sizes of restricted pSISI
and T jerrooxitians chromosome hybridized by the probe The lanes correspond to those in Fig 23a A DNA digested with Pst served as the molecular weight nlUkermiddot
50
DNA (Fig BamHI digests SHklC at 26 and 2S kb 1 2) HindIII
at 10 kb 3 and 4) and BgnI at 46 (lanes 5 and 6) The signals in
the 181 was because the purified KpnI-San probe from p81 had a small quantity
ofcontaminating vector DNA which has regions ofhomology to the cosmid
vector The observation that the band sizes of hybridizing
for both pS181 and the T ferrooxidans ATCC 33020 same
digest that the region from BgnI site at to HindIII site at 16
kb on -VJjUIU 1 represents unrearranged chromosomal DNA from T ferrooxidans
ATCC there were no hybridization signals in to those predicted
the map with the 2 4 and 6 one can that are no
multiple copies of the probe (a Tn7-like segment) in chromosome of
and Lferrooxidans
of plasmid pSI was found to fall entirely within a Tn7-like trallSDOS()n nrpn
on chromosome 33020 4) A Southern hybridizationUUAcpoundUU
was out to determine whether Tn7-like element is on other
ofT ferrooxidans of T and Lferrooxidans results of this
experiment are shown 24 Lanes D E and F 24 represent strains
19377 DSM and Lferrooxidans DSM 2705 digested to
with BgnI enzyme A hybridization was obtained for
the three strains (lane A) ATCC 19859 (lane B) and UUHUltn
51
(A) AAB CD E F kb
140
115 shy
284 -244 = 199 -
_ I
116 -109 -
08
os -
(8)) A B c o E F
46 kb -----
Fig 24 (a) Autoradiographic image of chromosomal DNA of T ferrooxidans strains ATCC 33020 19859 23270 (lanes A B C) Tthiooxidans strains ATCC 19377 and DSM 504 (D and E) and Lferrooxidans strain DSM 2705 (lane F) all restricted with Bgffi A Pst molecular weight marker was used for sizing (b) Hybridization of the 17 kb KpnI-San piece of p81852 (probe) to the chromosomal DNA of the organisms mentioned 1 Fig 24a The lanes in Fig 24a correspond to those in Fig 24b
(lane C) uuvu (Fig 24) The same size BgllI fragments (46 kb) were
lanes A Band C This result
different countries and grown on two different
they Tn7-like transposon in apparently the same location
no hybridization signal was obtained for T thiooxidans
1 504 or Lferrooxidans DSM 2705 (lanes D E and F of
T thiooxidans have been found to be very closely related based on 1
rRNA et 1992) Since all Tferrooxidans and no Tthiooxidans
~~AA~~ have it implies either T ferrooxidans and
diverged before Tn7-like transposon or Tthiooxidans does not have
an attTn7 attachment the Tn7-like element Though strains
T ferrooxidans were 10catH)uS as apart as the USA and Japan
it is difficult to estimate acquired the Tn7-like transposon as
bacteria get around the world are horizontally transmitted It
remains to be is a general property
of all Lferrooxidans T ferrooxidans strains
harbour this Tn7-like element their
CHAPTER 3
5431 Summary
54Introduction
56Materials and methods
56L Bacterial and plasmid vectors
Media and solutions
56333 DNA
57334 gel electrophoresis
Competent preparation
57336 Transformation of DNA into
58337 Recombinant DNA techniques
58ExonucleaseIII shortening
339 DNA sequencing
633310 Complementation studies
64Results discussion
1 DNA analysis
64Analysis of ORF-l
72343 Glucosamine gene
77344 Complementation of by T ferrooxidans glmS
57cosmid pSIS1 pSlS20
58Plasmidmap of p81816r
Fig Plasmidmap of lS16f
60Fig 34 Restriction digest p81S1Sr and pSIS16f with
63DNA kb BamHI-BamHI
Fig Codon and bias plot of the DNA sequence of pSlS16
Fig 37 Alignment of C-terminal sequences of and Bsubtilis
38 Alignment of amino sequences organisms with high
homology to that of T ferrooxidans 71
Fig Phylogenetic relationship organisms high glmS
sequence homology to Tferrooxidans
31 Summary
A 35 kb BamHI-BamHI p81820 was cloned into pUCBM20 and pUCBM21 to
produce constructs p818 and p81816r respectively This was completely
sequenced both rrelct1()ns and shown to cover the entire gmS (184 kb) Fig 31
and 34 The derived sequence of the T jerrooxidans synthetase was
compared to similar nrvnC ~ other organisms and found to have high sequence
homology was to the glucosamine the best studied
eubacterium EcoU Both constructs p81816f p81 the entire
glmS gene of T jerrooxidans also complemented an Ecoli glmS mutant for growth on medium
lacking N-acetyl glucosamine
32 =-====
Cell wall is vital to every microorganism In most procaryotes this process starts
with amino which are made from rructClsemiddotmiddotn-[)ll()S by the transfer of amide group
to a hexose sugar to form an This reaction is by
glucosamine synthetase the product of the gmS part of the catalysis
to the 40-residue N-terminal glutamine-binding domain (Denisot et al 1991)
participation of Cys 1 to generate a glutamyl thiol ester and nascent ammonia (Buchanan
368-residue C-terminal domain is responsible for the second part of the ClUUU
glucosamine 6-phosphate This been shown to require the ltgtttrltgtt1iltn
of Clpro-R hydrogen of a putative rrulctoseam 6-phosphate to fonn a cis-enolamine
intennediate which upon reprotonation to the gives rise to the product (Golinelli-
Pimpaneau et al 1989)
bacterial enzyme mn two can be separated by limited chymotryptic
proteolysis (Denisot et 199 binding domain encompassing residues 1 to
240 has the same capacity to hydrolyse (and the corresponding p-nitroanilide
derivative) into glutamate as amino acid porI11
binding domain is highly cOllserveu among members of the F-type
ases Enzymes in this include amidophosphophoribosyl et al
1982) asparagine synthetase (Andrulis et al 1987) glucosamine-6-P synmellase (Walker et
aI 1984) and the NodM protein of Rhizobium leguminosarum (Surin and 1988) The
368-residue carboxyl-tenninal domain retains the ability to bind fructose-6-phosphate
The complete DNA sequence of T ferrooxidans glmS a third of the
glmU gene (preceding glmS) sequences downstream of glmS are reported in this
chapter This contains a comparison of the VVA r glmS gene
and Mycobacterium (422 ) An interesting observation was that the T ferrooxidans
glmS gene had comparatively high homology sequences to the Rhizobium leguminosarum and
Rhizobium meliloti M the amino to was 44 and 436
respectively A consensus Dalgarno upstream of the start codon
(ATG) is in 35 No Ecoli a70-type n r~ consensus sequence was
detected in the bp preceding the start codon on intergenic distance
between the two and the absence of any promoter consensus sequence Plumbridge et
al (1993) that the Ecoli glmU and glmS were co-transcribed In the case
the glmU-glmS --n the T errooxidans the to be similar The sequences
homology to six other amidotransferases (Fig 38) which are
(named after the purF-encoded l phosphoribosylpyrophosphate
amidotransferase) and Weng (1987)
The glutamine amide transfer domain of approximately 194 amino acid residues is at the
of protein chain Zalkin and Mei (1989) using site-directed to
the 9 invariant amino acids in the glutamine amide transfer domain of
phosphoribosylpyrophosphated indicated in their a
catalytic triad is involved glutamine amide transfer function of
73
Comparison of the ammo acid sequence alignment of ghtcosamine-6-phosphate
amidotranferases (GFAT) of Rhizobium meliloti (R_m) Rhizobium leguminnosarum (RJ)
Ecoli (E_c) Hinjluenzae Mycobacterium leprae (M_I) Bsubtilis (B_s) and
Scerevisiae (S_c) to that of Tferrooxidans Amino acids are identified by their single
codes The asterisks () represent homologous amino acids of Tferrooxidans glmS to
at least two of the other Consensus ammo acids to all eight organisms are
highlighted (bold and underlined)
1 50 R m i bullbullbullbullbullbull hkps riag s tf bullbullbullbull R~l i bullbullbullbullbullbull hqps rvaep trod bullbullbullbull a
a bullbullbullbullbullbull aa 1r 1 d bullbullbull a a bullbullbullbullbullbull aa inh g bullbullbullbullbullbull sktIv pmilq 1 1g bullbullbull a MCGIVi V D Gli RI IYRGYPSAi AI D 1 gvmar s 1gsaks Ikek a bullbullbullbull qfcny Iversrgi tvq t dgd bullbull ead
51 100 R m hrae 19ktrIk bull bullbull bullbull s t ateR-l tqrae 19rkIk bullbullbull s t atrE-c htIrI qmaqae bull bull h bullbull h gt sv aae in qicI kads stbull bullbull k bullbull i gt T f- Ivsvr aetav bullbullbull r bullbull gq qg c
DL R R G V L AV E L G VGI lTRW E H ntvrrar Isesv1a mv bull sa nIgi rtr gihvfkekr adrvd bullbullbull bullbull nve aka g sy1stfiykqi saketk qnnrdvtv she reqv
101 150 R m ft g skka aevtkqt R 1 ft g ak agaqt E-c v hv hpr krtv aae in sg tf hrI ksrvlq T f- m i h q harah eatt
S E Eamp L A GY l SE TDTEVIAHL ratg eiavv av
qsaIg lqdvk vqv cqrs inkke cky
151 200 bullbull ltk bullbull dgcm hmcve fe a v n bull Iak bullbull dgrrm hmrvk fe st bull bull nweI bullbull kqgtr Iripqr gtvrh 1 s bull bull ewem bullbullbull tds kkvqt gvrh hlvs bull bull hh bullbull at rrvgdr iisg evm
V YR T DLF A A L AV S D PETV AR G M 1 eagvgs lvrrqh tvfana gvrs B s tef rkttIks iInn rvknk 5 c Ihlyntnlqn ~lt klvlees glckschy neitk
74
201 R m ph middot middot middot R 1 ah- middot middot middot middot middot middot E c 1 middot middot middot middot middot middot middot Hae in 1 middot middot middot middot middot T f - c11v middot middotmiddot middot middot middot middot middot M 1 tli middot middot middot middot middot middot middot middot middot B s 11 middot middot middot middot middot S c lvkse kk1kvdfvdv
251 R m middot middot middot middot middot middot middot middot middot middot middot middot middot middot
middot middot middot middot R-1 middot middot middot middot middot middot E c middot middot middot middot middot middot middot middot Hae in middot middot middot middot middot middot middot middot middot middot middot middot T f shy middot middot middot middot middot middot middot middot middot middot middot middot 1M middot middot middot middot middot middot middot middot middot middot middot middot middot middot B s middot middot middot middot middot middot middot middot middot middot middot middot middot middot middot S c feagsqnan1 1piaanefn1
301 R m fndtn wgkt R-1 fneti wgkt E c rf er Hae in srf er T f- rl mlqq
PVTR V YE DGDVA R M 1 ehqaeg qdqavad B s qneyem kmvdd S c khkkl dlhydg
351 R m nhpe v R-1 nhe v E c i nk Hae in k tli T f- lp hr
~ YRHlrM~ ~I EQP AVA M 1 geyl av B-s tpl tdvvrnr S-c pd stf
401 Rm slasa lk R-1 slasca lk E c hql nssr Hae in hq nar-T f f1s hytrq
RVL A SiIS A VG W M 1 ka slakt B s g kqS c lim scatrai
T ferrooxidans (Tf) Scerevisiae (Saee) Mleprae (Myele) and Bsublilis (Baesu)
tr1 ()-ltashyc 8 ~ er (11
-l l
CIgt
~ r (11-CIgt (11
-
$ -0 0shy
a c
omiddot s
S 0 0shyC iii omiddot $
ttl ~ () tI)
I-ltashyt ()
0
~ smiddot (11
81
CHAPTER 4
8341 Summary
8442 Introduction
8643 Materials and methods
431 Bacterial strains and plasmids 86
432 DNA constructs subclones and shortenings 86
864321 Contruct p81852
874322 Construct p81809
874323 Plasmid p8I809 and its shortenings
874324 p8I8II
8844 Results and discussion
441 Analysis of the sequences downstream the glmS gene 88
442 TnsBC homology 96
99443 Homology to the TnsD protein
118444 Analysis of DNA downstream of the region with TnsD homology
LIST OF FIGURES
87Fig 41 Alignment of Tn7-like sequence of T jerrooxidans to Ecoli Tn7
Fig 42 DNA sequences at the proximal end of Tn5468 and Ecoli glmS gene 88
Fig 43 Comparison of the inverted repeats of Tn7 and Tn5468 89
Fig 44 BLAST results of the sequence downtream of T jerrooxidans glmS 90
Fig 45 Alignment of the amino acid sequence of Tn5468 TnsA-like protein 92 to TnsA of Tn7
82
Fig 46 Restriction map of the region of cosmid p8181 with amino acid 95 sequence homology to TnsABC and part of TnsD of Tn7
Fig 47 Restriction map of cosmid p8181Llli with its subclonesmiddot and 96 shortenings
98 Fig 48 BLAST results and DNA sequence of p81852r
100 Fig 49 BLAST results and DNA sequence of p81852f
102 Fig 410 BLAST results and DNA sequence of p81809
Fig 411 Diagrammatic representation of the rearrangement of Tn5468 TnsDshy 106 like protein
107Fig 412 Restriction digests on p818lLlli
108 Fig 413 BLAST results and DNA sequence of p818lOEM
109 Fig 414 BLAST results on joined sequences of p818104 and p818103
111 Fig 415 BLAST results and DNA sequence of p818102
112 Fig 416 BLAST results and nucleotide sequence of p818101
Fig 417 BLAST results and DNA sequence of the combined sequence of 113p818l1f and p81810r
Fig 418 Results of the BLAST search on p81811r and the DNA sequence 117obtained from p81811r
Fig 419 Comparison of the spa operons of Ecali Hinjluenzae and 121 probable structure of T ferraaxidans spa operon
83
CHAPTER FOUR
TN7-LlKE TRANSPOSON OF TFERROOXIDANS
41 Summary
Various constructs and subclones including exonuclease ill shortenings were produced to gain
access to DNA fragments downstream of the T ferrooxidans glmS gene (Chapter 3) and
sequenced Homology searches were performed against sequences in GenBank and EMBL
databases in an attempt to find out how much of a Tn7-like transposon and its antibiotic
markers were present in the T ferrooxidans chromosome (downstream the glmS gene)
Sequences with high homology to the TnsA TnsB TnsC and TnsD proteins of Tn7 were
found covering a region of about 7 kb from the T ferrooxidans glmS gene
Further sequencing (plusmn 45 kb) beyond where TnsD protein homology had been found
revealed no sequences homologous to TnsE protein of Tn7 nor to any of the antibiotic
resistance markers associated with Tn7 However DNA sequences very homologous to Ecoli
ATP-dependentDNA helicase (RecG) protein (EC 361) and guanosine-35-bis (diphosphate)
3-pyrophosphohydrolase (EC 3172) stringent response protein of Ecoli HinJluenzae and
Vibrio sp were found about 15 kb and 4 kb respectively downstream of where homology to
the TnsD protein had been detected
84
42 Transposable insertion sequences in Thiobacillus ferrooxidizns
The presence of two families (family 1 and 2) of repetitive DNA sequences in the genome
of T ferrooxidans has been described previously (Yates and Holmes 1987) One member of
the family was shown to be a 15 kb insertion sequence (IST2) containing open reading
frames (ORFs) (Yates et al 1988) Sequence comparisons have shown that the putative
transposase encoded by IST2 has homology with the proteins encoded by IS256 and ISRm3
present in Staphylococcus aureus and Rhizobium meliloti respectively (Wheatcroft and
Laberge 1991) Restriction enzyme analysis and Southern hybridization of the genome of
T ferrooxidans is consistent with the concept that IST2 can transpose within Tferrooxidans
(Holmes and Haq 1990) Additionally it has been suggested that transposition of family 1
insertion sequences (lST1) might be involved in the phenotypic switching between iron and
sulphur oxidizing modes of growth including the reversible loss of the capacity of
T ferrooxidans to oxidise iron (Schrader and Holmes 1988)
The DNA sequence of IST2 has been determined and exhibits structural features of a typical
insertion sequence such as target-site duplications ORFs and imperfectly matched inverted
repeats (Yates et al 1988) A transposon-like element Tn5467 has been detected in
T ferrooxidans plasmid pTF-FC2 (Rawlings et al 1995) This transposon-like element is
bordered by 38 bp inverted repeat sequences which has sequence identity in 37 of 38 and in
38 of 38 to the tnpA distal and tnpA proximal inverted repeats of Tn21 respectively
Additionally Kusano et al (1991) showed that of the five potential ORFs containing merR
genes in T ferrooxidans strain E-15 ORFs 1 to 3 had significant homology to TnsA from
transposon Tn7
85
Analysis of the sequence at the tenninus of the 35 kb BamHI-BamHI fragment of p81820
revealed an ORF with very high homology to TnsA protein of the transposon Tn7 (Fig 44)
Further studies were carried out to detennine how much of the Tn7 transposition genes were
present in the region further downstream of the T ferroxidans glmS gene Tn7 possesses
trimethoprim streptomycin spectinomycin and streptothricin antibiotic resistance markers
Since T ferrooxidans is not exposed to a hospital environment it was of particular interest to
find out whether similar genes were present in the Tn7-like transposon In the Ecoli
chromosome the Tn7 insertion occurs between the glmS and the pho genes (pstS pstC pstA
pstB and phoU) It was also of interest to detennine whether atp-glm-pst operon order holds
for the T ferrooxidans chromosome It was these questions that motivated the study of this
region of the chromosome
43 OJII
strains and plasm ids used were the same as in Chapter Media and solutions (as
in Chapter 3) can in Appendix Plasmid DNA preparations agarose gel
electrophoresis competent cell preparations transformations and
were all carned out as Chapter 3 Probe preparation Southern blotting hybridization and
detection were as in Chapter 2 The same procedures of nucleotide
DNA sequence described 2 and 3 were
4321 ~lllu~~~
Plasmid p8I852 is a KpnI-SalI subclone p8I820 in the IngtUMnt vector kb) and
was described Chapter 2 where it was used to prepare the probe for Southern hybridization
DNA sequencing was from both (p81852r) and from the Sail sites
(p8I852f)
4322 ==---==
Construct p81809 was made by p8I820 The resulting fragment
(about 42 kb) was then ligated to a Bluescript vector KS+ with the same
enzymes) DNA sequencing was out from end of the construct fA
87
4323 ~mlJtLru1lbJmalpoundsectM~lllgsect
The 40 kb EeoRI-ClaI fragment p8181Llli into Bluescript was called p8181O
Exonuclease III shortening of the fragment was based on the method of Heinikoff (1984) The
vector was protected with and ClaI was as the susceptible for exonuclease III
Another construct p818IOEM was by digesting p81810 and pUCBM20 with MluI and
EeoRI and ligating the approximately 1 kb fragment to the vector
4324 p81811
A 25 ApaI-ClaI digest of p8181Llli was cloned into Bluescript vector KS+ and
sequenced both ends
88
44 Results and discussion
of glmS gene termini (approximately 170
downstream) between
comparison of the nucleotide
coli is shown in Fig 41 This region
site of Tn7 insertion within chromosome of E coli and the imperfect gtpound1
repeat sequences of was marked homology at both the andVI
gene but this homology decreased substantially
beyond the stop codon
amino acid sequence
been associated with duplication at
the insertion at attTn7 by (Lichtenstein and as
CCGGG in and underlined in Figs41
CCGGG and are almost equidistant from their respelt~tle
codons The and the Tn7-like transposon BU- there is
some homology transposons in the regions which includes
repeats
11 inverted
appears to be random thereafter 1) The inverted
repeats of to the inverted repeats of element of
(Fig 43) eight repeats
(four traJrlsposcm was registered with Stanford University
California as
A search on the (GenBank and EMBL) with
of 41 showed high homology to the Tn sA protein 44) Comparison
acid sequence of the TnsA-like protein Tn5468 to the predicted of the
89
Fig 41
Alignment of t DNA sequence of the Tn7-li e
Tferrooxi to E i Tn7 sequence at e of
insertion transcriptional t ion s e of
glmS The ent homology shown low occurs within
the repeats (underl of both the
Tn7-li transposon Tn7 (Fig 43) Homology
becomes before the end of t corresponding
impe s
T V E 10 20 30 40 50 60
Ec Tn 7
Tf7shy(like)
70 80 90 100 110 120
Tf7shy(1
130 140 150 160 170 180 Ec Tn 7 ~~~=-
Tf7shy(like)
90
Fig 42 DNA sequences at the 3 end of the Ecoll glmS and the tnsA proximal of Tn7 to the DNA the 3end of the Tferrooxidans gene and end of Tn7-like (a) DNA sequence determined in the Ecoll strain GD92Tn7 in which Tn been inserted into the glmS transcriptional terminator Walker at ai 1986 Nucleotide 38 onwards are the of the left end of Tn DNA
determined in T strain ATCC 33020 with the of 7-like at the termination site of the glmS
gene Also shown are the 22 bp of the Tn7-like transposon as well as the region where homology the protein begins A good Shine
sequence is shown immediately upstream of what appears to be the TTG initiation codon for the transposon
(a)
_ -35 PLE -10 I tr T~ruR~1 truvmnWCIGACUur ~~GCfuA
100 no 110 110 110
4 M S S bull S I Q I amp I I I I bull I I Q I I 1 I Y I W L ~Tnrcmuamaurmcrmrarr~GGQ1lIGTuaDACf~1lIGcrA
DO 200 amp10 220 DG Zlaquot UO ZIG riO
T Q IT I I 1 I I I I I Y sir r I I T I ILL I D L I ilGIIilJAUlAmrC~GlWllCCCAcarr~GGAWtCmCamp1tlnmcrArClGACTAGNJ
Fig 45 (a) Alignment of the amino acid sequence of Tn5468 (which had homology to TnsA of Tn7 Fig 44) to the amino acid sequence of ORF2 (between the merR genes of Tferrooxidans strain E-1S) ORF2 had been found to have significant homology to Tn sA of Tn7 (Kusano et ai 1991) (b) Alignment of the amino acids translation of Tn5468 (above) to Tn sA of Tn7 (c) Alignment of the amino acid sequence of TnsA of Tn7 to the hypothetical ORF2 protein of Tferrooxidans strain E-1S
(al
Percent Similarity 60000 Percent Identity 44444
Tf Tn5468 1 LARQRYGVDEDRVARFQKEGRGQGRGADYHPWLTIQDVPSQGRSHRLKGI SO 11 1111111 I 1111 1111 I I 111
Fig 49 (a) Results of the BLAST search on the inverted and complemented nucleotide sequence of 1852f (from the Sall site) Results indicate amino acid sequence to the TnsC of Tn7 (protein E is the same as TnsC protein) (b) The nucleotide sequence and open reading frame of p81852f
High Prob producing Segment Pairs Frame Score P(N)
IB255431QQECE7 protein E - Escher +1 176 15e-20 gplX044921lSTN7EUR 1 Tn7 E with +1 176 15e-20 splP058461 ECOLI TRANSPOSON TN7 TRANSPOSITION PR +1 176 64e-20 gplU410111 4 D20248 gene product [Caenorhab +3 59 17e-06
IB255431QQECE7 ical protein E - Escherichia coli transposon Tn7 (fragment)
417 (al BLAST results obtained from combined sequence of (inverted and complemented) and 1810r good sequence to Ecoli and Hinfluenzae DNA RecG (EC 361) was found (b) The combined sequence and open frame which was homologous to RecG
444 Analysis of DNA downstream of region with TnsD homology
The location of the of p81811 ApaI-CLaI construct is shown in Fig 47 The single strand
sequence from the C LaI site was joined to p8181Or and searched using BLAST against the
GenBank and EMBL databases Good homology to Ecoli and Hinjluezae A TP-dependent
DNA helicase recombinase proteins (EC 361) was obtained (Fig 417) The BLAST search
with the sequence from the ApaI end showed high sequence homology to the stringent
response protein guanosine-35-bis (diphosphate) 3-pyrophosphohydrolase (EC 3172) of
Ecoli Hinjluenzae and Scoelicolor (Fig 418) In both Ecoli and Hinjluenzae the two
proteins RecG helicase recombinase and ppGpp stringent response protein constitute part of
the spo operon
445 Spo operon
A brief description will be given on the spo operons of Ecoli and Hinjluenzae which appear
to differ in their arrangements In Ecoli spoT gene encodes guanosine-35-bis
pyrophosphohydrolase (ppGpp) which is synthesized during stringent response to amino acid
starvation It is also known to be responsible for cellular ppGpp degradation (Gentry and
Cashel 1995) The RecG protein is required for normal levels of recombination and DNA
repair RecG protein is a junction specific DNA helicase that acts post-synaptically to drive
branch migration of Holliday junction intermediate made by RecA during the strand
exchange stage of recombination (Whitby and Lloyd 1995)
The spoS (also called rpoZ) encodes the omega subunit of RNA polymerase which is found
associated with core and holoenzyme of RNA polymerase The physiological function of the
omega subunit is unknown Nevertheless it binds stoichiometrically to RNA polymerase
119
Fig 418 BLAST search nucleotide sequence of 1811r I restrict ) inverted and complement high sequence homology to st in s 3 5 1 -bis ( e) 3 I ase (EC 31 7 2) [(ppGpp)shyase] -3-pyropho ase) of Ecoli Hinfluenzae (b) The nucleot and the open frame with n
Add 4 ml 52 mix by inversion at room temp for 5 mins
Add 4 ml 53 Mix by to homogenous suspension
Spin at 15 K 40 mins at 4
remove to fresh tube
Equilibrate column with 2 ml N2
Load supernatant 2 to 4 ml amounts
Wash column 2 X 4 of N3 Elute the DNA the first bed
each) add 07
volumes of isopropanol
Spin at 4 Wash with 70 Ethanol Resuspended pellet in n rv 100 AU of TE and scan
volume of about 8 to 10 drops) To the eluent (two
to
140
SEQUITHERM CYCLE SEQUENCING
Alf-express Cy5 end labelled promer method
only DNA transformed into end- Ecoli
The label is sensitive to light do all with fluorescent lights off
3-5 kb
3-7 kb
7-10 kb
Thaw all reagents from kit at well before use and keep on
1) Label 200 PCR tubes on or little cap flap
(The heated lid removes from the top of the tubes)
2) Add 3 Jtl of termination mixes to labelled tubes
3) On ice with off using 12 ml Eppendorf DNA up to
125 lll with MilliQ water
Add 1 ltl
Add lll of lOX
polymeraseAdd 1 ltl
Mix well Aliquot 38 lll from the eppendorf to Spin down
Push caps on nrnnp1
141
Hybaid thermal Cycler
Program
93 DC for 5 mins 1 cycle
93degC for 30 sees
55 DC for 30 secs
70 DC for 60 secs 30 cycle 93 DC_ 30s 55 DC-30s
70degC for 5 mins 1 cycle
Primer must be min 20 bp long and min 50 GC content if the annealing step is to be
omitted Incubate at 95 DC for 5 mins to denature before running Spin down Load 3 U)
SEQUITHERM CYCLE SEQUENCING
Ordinary method
Use only DNA transformed into end- Ecoli strain
3-5 kb 3J
3-7 kb 44
7-10 kb 6J
Thaw all reagents from kit at RT mix well before use and keep on ice
1) Label 200 PCR tubes on side or little cap flap
(The heated lid removes markings from the top of the tubes)
2) Add 3 AU of termination mixes to labelled tubes
3) On ice using l2 ml Eppendorf tubes make DNA up to 125 41 with MilliQ water
142
Add I Jtl of Primer
Add 25 ttl of lOX sequencing buffer
Add 1 Jtl Sequitherm DNA polymerase
Mix well spin Aliquot 38 ttl from the eppendorf to each termination tube Spin down
Push caps on properly
Hybaid thermal Cycler
Program
93 DC for 5 mins 1 cycle
93 DC for 30 secs
55 DC for 30 secs
70 DC for 60 secs 30 cycles
93 DC_ 30s 55 DC-30s 70 DC for 5 mins 1 cycle
Primer must be minimum of 20 bp long and min 50 GC content if the annealing step is
to be omitted Incubate at 95 DC for 5 mins to denature before running
Spin down Load 3 ttl for short and medium gels 21t1 for long gel runs
143
REFERENCES
144
REFERENCES
Abrahams J P Lutter R Todd R J van Raaij M J Leslie A G W and Walker J E 1993 Inherent asymmetry of the structure of F1-ATPase from bovine heart mitochondria at 65 Aresolution EMBO J 121775-1780
Abrahams J P Leslie A G W Lutter R and Walker 1 E 1994 Structure at 28 A resolution of FeATPase from bovine heart mitochondria Nature 370621-628
Adzumah K and Mizuuchi K 1988 Target immunity of Mu transposition reflects a differential distribution of Mu B protein Cell 53257-266
Akey C W Crepeau R H Dunn S D McCarty R E and Edelstein S J 1983 Electron microscopy of single molecules and crystals of F1-ATPases EMBO J 21409-1415
Allet B 1979 Mu insertion duplicates a 5 bp sequence at the host inserted site Cell 16 123shy129
Amzel L M and Pedersen P L 1983 Proton ATP-ases structure and mechanism Ann Rev Biochem 52801-824
Andersson L Mac Neela J and Wolfenden R 1985 Use of secondary isotope effects and varying pH to investigate mode of binding of inhibitory amino aldehydes by leucine aminopeptidase Biochem 24330-333
Andrews G F Dugan R P and Stevens C J 1992 Combining physical and bacterial treatment for removing pyritic sulfur from coal In Processing and Utilization of High-sulfur coals IV Dugan P R D Quigley and Y Attia (eds) p 515 Elsevier New York
Andrulis I L Chen J and Ray P N 1987 Isolation of human cDNAs for asparagine synthetase and expression in Jansen rat sarcoama cells Mol Cell BioI 72435-2443
Andruszkiewicz R Milewsky S Zieniawa T and Borowski E 1990 Anticandidal properties of N3 -(4-methoxy-fumaroyl)-L-2 3-diamino propanoic acid oligopeptides 1 Med Chern 33132-135
Arciszewska L K Drake D and Craig NL 1989 Transposon Tn7 cis-acting sequences in transposition and transposition immunity J Mol BioI 20735-42
Bachmann B J 1983 Linkage map of Escherichia coli K-12 edition 7 Microbiol Rev 47180-230
145
B Plumbridge 1 A Cochet D Souza J M M M and Calcagno M 1988 Cordinated regulation of amino J
Bacteriol 1754951-4956
0 B Vermoote P V and Le Goffic F 1993 synthetase from Ecoli lUllL- mechanism and inhibition by N3-fumaroyl-2 3-diaminopropionic derivatives Biochem 27 2282-2287
Vermoote P Haumont PY syrlthl~ta~e from Escherichia coli purification site location Biochemistry 26 1940-1948
Badet B Inagaki K Soda K Walsh dependent inhibition of Bacillus stearothermophilus alanine racemase by phosphonate isomers by isomerization to non covalent enzyme-(1-aminoethyl) complexes Biochem 253275-3282
Badet-Denisot M A and Badet of glucoseamine-6shyphosphate synthetase by diethylpyrocarbonates histidine requirement for enzymatic activity Arch Biochem Biophys
Bainton R Gamas P and transposition in vitro proceeds through an excised transposon intermediate (JpnIPtlltpi1 111 breaks in DNA Cell 65805-816
Barry G 1986 Permanent insertion of v~u into the chromosomes of soil bacteria BioTechnology 4446-449
Barth P T Datta N Grinter N S 1976 Transposition a deoxyribonucleic acid sequence trimethoprim and streptomycin resistances from R483 to other replicons 1 Bacteriol 125800-810
Bates C J Adams W and R R 1968 Control of the formation of uri dine diphospho-N-acetyl and glycoprotein synthesis in rat liver J Chern 2411705-17
Benjamin N 1989 Intramolecular transposition of TnlD Cell 59373shy383
Bennet R L and Malamy M resistant mutants of Escgerichia coli and phosphate Commun 40496-503
Benneth1 1978 Bacterial leaching patterns on pyrite crystal J Bacteriol
146
Berg D E Davies 1 Allet B and Rochaix 1 D 1975 Transposition of R factor genes to bacteriophage lambda Proc Natl Acad Sci USA 723268-3632
Berg D E and Drummond M1978 Absence of DNA sequences homologous to transposable element Tn5 (Kan) in the chromosome of Ecoli K-12 J Bcateriol 136419-422
Birkenhager R Hoppert M Deckers-Hebestriet G Mayer F and Altendorf K 1995 The Fo complex of the Ecoli ATP synthase investigation by electron imaging and immunoelectron microscopy Eur J Biochem 23058-67
Bjqgtrbaek C Foersom V and Michelsen O 1990 The transmembrane topology of the a subunit from ATPase in Escherichia coli analysed by PhoA protein fusions FEBS lett 26031-34
Boekemia E J Berden JA and van Heel MG 1986 sructure of mitochondrial F1-ATPase studied by electron microscopy and image processing Biochim Biophys Acta 851353-360
Bolton E Glynn P and OGara F 1984 Site specific transposition of Tn7 into a Rhizobiwn meliloti megaplasmid Mol Gen Genet 193153-157
Boos W 1974 Bacterial transport Annu Rev Biochem 43123-146
Boursaux-Eude C Girons I S and Zuerner R 1995 IS1500 an IS3-like element from Leptospira interrogans Microbiol 1412165-2173
Boyer P D 1993 The binding change mechanism for ATP synthase-some probabilities and possibilities Biochim Biophys Acta 1140215-250
Brachet P Eisen H and Rambach A 1970 Mutations in coliphage lambda affecting the expression of replicative functions 0 and P Mol Gen Genet 108266-276
Brink J Boekemia E 1 and van Bruggen E F 1987 The structure of NADH ubiquitone oxidoreductase fron beef heart mitochondria Crystals containing an octameric arrangement of iron-sulphur protein fragments Eur J Biochem 166287-294
Brock T D and Gustafson J 1976 Ferric iron reduction by sulphur- and iron-oxidizing bacteria Appl Environ Microbiol 32 567-571
Brown L D Dennehy M E and Rawlings D E 1994 The FI genes of the FIFo ATP synthase from the acidophilic bacterium Thiobacillus jerrooxidans complement Escherichia coli FI unc mutants FEMS Microbiol Lett 12219-25
Buchanan 1 1 Adv The Amidotransferases Enzymol 3991-183
Buckley Science
Caruso M Pseudomonas nOVHrn
oxidation of pyrite Applied
1 1982 Interactions Mol Gen Genet
phage 1
Chmara H by Biophysica
synthetase from bacteria antibiotic tetaine Biochimica et
Microbiol Lett 11197-206 controls on the oxidation of refractory Barberton
genetic elements and evolution Nature 263731shyCohen S N 1976 738
Corbet C M and 1 Ingledew 1987 Is oxidation by Thiobacillus ferrooxidans
Couillard D and ~~b 118(5)808-81
Metallurgical residue V UlllLltHIH of metals from sewage sludge J
Cox G B a reassessment of the
F and Hatch L 1986 The mechanism of ATP synthase of the b and a subunits Biochim Acta 84962-69
Craig N L 1995 Unity in Science 270253-254
Craig N and Gamas P 1 Purification and characterization of a transposition protein that binds ATP DNA Nuc Acids Res
1 2873-97 C A 1990 in response to environmental stress Advances in
alUIUH~ on the activity subunit b-specific polyc1onal
G Simoni and Altendorf K 1992 Influence vlJnu~ of the ATP synthase of t-S(nel~lCn
1 BioI Chern 26712364shy
M A Le Goffic F and 1991 Glucosamine- 6P two proteins on limited proteolysis ltVU Biophys 288225-230
148
Dobrogosz W J 1968 _l in Escherichia coli and its to catabolite repression J
Doublet P 1 van Heijenoort and 1993 The murI gene of is an essential gene that encodes klUltUllU~v racemase activity J Bacteriol 1752970-2979
P J van Heijenoort 1992 Identification of the Ecoli which is required for the D-glutamic acid a specific component peptidoglycan 1 Bacteriol
Garren A Garen and Torri ani 1961 Genetic control of repression UCUlllV phosphatase in Ecoli 1 Mol BioI
D J and Boeke 1 D 1990 A 1-1-0- structure is required for Ty1 transposition Genes Develop 4324-330
1982 Transposition of Tn7 occurs at a on Caulobacter crescentus 10SIClmle 1 Bacteriol 151 1056-1 058
H 1990 Molecular IHovUUU)11 -transporting 345-391 In T 12 Bacterial
Press Ltd London
transport and coupling by the synthase insights mechanism of function J Bioenerg 24485-491
1992b Subunit c of FIFo ATP role m transduction Biochim Biophys Acta 1101 v--rJ
Eliopoulos E E Jackson P 1 Keen IN HUIUVI L Thompson P and 1 Structure of a 16 kDa integral AU-UUl that identity to
of the vacuolar H+ -ATPase Protein 15
Fling M 1 C 1985 Nucleotide sequence of encoding the amino glycoside-modifying enzyme 3(9)-O-nucleotidyltransferase Res 137095-7106
Fling M and C l-1UUJIU nucleotide sequence of dihydrofolate rhnrpri by Tn7 Nucleic Acids Res 115
Flores Llchtenstem C P 1990 DNA sequence anlysis of tnsA for the Tn7 transposition Nucleic Acids 18901 911
149
149
Foster D L and Fillingame R H 1982 Stoichiometry of subunits in the H+-ATPase complex of Escherichia coli J BioI Chern 2572009-2015
Fraga D Hermolin J Oldenburg M Miller MJ and Fillingame R H 1994 Arginine 41 of subunit c of Escherichia coli H+-A TP synthase is essential in binding and coupling of F to Fo J BioI Chern 2697532-7537
Friedl P Hoppe J Gunsalus R P Michelsen 0 von Meyenburg K and Schairer H U 1983 Membrane integration and function of the three Fo subunits of the A TP-synthase of Escherichia coli K12 EMBO 1 299-103
Frisa P S and Sonneborn D R 1982 Developmentally regulated interconversion between end product inhibitable and non-inhibitable forms of a first pathway-specific enzyme activity can be mimicked in vitro by dephosphorylation reactions Proc Natl Acad Sci USA 796289-6293
Fujiwara T and Mizuuchi K 1988 Retroviral DNA integration Structure of an integration intermediate Cell 54497-504
Galas D J and Chandler M 1989 Bacterial insertion sequences In Mobile DNA Berg D E and Howe M M (eds) Washington D C American Society for Microbiology pp 109shy162
Gay N J Tybulewicz V L1 Walker JE 1986 Insertion of transposon Tn7 into the Ecoli gmS transcriptional terminator Biochem J 234 111-117
Gentry D R and Cashel M 1995 Cellular localization of the Escherichia coli SpoT protein J Bacteriol 177 3890-3893
Gentry D R Xiao H Burgess R R and Cashel M 1991 The omega subunit of Ecoli K-12 RNA polymerase is not required for stringent RNA control in vivo J Bacteriol 173 3901-3903
Girvin M E and Fillingame R H 1993 Helical structure and folding of subunit c of FIFo ATP synthase IH NMR resonace assignments and NOE analysis Biochemistry 3212167shy12177
Gogol E P Lucken U Bork T and Capaldi R A 1989 Molecular architecture of Escherichia coli F Adenosinetriphosphatase Biochemistry 284709-4716
Gogol E P Aggeler R Sagermann M and Capaldi R A 1989 Cryoelectronic Microscopy of Escherichia coli FI Adenosinetriphosphatase Decorated with Monoclonal Antibodies to Individual Subunits of the complex Biochemistry 284717-4724
150
Heinikoff S 1984
Golinelli-Pimpaneaux B and Badet involvement of Lys603 from Ecoli glucosamoine-6-phosphate synthase substrate fructose-6-phosphate 1 Biochem 201175-182
Gottesman M M and Rosner 1 L of a detenninant of uvu_bullu
resistance by coliphage lambda Sci USA 725041-5045
Grunstein M and Hogness D
Colony hybridization a method for isolation cloned DNAs that contain a 1Jbullu Proc NatL Acad Sci USA 723961-3965
Hauer B and Shapiro 1 A 1984 Control of Tn7 transposition Mol Gen Genet 194
Hedges R W and Jacob Transposition of ampicillin resistance from to other replicons MoL 1-40
Hennolin 1 Gallant 1 psi subunit in the Fo sector of the H+ -ATPase of Ecoli J Bioi
R H 1983 Topology organization and function
Hennolin J and R 1989 Assembly of Fo sector of synthase Interdepenndence of subunit insertion into the membrane 1 2817
van Montagu M Holsters Zambryski P Beuckeleer M Willmitzer and Schell 1 M 1980 interaction of
DNA plant cells Proc Soc Lond 210351-365
P 1972 Insertion mutations control region of Physical characterization of the mutants Mol Gen Genet
115266-276
Holmes applications pI
UI Haq 1990 Adaptation of Thiobacillus vu)nuu for industrial In J Salley R G McCready Wichlacz (ed)
1989 CANMET Ottawa Canada
Holtje J V and U Schwarz 1985 Biosynthesis and growth murein sacculus p 77shy119 InN (ed) Molecular cytology of Escherichia Academic Press Inc New
Acad Sci USA
Heffron E Reubens C and which mediates ampicillin
Translocation of a plasmid DNA sequence nature and specificity of Natl
digestion with exonuclease III creates fl tr breakpoints 1-369
Hernalsteens J M Agrobacterium
Hirsch H the galactose nnnn fJu
151
Holzenburg A Jones P c Franklin T Padi J B and Finbow M E 1993 Evidence for a common structure 1 Biochem 21321-30
Hoppe 1 and Sebald W 1986 Topological pathway of the protons through Fo is provided by amino acid the lipid phase Biochimie (Paris) 68427-434
S Otsubo H Davidson N and 1975 Electron microscope heteroduplex of sequence relations among plasmids identification and mapping of the
insertion sequences IS] and IS2 in F and R plasmids J Bacteriol 22762-775
Inoue c Sugawara K and 1991 regulatory gene in Thiobacillus ferrooxidans is spaced apart from Mol Microbiol 52707-2718
Ish-Horowicz D and Burke and cosmid cloning Nucleic Acids Res 92989-2998
Johnston B Clennell M and D 1995 Structure and function of Tn5467 a Tn2l-like transposon located on T jerrooxidans broad host range plasmid Appl Envir Micro
Kahmann R and Kamp Nucleotide sequences of the attachment bacteriophage Mu DNA Nature 280247-250
Kahn K and Schaefer M R Characterization of trans po son 5469 from cyanobacterium Fremyella diplosiphon J 1777026-7032
Karavaiko G Golovacheva S Pivovarova T A Tzaplina I A and Vartanjan 1988 Thermophilic ltPM Sulfobacillus page 29-41 In Biohydrometallurgyshy87 Norris P and Science and Technology Letters Kew 1
Kennedy J and Humpphreys J D 1976 Microbial cells living immobilised on Nature 261242-244
Koonin 1993 SpoU protein of Escherichia coli belongs to a new family of Nucleic Acids Res 19
Kopecko J and site specific recA mdependtm recombination between bacterial Pt of palindromes at the N ad Acad Sci USA
on L-glutamine D-fructose ud()transtiera~e 1 BioI
152
Krumholz L R Esser U and Simoni RD 1989 Nucleotide sequence of the unc operon of Vibrio alginolyticus Nucleic Acids Res 177993-7994
Kubo K and Craig N 1990 Bacterial transposon Tn7 utilizes two classes of target sites 1 Bacteriol 1722774-2778
Kucharczyk N Denisot M A Le Goffic F and Badet B 1990 Glucosarnine-6-phosphate synthase from Ecoli detennination of the mechanism of inactivation by N3 fumaroyl-L-2-3 diarninoproprionic derivatives Biochemistry 293668-3676
Kusano M Takeshima T Inoue C and Sugawara K 1991 Evidence for two sets of structural genes coding for Ribulose biphosphate carboxylase in Thiobacillus ferrooxidans 1 Bacteriol 1737313-7323
Lane Dl A P Harrison lnr D Stahl B Pace SJ Giovannoni GJ Olsen and N R Pace 1992 Evolutionary relationship among sulphur- and iron-oxidizing eubacteria 1 Bacteriol 174267-278
Lee C H Bhagwhat A and Heffron F 1983 Identification of a transposon TnJ sequence required for transposition immunity Natl Acad Sci USA 806765-6769
Lewis M 1 Chang 1 A and Somoni R D 1990 A topological analysis of subunit a from Escherichia coli F1Fo-ATP synthase predicts eight transmembrane segments 1 BioI Chern 265 10541-10550
Lichtenstein C P and Brenner S 1982 Unique insertion site of Tn7 in the Ecoli chromosome Nature (London) 297601-603
Lichtenstein C P and Brenner S 1981 Site-specific properties of transposition to the Ecoli chromosome Mol Gen Genet 183380-387
Liu X Petersson S and Sandstrom A Mesophilic versus moderate thennophilic bioleaching Biohydrometallurgy Technologies Vol 1 A E Tonna 1 E Wey and Lakshmanan V 1 (ed) pp 29-38
Lizarna H M and Sankey B M 1993 Oxidation of H2S by Thiobacillus thiooxidans is inhibited by substrate and methane BiohydrometaHurgy Technologies Vol II A E Tonna 1 E Wey and C L Brierley (ed) pp 339-364
Lundgren D G and Silver M 1980 Ore leaching by bacteria Ann Rev Microbiol 34263shy268
Makino K Amemura M Shinagawa H Kobayashi A and Nakata A 1985 Sequence of the genes involved in phosphate transport and regulation of the phosphate regulon in Escherichia coli 1 Mol BioI 184231-240
Makino K Shinagawa Amemura M Kimura S Nakata A and Ishihama 1988 Euuv1J of the phosphate regulon of Ecoli Activation of pstS by the PhoB
protein in vitro 1 Mol BioI 20385-95
Makino K Shinagawa H Amemura M Yamada M and 1990 Signal transduction in the phosphate regulon of the Escherichia coli involves phosphotransfer nprlrJp~middotn PhoR and PhoB proteins J Mol BioI 21055
Malamy 1966 Frameshift mutations in the nnlnn of Escherichia coli Cold Harb Symp Quant BioI 31189-201
Malamy M H 1972 Electron microscopy insertions in the lac operon of Escherichia coli MoL Gen
Malamy M H and Bennett R L 1970 mutants of Ecoli and phosphate transport Biochem Biophys Res Commun
Maniatis T Fritsch EF Sambrook J 1982 nn A laboratory manual Cold Spring Harbor Laboratory Press Cold
McKnight G L S L Mudri SH Mathewest R S Marshall P O Sheppard and P J OHara 1990 Molecular cloning synthesis and bacterial expression of human glutaminefructose-6-phosphate UUAUU u J Chem 26725208-25212
Medveczky N and H Rosenburg 1970 phosphate-binding protein of Escherichia Biochim Biophys Acta 211 158-168
Mei B and Zalkin H 1989 A Cysteine-Histidine-Aspartate catalytic triad is involved in Glutamide Amide Transfer Function purF-type Glutamine amodotransferases 1 BioI Chem 264 16613-16619
Mengin-Lecreulx D and J van 1993 Identification of glmU gene encoding Nshyacetylglucosamine in Escherichia coli J Bacteriol 1756150shy6157
Michaelis M J and Criddle M J 1970 Mitochondrial DNA and mutants in Saccharomyces cerevisiae Biochem Genet 5487-495
Michaelis Starlinger P 1969 Two insertions in the jO v
operon having different homologous DNA sequences MoL Gen Genet 10437 377
Miller J H Calos Transposable elements Cell 20579-595
154
Miller J Oldenburg M and Fillingame R 1990 The essential carboxyl group of in subunit c the FIFo ATP synthase can be and H( +)-translocating function retained Proc NatL Acad 874900-4904
Mitcell P 1966 Chemiosmotic coupling in - and phosphorylation BioL Rev 41455-502 (1966)
Mizuuchi K 1984 Mechanism of transposition of bacteriophage Mu Polarity of the strand reaction at the initiation of the transposon Cell 39395-404
C Ph de Donato Berthelin J Surface oxidized species a key factor in the study of bioleaching processes Biohydrometallurgical In A J
Weyand V I Lakshmanan ed 1993 Vol 1 175-184
A Ishino Shinagawa H Makino K and M 1987 Nucleotide of the iap gene responsible for alkaline phosphatase isoenzyme conversion in Ecoli
identification of product 1 1695429-5433
K 1989 The tsr gene-coding prevents thiopeptin from inhibiting ppGpp synthesis in Streptomyces lividans FEMS Microbiol Lett
Ogasawara N and Yoshikawa H 1992 and their organIzatIon in the repnc()n of the bacterial chromosome Mol Microbiol 6(5)629-634
Ohtsubo Davidson N and 1975 microscope heteroduplex studies of sequence relations among plasmids identification and mapping of the insertion sequences 1 and IS2 in F and R plasmids 1 122746-775
Olson G J 1994 Microbial oxidation gold ores and gold bioleaching FEMS un Lett 119 1-6
Orle K A N L 1991 Identification of transposition prroteins by the bacterial transposon [corrected republished originally printed in Gene 1990 Nov 30 96(1) Gene 10425-31
Ouellette M Roy P Homology of ORFs from and from to site specific recombinases 1987 Nucl Acids 10055-10059
Murein synthesis p663-671ln Neidhardt J L Ingraham B Louw M~ M Schaechter H E Umbarger (ed) Escherichia coli and Salmonella
ryphimurium cellular and bioI voL 1 American Society Microbiology Washington
Perlin D S and Senior A Functional and cross-reactivity of antibody to purified subunit b (uncF protein) of Escherichia coli proton-ATPase Arch Biochem Biophys 236603-611
155
Plumbridge J A O Cochet Souza J M Altramirano M M Calcagno M L and Badet B 1993 Coordinated regulation of amino sugar-synthesizing and -degrading enzymes in Escherichia coli K-12 J Bacteriol 175(16)4951-4956
Pretorius I M Rawlings D E and Woods D R 1986 Identification and cloning of Thiobacillus jerrooxidans structural Nif genes in Escherichia coli Gene 4559-65
Qadri M I Flores C c Davis J and Lichtenstein C 1989 Genetic analysis of attTn7 the transposon Tn7 attachment site in Escherichia coli using a novel M13-based transduction assay 1 Mol BioI 20785-98
Radstrom P Skold 0 Swedberg J Roy P H and Sundstrom L 1994 Tn5090 of plasmid R751 which carries integron is related to Tn7 Mu and retroelements J Bacteriol 1763257-3268
Raetz C R H 1987 Structure and biosynthesis of lipid A in Escherichia coli pp 498-503 In F C Niedhardt J L Ingraham K B Low B Magasanik M Schaechter and H E Umbarger (ed) Escherichia coli and Salmonella typhimurium cellular and molecular biology vol 1 American Society for Microbiology Washington DC
Rao N N and Torriani A 1990 Molecular aspects of phosphate transport in Escherichia coli Mol Microbiol 4(7) 1083-1090
Rawlings DE D R Woods and NP Mjoli 1991 The cloning and structre of genes from the autotrophic biomining bacterium Thiobacillusjerrooxidans p 215-237 In PJ Greenaway (ed) Advances in gene technology vol 2 JAI Press London
Richet E and O Raibaud 1989 MalT the regulatory protein of the Escherichia coli maltose system is an A TP-dependent transcriptional activator EMBO 1 8981-987
Rogers M Ekaterrinaki N Nimmo E and Sherratt D 1986 Analysis of Tn7 transposition Mol Gen Genet 205550-556
Ronson C W Nixon B T Albright L M anf Ausubel F M 1987 Rhizobium meliloti ntrA (rpoN) gene is required for diverse metabolic functions J Bacteriol 1692424-2431
Rosenburg H Gerdes R G and Chegwidden K 1977 Two systems for the uptake of phosphate in Ecoli JBacterioL 131505-511
Rosenburg H 1987 In ion transport in Prokaryotes Rosen B P and Silver S (eds) New York Academic Press pp 205-248
Ross D G Swan 1 and N Klecker 1979 Physical structures of TnlO-promoted deletions and inversions role of 1400 base pairs inverted repetitions Cell 16721-731
156
Saedler H and P 19670 mutation in the galactose operon in Ecoli II Physiological characterization MoL Gen Genet 100 190-202
Saedler P 1968 00 and strong polar mutations in the Genet 102353-363
Sambrook J Fritsch Maniatis 1989 Molecular cloning a laboratory manual Cold Harbor Laboratory Cold Spring Harbor New York
Sand W Gehrke T Hallmann Rohde K Sobotke B and Wintzien S 1993 Bioleaching metal sulphides The importance of Leptospirillum ferrooxidans Biohydromemetallurgical Technologies Voll pp 15-25
Sanger Nicklen and Coulson A DNA sequencing with chain-tennination inhibitors Natl Acad USA 745463-5467
Schneider E and Altendorf K All the three subunits are required for an active proton channel (Fo) of Escherichia coli synthase (FIFo) ~-
518
Schneider E and Allendorf K 1987 Bacterial adenosine 5-triphosphate synthase purification and reconstitution complexes and biochemical and functional characterization of subunits Microbio Rev 51477-497
Schrader 1 A and D S Holmes 1988 Phenotypic switching Thiobacillus ferrooxidans J BacterioL 1703915-3023
Scordilis G E H and Lassie 1987 Identification of transposable elements which activates gene expression in Pseudomonas cepacia J Bacteriol 1698-13
Sekine Eisaki N and Ohtsubo E 1996 Identification and characterization of the lS3 molecules generated by breaks 1 BioL Chern 271197-202
Senior A E 1990 The proton-trans locating of Escherichia coli Annu Rev Biophys Chern 194-41
Shapiro 1 and Adhya S 1969 The jLU operon of K-12 n A deletion analysis of the structure polarity 62249-264
J A 1969 Mutations caused by insertion genenc material the galactose operon Escherichia 1 MoL BioI 4093-105
Silvennan M P 1967 Mechanism of bacterial pyrite oxidation 1 Bacteriol 941046-1051
157
C C ElIson and Levinson 1983 Identification of the typel trimethoprim resistance reductase specified by the R-plasmid R43 comparison with procaryotic and eucaryotic dihydrofolate reductase 1 Bacteriol 155 100 1-1008
Smith and Jones P transposition a multigene process Identification of a regulatory product 147915-7927
Sprague Jr Bell R M Cronan 1 A mutant of Ecoli auxotrophic for organic phosphates evidence two defects in phosphate Mol Gen Genet 14371-77
Starlinger and Michaelis 1968 Suppression the sequential aO[)ealame of the galactose enzymes in a transferase amber mutant coli Mol Genet 102367-369
Starlinger 1977 IS elements in microorganisms MicrobioL Immunol
Steffens K Schneider E Herkernhoff B Schmid Altendorf K portion of Escherichia coli A TP synthase resolution of from subunit b J BioI Chern 2625866-5869
Steffens A Deckers-hebestreit G and Altendorf K 1987b The and functional relationship of A TP synthases (FoFI) from Ecoii and the thermophilic bacterium PS3 J Biol 2626334-6338
Strominger J and M S Smith 1959 Uridine diphosphoacetylglucosamine pyrophosphorylase 1 BioI Chern 2341822-1827
Sundstrom Skold 1990 dhfrI trimethoprim resistance gene of can be found at in other genetic surroundings Antimicrob Agents Chemother 34642shy650
Sundstrom L Roy P H and Skold Site-specific of three cassettes in Tn7 J Bacteriol 1733025-3028
Surin B P and Downie JA 1988 of the Rhizobium leguminosarum nodLMN involved efficient host-specific nodulation Mol Microbiol 2 173-183
B P Dixon N and Rosenburg 1986 Purification PhoU protein a OClItnl
regulator of the pho regulon of Ecoli K-1 J Bacteriol 168631
B P D A Jans A L Fimmel Shaw GB Cox Rosenburg Structural gene for the phosphate-repressible phosphate binding of Escherichia coli
own promoter nucleotide of the phoS 1 Bacteriol
158
Suzuki I Chang W and Takeuchi 1994 Oxidation of organic compounds by Thiobacilli Symp Series 55060-67
Takeyama M S Y Noumi T Maeda Ishibashi S and Futai M 1988 Beta subunit of Ecoli amino acid replacement within a conserved sequence G-X-X-X-X-GshyK-TS) v~u binding proteins lett 218222-226
Thomson JA M Hendson and R M 1981 Mutagenesis by insertion of drug resistance Tn7 into a vibrio 11 J Bacteriol
Tichy R Grotenhuis J T c Janssen van Houten R Rulkens and Lettinga 1993 Application of the sulphur cycle bioremediation of soils polluted with heavy metals In Int Conf Contaminated 93 ed F Arendt G J Annokee R Bosman and W J van der Brink Kluwer Academic Publishers Dordrecht pp 1461-1462
G and Mayer An electron approach to the quaternary structure of mitochondrial Eur J Biochem 13237-45
J and Tschape streptothricin resistance transposons Tn1825 and Tn1826 and transposon Tn 7 Plasmidnuu
18246-249
Tso J Y Zalkin H van Cleemput M Yanofsky C and JM 1982 Nucleotide of Escherichia coli and deduced amino glutamine
phosphribosylpyrophosphate am idotransferase J BioI Chern
V Mesyanzhinova I V Koslov I A and Orlova 1984 Structure of studied by electron and image processing Lett 167285-289
P Barber C and transposons Tn5 and Tn7 in Xanthomonas campestris pv campestris MoL Gen 157
Ullrich J and van Putten P Identification of the Gonococcal glmU gene UVUUIJ
the enzyme N-acetylglucosamine 1-Phosphate Uridyltransferase involved in the synthesis UDP-GlcNAc J of Bact 1776902-6909
M 1992 Eight bacterial proteins includin]g UDP-N -acetylglucosamine acyltransferase three vother of Escherichia coli of a six-residue n tVI
theme FEMS MicrobioL 97249-254
van der Steen J J D Doddema J and de Uidoging van zware metalen afvalstromen met behulp van thiobacilli (Removal metals from waste streams
using thiobacilli) Ministry of Housing Physical and Enviromental (VROM) Directoraat-Generaal Milieubeheer Rapport 199210 Hague
Vermoote P 1988 Universite Paris VI Paris Hrllnro
159
Vignais P V Lunard Issartel J P and Dupuis A 1985 Interaction n l1f middotn
oligomycin sensitivity protein (OSCP) beef heart mitochondrial FeATPase 2 Identification of interacting Fl subunits cross-linking Biochem J
Vik S and Dao NN Prediction of transmembrane topology of Fo proteins from Ecoli ATP synthase variational hydrophobic moment analyses Biochim Biophys Acta 1140 199-207
von Meyenburg B B Jcentrgensen J Nielsen and F Hansen 1982 Promoters of the atp operon for the membrane-bound ATP synthase of Ecoli mapped by TnlO insertion mutations MoL Gen Genet 188240-248
von Meyenberg F G Hansen 1980 The origin of replication oriC the Ecoli chromosome near oriC and construction of oriC mutants ICN-UCLA Symp MoLCellBiol 191 159
Waddell S and Craig N 1989 Tn7 transposition recognition of the attTn7 Proc Natl Sci USA 863958-3962
Waddell C S and Craig N L 1988 transposition two transposition pathways directed by five Tn7-encoded genes Develop 21
J E Gay N J Saraste M and A N 1984 DNA sequence the Escherichia coli unc operon Completion of the sequence of a kilobase segment containing
oriC unc and phoS J224799-815
and Collinson 1 1994 the role the stalk in the coupling mechanism of FEBS 34639-43
Walker 1 E Gay N J and Tybulewicz V L 1986 of transposon Tn7 into the Escherichia glmS transcriptional terminator Biochem 1 243 111-1
Wanner Land McSharry 1982 Phosphate-controlled gene in Escherichia coli using Mudl-directed lacZ fusions J Mol BioI 158347-363
Weng M and Zalkin H 1987 Structural role for a region the CTP synthetase glutamide amide transfer domain J Bacteriol 1693023-3028
Wheatcroft R and S and nucleotide sequence of Rhizobiwn meliloti insertion between the transposase encoded by ISRm3 and those encoded by Staphylococcus aureus and Thiobacillus ferrooxidans IST2 J 1732530-2538
160
Whitby M C and Lloyd R 1995 Branch of three-strand recombination intennediates by RecG a possible pathway for securing middotIULl initiated by duplex DNA 1 143303-3310
Wilkens and Capaldi R A Assymmetry and changes in examined by cryoelectronmicroscopy BioI Chern Hoppe-Seyler 37543-51
and Malamy M 1974 The loss of the PhoS periplasmic protein leads to a the specificity a constitutive phosphate transport system in Escherichia coli Biochem Res Commun 60226-233
WiUsky G Rand Malamy M 1980 of two separable inorganic phosphate transport systems in Escherichia coli J BacterioL 144356-365
P J and and C F 1973 enzyme of metabolism and Stadtman R eds) pp 343-363 Academic Press New York
Winterbum P J and Phelps F 197L control of hexosamine biosynthesis by glucosamine synthetase Biochem 1 121711
Wolf-Watz H and Norquist A 1979 Deoxyribonucleic acid and outer membrane protein to outer membrane protein involves a protein J 14043-49
Wolf-Watz H and M 1979 Deoxyribonucleic acid and outer membrane strains for oriC elevated levels deoxyribonucleic acid-binding protein and
11 for specific UU6 of the oriC region to outer membrane J Bacteriol 14050-58
Wolf-Watz H 1984 Affinity of two different chromosome to the outer membrane of Ecoli J Bacteriol 157968-970
Wu C and Te Wu 197 L Isolation and characterization of a glucosamine-requiring mutant of Escherichia 12 defective in glucoamine-6-phosphate synthetase 1 Bacteriol 105455-466
Yamada M Makino Shinagawa Nakata A 1990 Regulation of the phosphate regulon of Escherichia coli properties the pooR mutants and subcellular localization of protein Mol Genet 220366-372
Yates J R and Holmes D S 1987 Two families of repeatcXl DNA sequences Thiobacillus 1 Bacteriol 169 1861-1870
Yates J R R P and D S 1988 an insertion sequence from Thiobacillus errooxidans Proc Natl 857284-7287
161
Youvan D c Elder 1 T Sandlin D E Zsebo K Alder D P Panopoulos N 1 Marrs B L and Hearst 1 E 1982 R-prime site-directed transposon Tn7 mutagenesis of the photosynthetic apparatus in Rhodopseudomonas capsulata 1 Mol BioI 162 17-41
Zalkin H and Mei B 1990 Amino terminal deletions define a glutamide transfer domain in glutamine phosphoribosylpyrophosphate ami do transferase and other purF-type amidotransferases 1 Bacteriol 1723512-3514
Zalkin H and Weng M L 1987 Structural role for a conserved region III the CTP synthetase glutamide amide transfer domain 1 Bacteriol 169 (7)3023-3028
Zhang Y and Fillingame R H 1994 Essential aspartate in subunit c of FIF0 ATP synthase Effect of position 61 substitutions in helix-2 on function of Asp24 in helix-I 1 BioI Chern 2695473-5479
The copyright of this thesis vests in the author No quotation from it or information derived from it is to be published without full acknowledgement of the source The thesis is to be used for private study or non-commercial research purposes only
Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author
Univers
ity of
Cap
e Tow
n
TABLE OF CONTENTS
ACKNOWLEOOEMENTS ii
ABBREVIATIONS iii
LIST OF TABLES iv
ABSTRACT v
Chapter 1 General Introduction 1
Chapter 2 Cosmid p8181 and its subclones 37
Chapter 3 T ferrooxidans glucosamine synthetase gene 53
Chapter 4 Tn7-like transposon of T ferrooxidans 81
Chapter 5 General Conclusions 124
Appendix 1 Media 130
Appendix 2 Buffers and Solutions 131
Appendix 3 Bacterial strains 138
Appendix 4 Standard Techniques 139
References 143
ii
ACKNOWLEDGEMENTS
I am most grateful to my supervisor Prof Doug Rawlings for his excellent supervision
guidance and encouragement throughout the research Without him this work would not
have been accomplished I am also indebted to my colleagues in the Thiobacillus Research
Unit Ant Smith Ros Powles Cliff Dominy Shelly Deane and the rest of my colleagues
in the department who in one way or the other helped me throughout the period of study
I am grateful to all the members of the Department of Microbiology for their assistance
and support during the course of this research Special thanks to Di James Anne Marie
who is always there when you need her and Di DeVillers for her amazing concern to let it
be there when you need it
My friends Kojo Joyce Pee Chateaux Vic Bob Felix Mawanda Moses the list of
which I cannot exhaust you have not been forgotten Thank you for your encouragement
which became the pillar of my strength Last but not the least I acknowledge the financial
support from Foundation for Research and Development of CSIR and the General Mining
Corp of South Africa
iii
A Ala amp Arg Asn Asp ATCC ATP
bp
C CGSC Cys
DNA dNTP(s) DSM
EDTA
9 G-6 P GFAT GIn Glu Gly
hr His
Ile IPTG
kb kD
I LA LB Leu Lys
M Met min ml NAcG-6-P
ORF (s) oriC
ABBREVIATIONS
adenine L alanine ampicillin L-Arginine L-Asparagine L-aspartic acid American Type Culture Collection adenos triphosphate
base pair(s)
cytosine Coli Genetic Stock Centre L-cysteine
deoxyribonucleic acid deoxyribonucleotide triphosphates Deutsche Sammlungvon Mikroorganismen (The German Collection) ethylenediamine tetraacetic acid
pSIS1 construct was further subcloned to plasm ids IS30 IS40 pSlS50
pS183S p81841 and p81 (Fig Some of subclones were extensively mapped
although not all sites are shown in 22 exact positions of the ends of
T jerrooxidans plasmids pTfatpl and pTfatp2 on the cosmid p8I81 were identified
21)
Tfeooxidans
that the cosmid was
unrearranged DNA digests of cosmid pSISI and T jerrooxidans chromosomal DNA were
with pSI The of the bands gave a positive hybridization signal were
identical each of the three different restriction enzyme digests of p8I8l and chromosomal
In order to confirm of pSI81 and to
49
kb kb ~
(A)
11g 50Sts5
middot 2S4
17 bull tU (probe)
- tosect 0S1
(e)
kb
+- 10 kb
- 46 kb
28---+ 26-+
17---
Fig 23 Hybrdization of cosmid pSISI and T jerrooxitians (A TCC 33020) chromosomal
DNA by the KpnI-SalI fragment of pSIS52 (probe) (a) Autoradiographic image of the
restriction digests Lane x contains the probe lane 13 and 5 contain pSISI restricted with BamHl HindIII and Bgffi respectively Lanes 2 4 and 6 contain T errooxitians chromosomal DNA also restricted with same enzymes in similar order (b) The sizes of restricted pSISI
and T jerrooxitians chromosome hybridized by the probe The lanes correspond to those in Fig 23a A DNA digested with Pst served as the molecular weight nlUkermiddot
50
DNA (Fig BamHI digests SHklC at 26 and 2S kb 1 2) HindIII
at 10 kb 3 and 4) and BgnI at 46 (lanes 5 and 6) The signals in
the 181 was because the purified KpnI-San probe from p81 had a small quantity
ofcontaminating vector DNA which has regions ofhomology to the cosmid
vector The observation that the band sizes of hybridizing
for both pS181 and the T ferrooxidans ATCC 33020 same
digest that the region from BgnI site at to HindIII site at 16
kb on -VJjUIU 1 represents unrearranged chromosomal DNA from T ferrooxidans
ATCC there were no hybridization signals in to those predicted
the map with the 2 4 and 6 one can that are no
multiple copies of the probe (a Tn7-like segment) in chromosome of
and Lferrooxidans
of plasmid pSI was found to fall entirely within a Tn7-like trallSDOS()n nrpn
on chromosome 33020 4) A Southern hybridizationUUAcpoundUU
was out to determine whether Tn7-like element is on other
ofT ferrooxidans of T and Lferrooxidans results of this
experiment are shown 24 Lanes D E and F 24 represent strains
19377 DSM and Lferrooxidans DSM 2705 digested to
with BgnI enzyme A hybridization was obtained for
the three strains (lane A) ATCC 19859 (lane B) and UUHUltn
51
(A) AAB CD E F kb
140
115 shy
284 -244 = 199 -
_ I
116 -109 -
08
os -
(8)) A B c o E F
46 kb -----
Fig 24 (a) Autoradiographic image of chromosomal DNA of T ferrooxidans strains ATCC 33020 19859 23270 (lanes A B C) Tthiooxidans strains ATCC 19377 and DSM 504 (D and E) and Lferrooxidans strain DSM 2705 (lane F) all restricted with Bgffi A Pst molecular weight marker was used for sizing (b) Hybridization of the 17 kb KpnI-San piece of p81852 (probe) to the chromosomal DNA of the organisms mentioned 1 Fig 24a The lanes in Fig 24a correspond to those in Fig 24b
(lane C) uuvu (Fig 24) The same size BgllI fragments (46 kb) were
lanes A Band C This result
different countries and grown on two different
they Tn7-like transposon in apparently the same location
no hybridization signal was obtained for T thiooxidans
1 504 or Lferrooxidans DSM 2705 (lanes D E and F of
T thiooxidans have been found to be very closely related based on 1
rRNA et 1992) Since all Tferrooxidans and no Tthiooxidans
~~AA~~ have it implies either T ferrooxidans and
diverged before Tn7-like transposon or Tthiooxidans does not have
an attTn7 attachment the Tn7-like element Though strains
T ferrooxidans were 10catH)uS as apart as the USA and Japan
it is difficult to estimate acquired the Tn7-like transposon as
bacteria get around the world are horizontally transmitted It
remains to be is a general property
of all Lferrooxidans T ferrooxidans strains
harbour this Tn7-like element their
CHAPTER 3
5431 Summary
54Introduction
56Materials and methods
56L Bacterial and plasmid vectors
Media and solutions
56333 DNA
57334 gel electrophoresis
Competent preparation
57336 Transformation of DNA into
58337 Recombinant DNA techniques
58ExonucleaseIII shortening
339 DNA sequencing
633310 Complementation studies
64Results discussion
1 DNA analysis
64Analysis of ORF-l
72343 Glucosamine gene
77344 Complementation of by T ferrooxidans glmS
57cosmid pSIS1 pSlS20
58Plasmidmap of p81816r
Fig Plasmidmap of lS16f
60Fig 34 Restriction digest p81S1Sr and pSIS16f with
63DNA kb BamHI-BamHI
Fig Codon and bias plot of the DNA sequence of pSlS16
Fig 37 Alignment of C-terminal sequences of and Bsubtilis
38 Alignment of amino sequences organisms with high
homology to that of T ferrooxidans 71
Fig Phylogenetic relationship organisms high glmS
sequence homology to Tferrooxidans
31 Summary
A 35 kb BamHI-BamHI p81820 was cloned into pUCBM20 and pUCBM21 to
produce constructs p818 and p81816r respectively This was completely
sequenced both rrelct1()ns and shown to cover the entire gmS (184 kb) Fig 31
and 34 The derived sequence of the T jerrooxidans synthetase was
compared to similar nrvnC ~ other organisms and found to have high sequence
homology was to the glucosamine the best studied
eubacterium EcoU Both constructs p81816f p81 the entire
glmS gene of T jerrooxidans also complemented an Ecoli glmS mutant for growth on medium
lacking N-acetyl glucosamine
32 =-====
Cell wall is vital to every microorganism In most procaryotes this process starts
with amino which are made from rructClsemiddotmiddotn-[)ll()S by the transfer of amide group
to a hexose sugar to form an This reaction is by
glucosamine synthetase the product of the gmS part of the catalysis
to the 40-residue N-terminal glutamine-binding domain (Denisot et al 1991)
participation of Cys 1 to generate a glutamyl thiol ester and nascent ammonia (Buchanan
368-residue C-terminal domain is responsible for the second part of the ClUUU
glucosamine 6-phosphate This been shown to require the ltgtttrltgtt1iltn
of Clpro-R hydrogen of a putative rrulctoseam 6-phosphate to fonn a cis-enolamine
intennediate which upon reprotonation to the gives rise to the product (Golinelli-
Pimpaneau et al 1989)
bacterial enzyme mn two can be separated by limited chymotryptic
proteolysis (Denisot et 199 binding domain encompassing residues 1 to
240 has the same capacity to hydrolyse (and the corresponding p-nitroanilide
derivative) into glutamate as amino acid porI11
binding domain is highly cOllserveu among members of the F-type
ases Enzymes in this include amidophosphophoribosyl et al
1982) asparagine synthetase (Andrulis et al 1987) glucosamine-6-P synmellase (Walker et
aI 1984) and the NodM protein of Rhizobium leguminosarum (Surin and 1988) The
368-residue carboxyl-tenninal domain retains the ability to bind fructose-6-phosphate
The complete DNA sequence of T ferrooxidans glmS a third of the
glmU gene (preceding glmS) sequences downstream of glmS are reported in this
chapter This contains a comparison of the VVA r glmS gene
and Mycobacterium (422 ) An interesting observation was that the T ferrooxidans
glmS gene had comparatively high homology sequences to the Rhizobium leguminosarum and
Rhizobium meliloti M the amino to was 44 and 436
respectively A consensus Dalgarno upstream of the start codon
(ATG) is in 35 No Ecoli a70-type n r~ consensus sequence was
detected in the bp preceding the start codon on intergenic distance
between the two and the absence of any promoter consensus sequence Plumbridge et
al (1993) that the Ecoli glmU and glmS were co-transcribed In the case
the glmU-glmS --n the T errooxidans the to be similar The sequences
homology to six other amidotransferases (Fig 38) which are
(named after the purF-encoded l phosphoribosylpyrophosphate
amidotransferase) and Weng (1987)
The glutamine amide transfer domain of approximately 194 amino acid residues is at the
of protein chain Zalkin and Mei (1989) using site-directed to
the 9 invariant amino acids in the glutamine amide transfer domain of
phosphoribosylpyrophosphated indicated in their a
catalytic triad is involved glutamine amide transfer function of
73
Comparison of the ammo acid sequence alignment of ghtcosamine-6-phosphate
amidotranferases (GFAT) of Rhizobium meliloti (R_m) Rhizobium leguminnosarum (RJ)
Ecoli (E_c) Hinjluenzae Mycobacterium leprae (M_I) Bsubtilis (B_s) and
Scerevisiae (S_c) to that of Tferrooxidans Amino acids are identified by their single
codes The asterisks () represent homologous amino acids of Tferrooxidans glmS to
at least two of the other Consensus ammo acids to all eight organisms are
highlighted (bold and underlined)
1 50 R m i bullbullbullbullbullbull hkps riag s tf bullbullbullbull R~l i bullbullbullbullbullbull hqps rvaep trod bullbullbullbull a
a bullbullbullbullbullbull aa 1r 1 d bullbullbull a a bullbullbullbullbullbull aa inh g bullbullbullbullbullbull sktIv pmilq 1 1g bullbullbull a MCGIVi V D Gli RI IYRGYPSAi AI D 1 gvmar s 1gsaks Ikek a bullbullbullbull qfcny Iversrgi tvq t dgd bullbull ead
51 100 R m hrae 19ktrIk bull bullbull bullbull s t ateR-l tqrae 19rkIk bullbullbull s t atrE-c htIrI qmaqae bull bull h bullbull h gt sv aae in qicI kads stbull bullbull k bullbull i gt T f- Ivsvr aetav bullbullbull r bullbull gq qg c
DL R R G V L AV E L G VGI lTRW E H ntvrrar Isesv1a mv bull sa nIgi rtr gihvfkekr adrvd bullbullbull bullbull nve aka g sy1stfiykqi saketk qnnrdvtv she reqv
101 150 R m ft g skka aevtkqt R 1 ft g ak agaqt E-c v hv hpr krtv aae in sg tf hrI ksrvlq T f- m i h q harah eatt
S E Eamp L A GY l SE TDTEVIAHL ratg eiavv av
qsaIg lqdvk vqv cqrs inkke cky
151 200 bullbull ltk bullbull dgcm hmcve fe a v n bull Iak bullbull dgrrm hmrvk fe st bull bull nweI bullbull kqgtr Iripqr gtvrh 1 s bull bull ewem bullbullbull tds kkvqt gvrh hlvs bull bull hh bullbull at rrvgdr iisg evm
V YR T DLF A A L AV S D PETV AR G M 1 eagvgs lvrrqh tvfana gvrs B s tef rkttIks iInn rvknk 5 c Ihlyntnlqn ~lt klvlees glckschy neitk
74
201 R m ph middot middot middot R 1 ah- middot middot middot middot middot middot E c 1 middot middot middot middot middot middot middot Hae in 1 middot middot middot middot middot T f - c11v middot middotmiddot middot middot middot middot middot M 1 tli middot middot middot middot middot middot middot middot middot B s 11 middot middot middot middot middot S c lvkse kk1kvdfvdv
251 R m middot middot middot middot middot middot middot middot middot middot middot middot middot middot
middot middot middot middot R-1 middot middot middot middot middot middot E c middot middot middot middot middot middot middot middot Hae in middot middot middot middot middot middot middot middot middot middot middot middot T f shy middot middot middot middot middot middot middot middot middot middot middot middot 1M middot middot middot middot middot middot middot middot middot middot middot middot middot middot B s middot middot middot middot middot middot middot middot middot middot middot middot middot middot middot S c feagsqnan1 1piaanefn1
301 R m fndtn wgkt R-1 fneti wgkt E c rf er Hae in srf er T f- rl mlqq
PVTR V YE DGDVA R M 1 ehqaeg qdqavad B s qneyem kmvdd S c khkkl dlhydg
351 R m nhpe v R-1 nhe v E c i nk Hae in k tli T f- lp hr
~ YRHlrM~ ~I EQP AVA M 1 geyl av B-s tpl tdvvrnr S-c pd stf
401 Rm slasa lk R-1 slasca lk E c hql nssr Hae in hq nar-T f f1s hytrq
RVL A SiIS A VG W M 1 ka slakt B s g kqS c lim scatrai
T ferrooxidans (Tf) Scerevisiae (Saee) Mleprae (Myele) and Bsublilis (Baesu)
tr1 ()-ltashyc 8 ~ er (11
-l l
CIgt
~ r (11-CIgt (11
-
$ -0 0shy
a c
omiddot s
S 0 0shyC iii omiddot $
ttl ~ () tI)
I-ltashyt ()
0
~ smiddot (11
81
CHAPTER 4
8341 Summary
8442 Introduction
8643 Materials and methods
431 Bacterial strains and plasmids 86
432 DNA constructs subclones and shortenings 86
864321 Contruct p81852
874322 Construct p81809
874323 Plasmid p8I809 and its shortenings
874324 p8I8II
8844 Results and discussion
441 Analysis of the sequences downstream the glmS gene 88
442 TnsBC homology 96
99443 Homology to the TnsD protein
118444 Analysis of DNA downstream of the region with TnsD homology
LIST OF FIGURES
87Fig 41 Alignment of Tn7-like sequence of T jerrooxidans to Ecoli Tn7
Fig 42 DNA sequences at the proximal end of Tn5468 and Ecoli glmS gene 88
Fig 43 Comparison of the inverted repeats of Tn7 and Tn5468 89
Fig 44 BLAST results of the sequence downtream of T jerrooxidans glmS 90
Fig 45 Alignment of the amino acid sequence of Tn5468 TnsA-like protein 92 to TnsA of Tn7
82
Fig 46 Restriction map of the region of cosmid p8181 with amino acid 95 sequence homology to TnsABC and part of TnsD of Tn7
Fig 47 Restriction map of cosmid p8181Llli with its subclonesmiddot and 96 shortenings
98 Fig 48 BLAST results and DNA sequence of p81852r
100 Fig 49 BLAST results and DNA sequence of p81852f
102 Fig 410 BLAST results and DNA sequence of p81809
Fig 411 Diagrammatic representation of the rearrangement of Tn5468 TnsDshy 106 like protein
107Fig 412 Restriction digests on p818lLlli
108 Fig 413 BLAST results and DNA sequence of p818lOEM
109 Fig 414 BLAST results on joined sequences of p818104 and p818103
111 Fig 415 BLAST results and DNA sequence of p818102
112 Fig 416 BLAST results and nucleotide sequence of p818101
Fig 417 BLAST results and DNA sequence of the combined sequence of 113p818l1f and p81810r
Fig 418 Results of the BLAST search on p81811r and the DNA sequence 117obtained from p81811r
Fig 419 Comparison of the spa operons of Ecali Hinjluenzae and 121 probable structure of T ferraaxidans spa operon
83
CHAPTER FOUR
TN7-LlKE TRANSPOSON OF TFERROOXIDANS
41 Summary
Various constructs and subclones including exonuclease ill shortenings were produced to gain
access to DNA fragments downstream of the T ferrooxidans glmS gene (Chapter 3) and
sequenced Homology searches were performed against sequences in GenBank and EMBL
databases in an attempt to find out how much of a Tn7-like transposon and its antibiotic
markers were present in the T ferrooxidans chromosome (downstream the glmS gene)
Sequences with high homology to the TnsA TnsB TnsC and TnsD proteins of Tn7 were
found covering a region of about 7 kb from the T ferrooxidans glmS gene
Further sequencing (plusmn 45 kb) beyond where TnsD protein homology had been found
revealed no sequences homologous to TnsE protein of Tn7 nor to any of the antibiotic
resistance markers associated with Tn7 However DNA sequences very homologous to Ecoli
ATP-dependentDNA helicase (RecG) protein (EC 361) and guanosine-35-bis (diphosphate)
3-pyrophosphohydrolase (EC 3172) stringent response protein of Ecoli HinJluenzae and
Vibrio sp were found about 15 kb and 4 kb respectively downstream of where homology to
the TnsD protein had been detected
84
42 Transposable insertion sequences in Thiobacillus ferrooxidizns
The presence of two families (family 1 and 2) of repetitive DNA sequences in the genome
of T ferrooxidans has been described previously (Yates and Holmes 1987) One member of
the family was shown to be a 15 kb insertion sequence (IST2) containing open reading
frames (ORFs) (Yates et al 1988) Sequence comparisons have shown that the putative
transposase encoded by IST2 has homology with the proteins encoded by IS256 and ISRm3
present in Staphylococcus aureus and Rhizobium meliloti respectively (Wheatcroft and
Laberge 1991) Restriction enzyme analysis and Southern hybridization of the genome of
T ferrooxidans is consistent with the concept that IST2 can transpose within Tferrooxidans
(Holmes and Haq 1990) Additionally it has been suggested that transposition of family 1
insertion sequences (lST1) might be involved in the phenotypic switching between iron and
sulphur oxidizing modes of growth including the reversible loss of the capacity of
T ferrooxidans to oxidise iron (Schrader and Holmes 1988)
The DNA sequence of IST2 has been determined and exhibits structural features of a typical
insertion sequence such as target-site duplications ORFs and imperfectly matched inverted
repeats (Yates et al 1988) A transposon-like element Tn5467 has been detected in
T ferrooxidans plasmid pTF-FC2 (Rawlings et al 1995) This transposon-like element is
bordered by 38 bp inverted repeat sequences which has sequence identity in 37 of 38 and in
38 of 38 to the tnpA distal and tnpA proximal inverted repeats of Tn21 respectively
Additionally Kusano et al (1991) showed that of the five potential ORFs containing merR
genes in T ferrooxidans strain E-15 ORFs 1 to 3 had significant homology to TnsA from
transposon Tn7
85
Analysis of the sequence at the tenninus of the 35 kb BamHI-BamHI fragment of p81820
revealed an ORF with very high homology to TnsA protein of the transposon Tn7 (Fig 44)
Further studies were carried out to detennine how much of the Tn7 transposition genes were
present in the region further downstream of the T ferroxidans glmS gene Tn7 possesses
trimethoprim streptomycin spectinomycin and streptothricin antibiotic resistance markers
Since T ferrooxidans is not exposed to a hospital environment it was of particular interest to
find out whether similar genes were present in the Tn7-like transposon In the Ecoli
chromosome the Tn7 insertion occurs between the glmS and the pho genes (pstS pstC pstA
pstB and phoU) It was also of interest to detennine whether atp-glm-pst operon order holds
for the T ferrooxidans chromosome It was these questions that motivated the study of this
region of the chromosome
43 OJII
strains and plasm ids used were the same as in Chapter Media and solutions (as
in Chapter 3) can in Appendix Plasmid DNA preparations agarose gel
electrophoresis competent cell preparations transformations and
were all carned out as Chapter 3 Probe preparation Southern blotting hybridization and
detection were as in Chapter 2 The same procedures of nucleotide
DNA sequence described 2 and 3 were
4321 ~lllu~~~
Plasmid p8I852 is a KpnI-SalI subclone p8I820 in the IngtUMnt vector kb) and
was described Chapter 2 where it was used to prepare the probe for Southern hybridization
DNA sequencing was from both (p81852r) and from the Sail sites
(p8I852f)
4322 ==---==
Construct p81809 was made by p8I820 The resulting fragment
(about 42 kb) was then ligated to a Bluescript vector KS+ with the same
enzymes) DNA sequencing was out from end of the construct fA
87
4323 ~mlJtLru1lbJmalpoundsectM~lllgsect
The 40 kb EeoRI-ClaI fragment p8181Llli into Bluescript was called p8181O
Exonuclease III shortening of the fragment was based on the method of Heinikoff (1984) The
vector was protected with and ClaI was as the susceptible for exonuclease III
Another construct p818IOEM was by digesting p81810 and pUCBM20 with MluI and
EeoRI and ligating the approximately 1 kb fragment to the vector
4324 p81811
A 25 ApaI-ClaI digest of p8181Llli was cloned into Bluescript vector KS+ and
sequenced both ends
88
44 Results and discussion
of glmS gene termini (approximately 170
downstream) between
comparison of the nucleotide
coli is shown in Fig 41 This region
site of Tn7 insertion within chromosome of E coli and the imperfect gtpound1
repeat sequences of was marked homology at both the andVI
gene but this homology decreased substantially
beyond the stop codon
amino acid sequence
been associated with duplication at
the insertion at attTn7 by (Lichtenstein and as
CCGGG in and underlined in Figs41
CCGGG and are almost equidistant from their respelt~tle
codons The and the Tn7-like transposon BU- there is
some homology transposons in the regions which includes
repeats
11 inverted
appears to be random thereafter 1) The inverted
repeats of to the inverted repeats of element of
(Fig 43) eight repeats
(four traJrlsposcm was registered with Stanford University
California as
A search on the (GenBank and EMBL) with
of 41 showed high homology to the Tn sA protein 44) Comparison
acid sequence of the TnsA-like protein Tn5468 to the predicted of the
89
Fig 41
Alignment of t DNA sequence of the Tn7-li e
Tferrooxi to E i Tn7 sequence at e of
insertion transcriptional t ion s e of
glmS The ent homology shown low occurs within
the repeats (underl of both the
Tn7-li transposon Tn7 (Fig 43) Homology
becomes before the end of t corresponding
impe s
T V E 10 20 30 40 50 60
Ec Tn 7
Tf7shy(like)
70 80 90 100 110 120
Tf7shy(1
130 140 150 160 170 180 Ec Tn 7 ~~~=-
Tf7shy(like)
90
Fig 42 DNA sequences at the 3 end of the Ecoll glmS and the tnsA proximal of Tn7 to the DNA the 3end of the Tferrooxidans gene and end of Tn7-like (a) DNA sequence determined in the Ecoll strain GD92Tn7 in which Tn been inserted into the glmS transcriptional terminator Walker at ai 1986 Nucleotide 38 onwards are the of the left end of Tn DNA
determined in T strain ATCC 33020 with the of 7-like at the termination site of the glmS
gene Also shown are the 22 bp of the Tn7-like transposon as well as the region where homology the protein begins A good Shine
sequence is shown immediately upstream of what appears to be the TTG initiation codon for the transposon
(a)
_ -35 PLE -10 I tr T~ruR~1 truvmnWCIGACUur ~~GCfuA
100 no 110 110 110
4 M S S bull S I Q I amp I I I I bull I I Q I I 1 I Y I W L ~Tnrcmuamaurmcrmrarr~GGQ1lIGTuaDACf~1lIGcrA
DO 200 amp10 220 DG Zlaquot UO ZIG riO
T Q IT I I 1 I I I I I Y sir r I I T I ILL I D L I ilGIIilJAUlAmrC~GlWllCCCAcarr~GGAWtCmCamp1tlnmcrArClGACTAGNJ
Fig 45 (a) Alignment of the amino acid sequence of Tn5468 (which had homology to TnsA of Tn7 Fig 44) to the amino acid sequence of ORF2 (between the merR genes of Tferrooxidans strain E-1S) ORF2 had been found to have significant homology to Tn sA of Tn7 (Kusano et ai 1991) (b) Alignment of the amino acids translation of Tn5468 (above) to Tn sA of Tn7 (c) Alignment of the amino acid sequence of TnsA of Tn7 to the hypothetical ORF2 protein of Tferrooxidans strain E-1S
(al
Percent Similarity 60000 Percent Identity 44444
Tf Tn5468 1 LARQRYGVDEDRVARFQKEGRGQGRGADYHPWLTIQDVPSQGRSHRLKGI SO 11 1111111 I 1111 1111 I I 111
Fig 49 (a) Results of the BLAST search on the inverted and complemented nucleotide sequence of 1852f (from the Sall site) Results indicate amino acid sequence to the TnsC of Tn7 (protein E is the same as TnsC protein) (b) The nucleotide sequence and open reading frame of p81852f
High Prob producing Segment Pairs Frame Score P(N)
IB255431QQECE7 protein E - Escher +1 176 15e-20 gplX044921lSTN7EUR 1 Tn7 E with +1 176 15e-20 splP058461 ECOLI TRANSPOSON TN7 TRANSPOSITION PR +1 176 64e-20 gplU410111 4 D20248 gene product [Caenorhab +3 59 17e-06
IB255431QQECE7 ical protein E - Escherichia coli transposon Tn7 (fragment)
417 (al BLAST results obtained from combined sequence of (inverted and complemented) and 1810r good sequence to Ecoli and Hinfluenzae DNA RecG (EC 361) was found (b) The combined sequence and open frame which was homologous to RecG
444 Analysis of DNA downstream of region with TnsD homology
The location of the of p81811 ApaI-CLaI construct is shown in Fig 47 The single strand
sequence from the C LaI site was joined to p8181Or and searched using BLAST against the
GenBank and EMBL databases Good homology to Ecoli and Hinjluezae A TP-dependent
DNA helicase recombinase proteins (EC 361) was obtained (Fig 417) The BLAST search
with the sequence from the ApaI end showed high sequence homology to the stringent
response protein guanosine-35-bis (diphosphate) 3-pyrophosphohydrolase (EC 3172) of
Ecoli Hinjluenzae and Scoelicolor (Fig 418) In both Ecoli and Hinjluenzae the two
proteins RecG helicase recombinase and ppGpp stringent response protein constitute part of
the spo operon
445 Spo operon
A brief description will be given on the spo operons of Ecoli and Hinjluenzae which appear
to differ in their arrangements In Ecoli spoT gene encodes guanosine-35-bis
pyrophosphohydrolase (ppGpp) which is synthesized during stringent response to amino acid
starvation It is also known to be responsible for cellular ppGpp degradation (Gentry and
Cashel 1995) The RecG protein is required for normal levels of recombination and DNA
repair RecG protein is a junction specific DNA helicase that acts post-synaptically to drive
branch migration of Holliday junction intermediate made by RecA during the strand
exchange stage of recombination (Whitby and Lloyd 1995)
The spoS (also called rpoZ) encodes the omega subunit of RNA polymerase which is found
associated with core and holoenzyme of RNA polymerase The physiological function of the
omega subunit is unknown Nevertheless it binds stoichiometrically to RNA polymerase
119
Fig 418 BLAST search nucleotide sequence of 1811r I restrict ) inverted and complement high sequence homology to st in s 3 5 1 -bis ( e) 3 I ase (EC 31 7 2) [(ppGpp)shyase] -3-pyropho ase) of Ecoli Hinfluenzae (b) The nucleot and the open frame with n
Add 4 ml 52 mix by inversion at room temp for 5 mins
Add 4 ml 53 Mix by to homogenous suspension
Spin at 15 K 40 mins at 4
remove to fresh tube
Equilibrate column with 2 ml N2
Load supernatant 2 to 4 ml amounts
Wash column 2 X 4 of N3 Elute the DNA the first bed
each) add 07
volumes of isopropanol
Spin at 4 Wash with 70 Ethanol Resuspended pellet in n rv 100 AU of TE and scan
volume of about 8 to 10 drops) To the eluent (two
to
140
SEQUITHERM CYCLE SEQUENCING
Alf-express Cy5 end labelled promer method
only DNA transformed into end- Ecoli
The label is sensitive to light do all with fluorescent lights off
3-5 kb
3-7 kb
7-10 kb
Thaw all reagents from kit at well before use and keep on
1) Label 200 PCR tubes on or little cap flap
(The heated lid removes from the top of the tubes)
2) Add 3 Jtl of termination mixes to labelled tubes
3) On ice with off using 12 ml Eppendorf DNA up to
125 lll with MilliQ water
Add 1 ltl
Add lll of lOX
polymeraseAdd 1 ltl
Mix well Aliquot 38 lll from the eppendorf to Spin down
Push caps on nrnnp1
141
Hybaid thermal Cycler
Program
93 DC for 5 mins 1 cycle
93degC for 30 sees
55 DC for 30 secs
70 DC for 60 secs 30 cycle 93 DC_ 30s 55 DC-30s
70degC for 5 mins 1 cycle
Primer must be min 20 bp long and min 50 GC content if the annealing step is to be
omitted Incubate at 95 DC for 5 mins to denature before running Spin down Load 3 U)
SEQUITHERM CYCLE SEQUENCING
Ordinary method
Use only DNA transformed into end- Ecoli strain
3-5 kb 3J
3-7 kb 44
7-10 kb 6J
Thaw all reagents from kit at RT mix well before use and keep on ice
1) Label 200 PCR tubes on side or little cap flap
(The heated lid removes markings from the top of the tubes)
2) Add 3 AU of termination mixes to labelled tubes
3) On ice using l2 ml Eppendorf tubes make DNA up to 125 41 with MilliQ water
142
Add I Jtl of Primer
Add 25 ttl of lOX sequencing buffer
Add 1 Jtl Sequitherm DNA polymerase
Mix well spin Aliquot 38 ttl from the eppendorf to each termination tube Spin down
Push caps on properly
Hybaid thermal Cycler
Program
93 DC for 5 mins 1 cycle
93 DC for 30 secs
55 DC for 30 secs
70 DC for 60 secs 30 cycles
93 DC_ 30s 55 DC-30s 70 DC for 5 mins 1 cycle
Primer must be minimum of 20 bp long and min 50 GC content if the annealing step is
to be omitted Incubate at 95 DC for 5 mins to denature before running
Spin down Load 3 ttl for short and medium gels 21t1 for long gel runs
143
REFERENCES
144
REFERENCES
Abrahams J P Lutter R Todd R J van Raaij M J Leslie A G W and Walker J E 1993 Inherent asymmetry of the structure of F1-ATPase from bovine heart mitochondria at 65 Aresolution EMBO J 121775-1780
Abrahams J P Leslie A G W Lutter R and Walker 1 E 1994 Structure at 28 A resolution of FeATPase from bovine heart mitochondria Nature 370621-628
Adzumah K and Mizuuchi K 1988 Target immunity of Mu transposition reflects a differential distribution of Mu B protein Cell 53257-266
Akey C W Crepeau R H Dunn S D McCarty R E and Edelstein S J 1983 Electron microscopy of single molecules and crystals of F1-ATPases EMBO J 21409-1415
Allet B 1979 Mu insertion duplicates a 5 bp sequence at the host inserted site Cell 16 123shy129
Amzel L M and Pedersen P L 1983 Proton ATP-ases structure and mechanism Ann Rev Biochem 52801-824
Andersson L Mac Neela J and Wolfenden R 1985 Use of secondary isotope effects and varying pH to investigate mode of binding of inhibitory amino aldehydes by leucine aminopeptidase Biochem 24330-333
Andrews G F Dugan R P and Stevens C J 1992 Combining physical and bacterial treatment for removing pyritic sulfur from coal In Processing and Utilization of High-sulfur coals IV Dugan P R D Quigley and Y Attia (eds) p 515 Elsevier New York
Andrulis I L Chen J and Ray P N 1987 Isolation of human cDNAs for asparagine synthetase and expression in Jansen rat sarcoama cells Mol Cell BioI 72435-2443
Andruszkiewicz R Milewsky S Zieniawa T and Borowski E 1990 Anticandidal properties of N3 -(4-methoxy-fumaroyl)-L-2 3-diamino propanoic acid oligopeptides 1 Med Chern 33132-135
Arciszewska L K Drake D and Craig NL 1989 Transposon Tn7 cis-acting sequences in transposition and transposition immunity J Mol BioI 20735-42
Bachmann B J 1983 Linkage map of Escherichia coli K-12 edition 7 Microbiol Rev 47180-230
145
B Plumbridge 1 A Cochet D Souza J M M M and Calcagno M 1988 Cordinated regulation of amino J
Bacteriol 1754951-4956
0 B Vermoote P V and Le Goffic F 1993 synthetase from Ecoli lUllL- mechanism and inhibition by N3-fumaroyl-2 3-diaminopropionic derivatives Biochem 27 2282-2287
Vermoote P Haumont PY syrlthl~ta~e from Escherichia coli purification site location Biochemistry 26 1940-1948
Badet B Inagaki K Soda K Walsh dependent inhibition of Bacillus stearothermophilus alanine racemase by phosphonate isomers by isomerization to non covalent enzyme-(1-aminoethyl) complexes Biochem 253275-3282
Badet-Denisot M A and Badet of glucoseamine-6shyphosphate synthetase by diethylpyrocarbonates histidine requirement for enzymatic activity Arch Biochem Biophys
Bainton R Gamas P and transposition in vitro proceeds through an excised transposon intermediate (JpnIPtlltpi1 111 breaks in DNA Cell 65805-816
Barry G 1986 Permanent insertion of v~u into the chromosomes of soil bacteria BioTechnology 4446-449
Barth P T Datta N Grinter N S 1976 Transposition a deoxyribonucleic acid sequence trimethoprim and streptomycin resistances from R483 to other replicons 1 Bacteriol 125800-810
Bates C J Adams W and R R 1968 Control of the formation of uri dine diphospho-N-acetyl and glycoprotein synthesis in rat liver J Chern 2411705-17
Benjamin N 1989 Intramolecular transposition of TnlD Cell 59373shy383
Bennet R L and Malamy M resistant mutants of Escgerichia coli and phosphate Commun 40496-503
Benneth1 1978 Bacterial leaching patterns on pyrite crystal J Bacteriol
146
Berg D E Davies 1 Allet B and Rochaix 1 D 1975 Transposition of R factor genes to bacteriophage lambda Proc Natl Acad Sci USA 723268-3632
Berg D E and Drummond M1978 Absence of DNA sequences homologous to transposable element Tn5 (Kan) in the chromosome of Ecoli K-12 J Bcateriol 136419-422
Birkenhager R Hoppert M Deckers-Hebestriet G Mayer F and Altendorf K 1995 The Fo complex of the Ecoli ATP synthase investigation by electron imaging and immunoelectron microscopy Eur J Biochem 23058-67
Bjqgtrbaek C Foersom V and Michelsen O 1990 The transmembrane topology of the a subunit from ATPase in Escherichia coli analysed by PhoA protein fusions FEBS lett 26031-34
Boekemia E J Berden JA and van Heel MG 1986 sructure of mitochondrial F1-ATPase studied by electron microscopy and image processing Biochim Biophys Acta 851353-360
Bolton E Glynn P and OGara F 1984 Site specific transposition of Tn7 into a Rhizobiwn meliloti megaplasmid Mol Gen Genet 193153-157
Boos W 1974 Bacterial transport Annu Rev Biochem 43123-146
Boursaux-Eude C Girons I S and Zuerner R 1995 IS1500 an IS3-like element from Leptospira interrogans Microbiol 1412165-2173
Boyer P D 1993 The binding change mechanism for ATP synthase-some probabilities and possibilities Biochim Biophys Acta 1140215-250
Brachet P Eisen H and Rambach A 1970 Mutations in coliphage lambda affecting the expression of replicative functions 0 and P Mol Gen Genet 108266-276
Brink J Boekemia E 1 and van Bruggen E F 1987 The structure of NADH ubiquitone oxidoreductase fron beef heart mitochondria Crystals containing an octameric arrangement of iron-sulphur protein fragments Eur J Biochem 166287-294
Brock T D and Gustafson J 1976 Ferric iron reduction by sulphur- and iron-oxidizing bacteria Appl Environ Microbiol 32 567-571
Brown L D Dennehy M E and Rawlings D E 1994 The FI genes of the FIFo ATP synthase from the acidophilic bacterium Thiobacillus jerrooxidans complement Escherichia coli FI unc mutants FEMS Microbiol Lett 12219-25
Buchanan 1 1 Adv The Amidotransferases Enzymol 3991-183
Buckley Science
Caruso M Pseudomonas nOVHrn
oxidation of pyrite Applied
1 1982 Interactions Mol Gen Genet
phage 1
Chmara H by Biophysica
synthetase from bacteria antibiotic tetaine Biochimica et
Microbiol Lett 11197-206 controls on the oxidation of refractory Barberton
genetic elements and evolution Nature 263731shyCohen S N 1976 738
Corbet C M and 1 Ingledew 1987 Is oxidation by Thiobacillus ferrooxidans
Couillard D and ~~b 118(5)808-81
Metallurgical residue V UlllLltHIH of metals from sewage sludge J
Cox G B a reassessment of the
F and Hatch L 1986 The mechanism of ATP synthase of the b and a subunits Biochim Acta 84962-69
Craig N L 1995 Unity in Science 270253-254
Craig N and Gamas P 1 Purification and characterization of a transposition protein that binds ATP DNA Nuc Acids Res
1 2873-97 C A 1990 in response to environmental stress Advances in
alUIUH~ on the activity subunit b-specific polyc1onal
G Simoni and Altendorf K 1992 Influence vlJnu~ of the ATP synthase of t-S(nel~lCn
1 BioI Chern 26712364shy
M A Le Goffic F and 1991 Glucosamine- 6P two proteins on limited proteolysis ltVU Biophys 288225-230
148
Dobrogosz W J 1968 _l in Escherichia coli and its to catabolite repression J
Doublet P 1 van Heijenoort and 1993 The murI gene of is an essential gene that encodes klUltUllU~v racemase activity J Bacteriol 1752970-2979
P J van Heijenoort 1992 Identification of the Ecoli which is required for the D-glutamic acid a specific component peptidoglycan 1 Bacteriol
Garren A Garen and Torri ani 1961 Genetic control of repression UCUlllV phosphatase in Ecoli 1 Mol BioI
D J and Boeke 1 D 1990 A 1-1-0- structure is required for Ty1 transposition Genes Develop 4324-330
1982 Transposition of Tn7 occurs at a on Caulobacter crescentus 10SIClmle 1 Bacteriol 151 1056-1 058
H 1990 Molecular IHovUUU)11 -transporting 345-391 In T 12 Bacterial
Press Ltd London
transport and coupling by the synthase insights mechanism of function J Bioenerg 24485-491
1992b Subunit c of FIFo ATP role m transduction Biochim Biophys Acta 1101 v--rJ
Eliopoulos E E Jackson P 1 Keen IN HUIUVI L Thompson P and 1 Structure of a 16 kDa integral AU-UUl that identity to
of the vacuolar H+ -ATPase Protein 15
Fling M 1 C 1985 Nucleotide sequence of encoding the amino glycoside-modifying enzyme 3(9)-O-nucleotidyltransferase Res 137095-7106
Fling M and C l-1UUJIU nucleotide sequence of dihydrofolate rhnrpri by Tn7 Nucleic Acids Res 115
Flores Llchtenstem C P 1990 DNA sequence anlysis of tnsA for the Tn7 transposition Nucleic Acids 18901 911
149
149
Foster D L and Fillingame R H 1982 Stoichiometry of subunits in the H+-ATPase complex of Escherichia coli J BioI Chern 2572009-2015
Fraga D Hermolin J Oldenburg M Miller MJ and Fillingame R H 1994 Arginine 41 of subunit c of Escherichia coli H+-A TP synthase is essential in binding and coupling of F to Fo J BioI Chern 2697532-7537
Friedl P Hoppe J Gunsalus R P Michelsen 0 von Meyenburg K and Schairer H U 1983 Membrane integration and function of the three Fo subunits of the A TP-synthase of Escherichia coli K12 EMBO 1 299-103
Frisa P S and Sonneborn D R 1982 Developmentally regulated interconversion between end product inhibitable and non-inhibitable forms of a first pathway-specific enzyme activity can be mimicked in vitro by dephosphorylation reactions Proc Natl Acad Sci USA 796289-6293
Fujiwara T and Mizuuchi K 1988 Retroviral DNA integration Structure of an integration intermediate Cell 54497-504
Galas D J and Chandler M 1989 Bacterial insertion sequences In Mobile DNA Berg D E and Howe M M (eds) Washington D C American Society for Microbiology pp 109shy162
Gay N J Tybulewicz V L1 Walker JE 1986 Insertion of transposon Tn7 into the Ecoli gmS transcriptional terminator Biochem J 234 111-117
Gentry D R and Cashel M 1995 Cellular localization of the Escherichia coli SpoT protein J Bacteriol 177 3890-3893
Gentry D R Xiao H Burgess R R and Cashel M 1991 The omega subunit of Ecoli K-12 RNA polymerase is not required for stringent RNA control in vivo J Bacteriol 173 3901-3903
Girvin M E and Fillingame R H 1993 Helical structure and folding of subunit c of FIFo ATP synthase IH NMR resonace assignments and NOE analysis Biochemistry 3212167shy12177
Gogol E P Lucken U Bork T and Capaldi R A 1989 Molecular architecture of Escherichia coli F Adenosinetriphosphatase Biochemistry 284709-4716
Gogol E P Aggeler R Sagermann M and Capaldi R A 1989 Cryoelectronic Microscopy of Escherichia coli FI Adenosinetriphosphatase Decorated with Monoclonal Antibodies to Individual Subunits of the complex Biochemistry 284717-4724
150
Heinikoff S 1984
Golinelli-Pimpaneaux B and Badet involvement of Lys603 from Ecoli glucosamoine-6-phosphate synthase substrate fructose-6-phosphate 1 Biochem 201175-182
Gottesman M M and Rosner 1 L of a detenninant of uvu_bullu
resistance by coliphage lambda Sci USA 725041-5045
Grunstein M and Hogness D
Colony hybridization a method for isolation cloned DNAs that contain a 1Jbullu Proc NatL Acad Sci USA 723961-3965
Hauer B and Shapiro 1 A 1984 Control of Tn7 transposition Mol Gen Genet 194
Hedges R W and Jacob Transposition of ampicillin resistance from to other replicons MoL 1-40
Hennolin 1 Gallant 1 psi subunit in the Fo sector of the H+ -ATPase of Ecoli J Bioi
R H 1983 Topology organization and function
Hennolin J and R 1989 Assembly of Fo sector of synthase Interdepenndence of subunit insertion into the membrane 1 2817
van Montagu M Holsters Zambryski P Beuckeleer M Willmitzer and Schell 1 M 1980 interaction of
DNA plant cells Proc Soc Lond 210351-365
P 1972 Insertion mutations control region of Physical characterization of the mutants Mol Gen Genet
115266-276
Holmes applications pI
UI Haq 1990 Adaptation of Thiobacillus vu)nuu for industrial In J Salley R G McCready Wichlacz (ed)
1989 CANMET Ottawa Canada
Holtje J V and U Schwarz 1985 Biosynthesis and growth murein sacculus p 77shy119 InN (ed) Molecular cytology of Escherichia Academic Press Inc New
Acad Sci USA
Heffron E Reubens C and which mediates ampicillin
Translocation of a plasmid DNA sequence nature and specificity of Natl
digestion with exonuclease III creates fl tr breakpoints 1-369
Hernalsteens J M Agrobacterium
Hirsch H the galactose nnnn fJu
151
Holzenburg A Jones P c Franklin T Padi J B and Finbow M E 1993 Evidence for a common structure 1 Biochem 21321-30
Hoppe 1 and Sebald W 1986 Topological pathway of the protons through Fo is provided by amino acid the lipid phase Biochimie (Paris) 68427-434
S Otsubo H Davidson N and 1975 Electron microscope heteroduplex of sequence relations among plasmids identification and mapping of the
insertion sequences IS] and IS2 in F and R plasmids J Bacteriol 22762-775
Inoue c Sugawara K and 1991 regulatory gene in Thiobacillus ferrooxidans is spaced apart from Mol Microbiol 52707-2718
Ish-Horowicz D and Burke and cosmid cloning Nucleic Acids Res 92989-2998
Johnston B Clennell M and D 1995 Structure and function of Tn5467 a Tn2l-like transposon located on T jerrooxidans broad host range plasmid Appl Envir Micro
Kahmann R and Kamp Nucleotide sequences of the attachment bacteriophage Mu DNA Nature 280247-250
Kahn K and Schaefer M R Characterization of trans po son 5469 from cyanobacterium Fremyella diplosiphon J 1777026-7032
Karavaiko G Golovacheva S Pivovarova T A Tzaplina I A and Vartanjan 1988 Thermophilic ltPM Sulfobacillus page 29-41 In Biohydrometallurgyshy87 Norris P and Science and Technology Letters Kew 1
Kennedy J and Humpphreys J D 1976 Microbial cells living immobilised on Nature 261242-244
Koonin 1993 SpoU protein of Escherichia coli belongs to a new family of Nucleic Acids Res 19
Kopecko J and site specific recA mdependtm recombination between bacterial Pt of palindromes at the N ad Acad Sci USA
on L-glutamine D-fructose ud()transtiera~e 1 BioI
152
Krumholz L R Esser U and Simoni RD 1989 Nucleotide sequence of the unc operon of Vibrio alginolyticus Nucleic Acids Res 177993-7994
Kubo K and Craig N 1990 Bacterial transposon Tn7 utilizes two classes of target sites 1 Bacteriol 1722774-2778
Kucharczyk N Denisot M A Le Goffic F and Badet B 1990 Glucosarnine-6-phosphate synthase from Ecoli detennination of the mechanism of inactivation by N3 fumaroyl-L-2-3 diarninoproprionic derivatives Biochemistry 293668-3676
Kusano M Takeshima T Inoue C and Sugawara K 1991 Evidence for two sets of structural genes coding for Ribulose biphosphate carboxylase in Thiobacillus ferrooxidans 1 Bacteriol 1737313-7323
Lane Dl A P Harrison lnr D Stahl B Pace SJ Giovannoni GJ Olsen and N R Pace 1992 Evolutionary relationship among sulphur- and iron-oxidizing eubacteria 1 Bacteriol 174267-278
Lee C H Bhagwhat A and Heffron F 1983 Identification of a transposon TnJ sequence required for transposition immunity Natl Acad Sci USA 806765-6769
Lewis M 1 Chang 1 A and Somoni R D 1990 A topological analysis of subunit a from Escherichia coli F1Fo-ATP synthase predicts eight transmembrane segments 1 BioI Chern 265 10541-10550
Lichtenstein C P and Brenner S 1982 Unique insertion site of Tn7 in the Ecoli chromosome Nature (London) 297601-603
Lichtenstein C P and Brenner S 1981 Site-specific properties of transposition to the Ecoli chromosome Mol Gen Genet 183380-387
Liu X Petersson S and Sandstrom A Mesophilic versus moderate thennophilic bioleaching Biohydrometallurgy Technologies Vol 1 A E Tonna 1 E Wey and Lakshmanan V 1 (ed) pp 29-38
Lizarna H M and Sankey B M 1993 Oxidation of H2S by Thiobacillus thiooxidans is inhibited by substrate and methane BiohydrometaHurgy Technologies Vol II A E Tonna 1 E Wey and C L Brierley (ed) pp 339-364
Lundgren D G and Silver M 1980 Ore leaching by bacteria Ann Rev Microbiol 34263shy268
Makino K Amemura M Shinagawa H Kobayashi A and Nakata A 1985 Sequence of the genes involved in phosphate transport and regulation of the phosphate regulon in Escherichia coli 1 Mol BioI 184231-240
Makino K Shinagawa Amemura M Kimura S Nakata A and Ishihama 1988 Euuv1J of the phosphate regulon of Ecoli Activation of pstS by the PhoB
protein in vitro 1 Mol BioI 20385-95
Makino K Shinagawa H Amemura M Yamada M and 1990 Signal transduction in the phosphate regulon of the Escherichia coli involves phosphotransfer nprlrJp~middotn PhoR and PhoB proteins J Mol BioI 21055
Malamy 1966 Frameshift mutations in the nnlnn of Escherichia coli Cold Harb Symp Quant BioI 31189-201
Malamy M H 1972 Electron microscopy insertions in the lac operon of Escherichia coli MoL Gen
Malamy M H and Bennett R L 1970 mutants of Ecoli and phosphate transport Biochem Biophys Res Commun
Maniatis T Fritsch EF Sambrook J 1982 nn A laboratory manual Cold Spring Harbor Laboratory Press Cold
McKnight G L S L Mudri SH Mathewest R S Marshall P O Sheppard and P J OHara 1990 Molecular cloning synthesis and bacterial expression of human glutaminefructose-6-phosphate UUAUU u J Chem 26725208-25212
Medveczky N and H Rosenburg 1970 phosphate-binding protein of Escherichia Biochim Biophys Acta 211 158-168
Mei B and Zalkin H 1989 A Cysteine-Histidine-Aspartate catalytic triad is involved in Glutamide Amide Transfer Function purF-type Glutamine amodotransferases 1 BioI Chem 264 16613-16619
Mengin-Lecreulx D and J van 1993 Identification of glmU gene encoding Nshyacetylglucosamine in Escherichia coli J Bacteriol 1756150shy6157
Michaelis M J and Criddle M J 1970 Mitochondrial DNA and mutants in Saccharomyces cerevisiae Biochem Genet 5487-495
Michaelis Starlinger P 1969 Two insertions in the jO v
operon having different homologous DNA sequences MoL Gen Genet 10437 377
Miller J H Calos Transposable elements Cell 20579-595
154
Miller J Oldenburg M and Fillingame R 1990 The essential carboxyl group of in subunit c the FIFo ATP synthase can be and H( +)-translocating function retained Proc NatL Acad 874900-4904
Mitcell P 1966 Chemiosmotic coupling in - and phosphorylation BioL Rev 41455-502 (1966)
Mizuuchi K 1984 Mechanism of transposition of bacteriophage Mu Polarity of the strand reaction at the initiation of the transposon Cell 39395-404
C Ph de Donato Berthelin J Surface oxidized species a key factor in the study of bioleaching processes Biohydrometallurgical In A J
Weyand V I Lakshmanan ed 1993 Vol 1 175-184
A Ishino Shinagawa H Makino K and M 1987 Nucleotide of the iap gene responsible for alkaline phosphatase isoenzyme conversion in Ecoli
identification of product 1 1695429-5433
K 1989 The tsr gene-coding prevents thiopeptin from inhibiting ppGpp synthesis in Streptomyces lividans FEMS Microbiol Lett
Ogasawara N and Yoshikawa H 1992 and their organIzatIon in the repnc()n of the bacterial chromosome Mol Microbiol 6(5)629-634
Ohtsubo Davidson N and 1975 microscope heteroduplex studies of sequence relations among plasmids identification and mapping of the insertion sequences 1 and IS2 in F and R plasmids 1 122746-775
Olson G J 1994 Microbial oxidation gold ores and gold bioleaching FEMS un Lett 119 1-6
Orle K A N L 1991 Identification of transposition prroteins by the bacterial transposon [corrected republished originally printed in Gene 1990 Nov 30 96(1) Gene 10425-31
Ouellette M Roy P Homology of ORFs from and from to site specific recombinases 1987 Nucl Acids 10055-10059
Murein synthesis p663-671ln Neidhardt J L Ingraham B Louw M~ M Schaechter H E Umbarger (ed) Escherichia coli and Salmonella
ryphimurium cellular and bioI voL 1 American Society Microbiology Washington
Perlin D S and Senior A Functional and cross-reactivity of antibody to purified subunit b (uncF protein) of Escherichia coli proton-ATPase Arch Biochem Biophys 236603-611
155
Plumbridge J A O Cochet Souza J M Altramirano M M Calcagno M L and Badet B 1993 Coordinated regulation of amino sugar-synthesizing and -degrading enzymes in Escherichia coli K-12 J Bacteriol 175(16)4951-4956
Pretorius I M Rawlings D E and Woods D R 1986 Identification and cloning of Thiobacillus jerrooxidans structural Nif genes in Escherichia coli Gene 4559-65
Qadri M I Flores C c Davis J and Lichtenstein C 1989 Genetic analysis of attTn7 the transposon Tn7 attachment site in Escherichia coli using a novel M13-based transduction assay 1 Mol BioI 20785-98
Radstrom P Skold 0 Swedberg J Roy P H and Sundstrom L 1994 Tn5090 of plasmid R751 which carries integron is related to Tn7 Mu and retroelements J Bacteriol 1763257-3268
Raetz C R H 1987 Structure and biosynthesis of lipid A in Escherichia coli pp 498-503 In F C Niedhardt J L Ingraham K B Low B Magasanik M Schaechter and H E Umbarger (ed) Escherichia coli and Salmonella typhimurium cellular and molecular biology vol 1 American Society for Microbiology Washington DC
Rao N N and Torriani A 1990 Molecular aspects of phosphate transport in Escherichia coli Mol Microbiol 4(7) 1083-1090
Rawlings DE D R Woods and NP Mjoli 1991 The cloning and structre of genes from the autotrophic biomining bacterium Thiobacillusjerrooxidans p 215-237 In PJ Greenaway (ed) Advances in gene technology vol 2 JAI Press London
Richet E and O Raibaud 1989 MalT the regulatory protein of the Escherichia coli maltose system is an A TP-dependent transcriptional activator EMBO 1 8981-987
Rogers M Ekaterrinaki N Nimmo E and Sherratt D 1986 Analysis of Tn7 transposition Mol Gen Genet 205550-556
Ronson C W Nixon B T Albright L M anf Ausubel F M 1987 Rhizobium meliloti ntrA (rpoN) gene is required for diverse metabolic functions J Bacteriol 1692424-2431
Rosenburg H Gerdes R G and Chegwidden K 1977 Two systems for the uptake of phosphate in Ecoli JBacterioL 131505-511
Rosenburg H 1987 In ion transport in Prokaryotes Rosen B P and Silver S (eds) New York Academic Press pp 205-248
Ross D G Swan 1 and N Klecker 1979 Physical structures of TnlO-promoted deletions and inversions role of 1400 base pairs inverted repetitions Cell 16721-731
156
Saedler H and P 19670 mutation in the galactose operon in Ecoli II Physiological characterization MoL Gen Genet 100 190-202
Saedler P 1968 00 and strong polar mutations in the Genet 102353-363
Sambrook J Fritsch Maniatis 1989 Molecular cloning a laboratory manual Cold Harbor Laboratory Cold Spring Harbor New York
Sand W Gehrke T Hallmann Rohde K Sobotke B and Wintzien S 1993 Bioleaching metal sulphides The importance of Leptospirillum ferrooxidans Biohydromemetallurgical Technologies Voll pp 15-25
Sanger Nicklen and Coulson A DNA sequencing with chain-tennination inhibitors Natl Acad USA 745463-5467
Schneider E and Altendorf K All the three subunits are required for an active proton channel (Fo) of Escherichia coli synthase (FIFo) ~-
518
Schneider E and Allendorf K 1987 Bacterial adenosine 5-triphosphate synthase purification and reconstitution complexes and biochemical and functional characterization of subunits Microbio Rev 51477-497
Schrader 1 A and D S Holmes 1988 Phenotypic switching Thiobacillus ferrooxidans J BacterioL 1703915-3023
Scordilis G E H and Lassie 1987 Identification of transposable elements which activates gene expression in Pseudomonas cepacia J Bacteriol 1698-13
Sekine Eisaki N and Ohtsubo E 1996 Identification and characterization of the lS3 molecules generated by breaks 1 BioL Chern 271197-202
Senior A E 1990 The proton-trans locating of Escherichia coli Annu Rev Biophys Chern 194-41
Shapiro 1 and Adhya S 1969 The jLU operon of K-12 n A deletion analysis of the structure polarity 62249-264
J A 1969 Mutations caused by insertion genenc material the galactose operon Escherichia 1 MoL BioI 4093-105
Silvennan M P 1967 Mechanism of bacterial pyrite oxidation 1 Bacteriol 941046-1051
157
C C ElIson and Levinson 1983 Identification of the typel trimethoprim resistance reductase specified by the R-plasmid R43 comparison with procaryotic and eucaryotic dihydrofolate reductase 1 Bacteriol 155 100 1-1008
Smith and Jones P transposition a multigene process Identification of a regulatory product 147915-7927
Sprague Jr Bell R M Cronan 1 A mutant of Ecoli auxotrophic for organic phosphates evidence two defects in phosphate Mol Gen Genet 14371-77
Starlinger and Michaelis 1968 Suppression the sequential aO[)ealame of the galactose enzymes in a transferase amber mutant coli Mol Genet 102367-369
Starlinger 1977 IS elements in microorganisms MicrobioL Immunol
Steffens K Schneider E Herkernhoff B Schmid Altendorf K portion of Escherichia coli A TP synthase resolution of from subunit b J BioI Chern 2625866-5869
Steffens A Deckers-hebestreit G and Altendorf K 1987b The and functional relationship of A TP synthases (FoFI) from Ecoii and the thermophilic bacterium PS3 J Biol 2626334-6338
Strominger J and M S Smith 1959 Uridine diphosphoacetylglucosamine pyrophosphorylase 1 BioI Chern 2341822-1827
Sundstrom Skold 1990 dhfrI trimethoprim resistance gene of can be found at in other genetic surroundings Antimicrob Agents Chemother 34642shy650
Sundstrom L Roy P H and Skold Site-specific of three cassettes in Tn7 J Bacteriol 1733025-3028
Surin B P and Downie JA 1988 of the Rhizobium leguminosarum nodLMN involved efficient host-specific nodulation Mol Microbiol 2 173-183
B P Dixon N and Rosenburg 1986 Purification PhoU protein a OClItnl
regulator of the pho regulon of Ecoli K-1 J Bacteriol 168631
B P D A Jans A L Fimmel Shaw GB Cox Rosenburg Structural gene for the phosphate-repressible phosphate binding of Escherichia coli
own promoter nucleotide of the phoS 1 Bacteriol
158
Suzuki I Chang W and Takeuchi 1994 Oxidation of organic compounds by Thiobacilli Symp Series 55060-67
Takeyama M S Y Noumi T Maeda Ishibashi S and Futai M 1988 Beta subunit of Ecoli amino acid replacement within a conserved sequence G-X-X-X-X-GshyK-TS) v~u binding proteins lett 218222-226
Thomson JA M Hendson and R M 1981 Mutagenesis by insertion of drug resistance Tn7 into a vibrio 11 J Bacteriol
Tichy R Grotenhuis J T c Janssen van Houten R Rulkens and Lettinga 1993 Application of the sulphur cycle bioremediation of soils polluted with heavy metals In Int Conf Contaminated 93 ed F Arendt G J Annokee R Bosman and W J van der Brink Kluwer Academic Publishers Dordrecht pp 1461-1462
G and Mayer An electron approach to the quaternary structure of mitochondrial Eur J Biochem 13237-45
J and Tschape streptothricin resistance transposons Tn1825 and Tn1826 and transposon Tn 7 Plasmidnuu
18246-249
Tso J Y Zalkin H van Cleemput M Yanofsky C and JM 1982 Nucleotide of Escherichia coli and deduced amino glutamine
phosphribosylpyrophosphate am idotransferase J BioI Chern
V Mesyanzhinova I V Koslov I A and Orlova 1984 Structure of studied by electron and image processing Lett 167285-289
P Barber C and transposons Tn5 and Tn7 in Xanthomonas campestris pv campestris MoL Gen 157
Ullrich J and van Putten P Identification of the Gonococcal glmU gene UVUUIJ
the enzyme N-acetylglucosamine 1-Phosphate Uridyltransferase involved in the synthesis UDP-GlcNAc J of Bact 1776902-6909
M 1992 Eight bacterial proteins includin]g UDP-N -acetylglucosamine acyltransferase three vother of Escherichia coli of a six-residue n tVI
theme FEMS MicrobioL 97249-254
van der Steen J J D Doddema J and de Uidoging van zware metalen afvalstromen met behulp van thiobacilli (Removal metals from waste streams
using thiobacilli) Ministry of Housing Physical and Enviromental (VROM) Directoraat-Generaal Milieubeheer Rapport 199210 Hague
Vermoote P 1988 Universite Paris VI Paris Hrllnro
159
Vignais P V Lunard Issartel J P and Dupuis A 1985 Interaction n l1f middotn
oligomycin sensitivity protein (OSCP) beef heart mitochondrial FeATPase 2 Identification of interacting Fl subunits cross-linking Biochem J
Vik S and Dao NN Prediction of transmembrane topology of Fo proteins from Ecoli ATP synthase variational hydrophobic moment analyses Biochim Biophys Acta 1140 199-207
von Meyenburg B B Jcentrgensen J Nielsen and F Hansen 1982 Promoters of the atp operon for the membrane-bound ATP synthase of Ecoli mapped by TnlO insertion mutations MoL Gen Genet 188240-248
von Meyenberg F G Hansen 1980 The origin of replication oriC the Ecoli chromosome near oriC and construction of oriC mutants ICN-UCLA Symp MoLCellBiol 191 159
Waddell S and Craig N 1989 Tn7 transposition recognition of the attTn7 Proc Natl Sci USA 863958-3962
Waddell C S and Craig N L 1988 transposition two transposition pathways directed by five Tn7-encoded genes Develop 21
J E Gay N J Saraste M and A N 1984 DNA sequence the Escherichia coli unc operon Completion of the sequence of a kilobase segment containing
oriC unc and phoS J224799-815
and Collinson 1 1994 the role the stalk in the coupling mechanism of FEBS 34639-43
Walker 1 E Gay N J and Tybulewicz V L 1986 of transposon Tn7 into the Escherichia glmS transcriptional terminator Biochem 1 243 111-1
Wanner Land McSharry 1982 Phosphate-controlled gene in Escherichia coli using Mudl-directed lacZ fusions J Mol BioI 158347-363
Weng M and Zalkin H 1987 Structural role for a region the CTP synthetase glutamide amide transfer domain J Bacteriol 1693023-3028
Wheatcroft R and S and nucleotide sequence of Rhizobiwn meliloti insertion between the transposase encoded by ISRm3 and those encoded by Staphylococcus aureus and Thiobacillus ferrooxidans IST2 J 1732530-2538
160
Whitby M C and Lloyd R 1995 Branch of three-strand recombination intennediates by RecG a possible pathway for securing middotIULl initiated by duplex DNA 1 143303-3310
Wilkens and Capaldi R A Assymmetry and changes in examined by cryoelectronmicroscopy BioI Chern Hoppe-Seyler 37543-51
and Malamy M 1974 The loss of the PhoS periplasmic protein leads to a the specificity a constitutive phosphate transport system in Escherichia coli Biochem Res Commun 60226-233
WiUsky G Rand Malamy M 1980 of two separable inorganic phosphate transport systems in Escherichia coli J BacterioL 144356-365
P J and and C F 1973 enzyme of metabolism and Stadtman R eds) pp 343-363 Academic Press New York
Winterbum P J and Phelps F 197L control of hexosamine biosynthesis by glucosamine synthetase Biochem 1 121711
Wolf-Watz H and Norquist A 1979 Deoxyribonucleic acid and outer membrane protein to outer membrane protein involves a protein J 14043-49
Wolf-Watz H and M 1979 Deoxyribonucleic acid and outer membrane strains for oriC elevated levels deoxyribonucleic acid-binding protein and
11 for specific UU6 of the oriC region to outer membrane J Bacteriol 14050-58
Wolf-Watz H 1984 Affinity of two different chromosome to the outer membrane of Ecoli J Bacteriol 157968-970
Wu C and Te Wu 197 L Isolation and characterization of a glucosamine-requiring mutant of Escherichia 12 defective in glucoamine-6-phosphate synthetase 1 Bacteriol 105455-466
Yamada M Makino Shinagawa Nakata A 1990 Regulation of the phosphate regulon of Escherichia coli properties the pooR mutants and subcellular localization of protein Mol Genet 220366-372
Yates J R and Holmes D S 1987 Two families of repeatcXl DNA sequences Thiobacillus 1 Bacteriol 169 1861-1870
Yates J R R P and D S 1988 an insertion sequence from Thiobacillus errooxidans Proc Natl 857284-7287
161
Youvan D c Elder 1 T Sandlin D E Zsebo K Alder D P Panopoulos N 1 Marrs B L and Hearst 1 E 1982 R-prime site-directed transposon Tn7 mutagenesis of the photosynthetic apparatus in Rhodopseudomonas capsulata 1 Mol BioI 162 17-41
Zalkin H and Mei B 1990 Amino terminal deletions define a glutamide transfer domain in glutamine phosphoribosylpyrophosphate ami do transferase and other purF-type amidotransferases 1 Bacteriol 1723512-3514
Zalkin H and Weng M L 1987 Structural role for a conserved region III the CTP synthetase glutamide amide transfer domain 1 Bacteriol 169 (7)3023-3028
Zhang Y and Fillingame R H 1994 Essential aspartate in subunit c of FIF0 ATP synthase Effect of position 61 substitutions in helix-2 on function of Asp24 in helix-I 1 BioI Chern 2695473-5479
TABLE OF CONTENTS
ACKNOWLEOOEMENTS ii
ABBREVIATIONS iii
LIST OF TABLES iv
ABSTRACT v
Chapter 1 General Introduction 1
Chapter 2 Cosmid p8181 and its subclones 37
Chapter 3 T ferrooxidans glucosamine synthetase gene 53
Chapter 4 Tn7-like transposon of T ferrooxidans 81
Chapter 5 General Conclusions 124
Appendix 1 Media 130
Appendix 2 Buffers and Solutions 131
Appendix 3 Bacterial strains 138
Appendix 4 Standard Techniques 139
References 143
ii
ACKNOWLEDGEMENTS
I am most grateful to my supervisor Prof Doug Rawlings for his excellent supervision
guidance and encouragement throughout the research Without him this work would not
have been accomplished I am also indebted to my colleagues in the Thiobacillus Research
Unit Ant Smith Ros Powles Cliff Dominy Shelly Deane and the rest of my colleagues
in the department who in one way or the other helped me throughout the period of study
I am grateful to all the members of the Department of Microbiology for their assistance
and support during the course of this research Special thanks to Di James Anne Marie
who is always there when you need her and Di DeVillers for her amazing concern to let it
be there when you need it
My friends Kojo Joyce Pee Chateaux Vic Bob Felix Mawanda Moses the list of
which I cannot exhaust you have not been forgotten Thank you for your encouragement
which became the pillar of my strength Last but not the least I acknowledge the financial
support from Foundation for Research and Development of CSIR and the General Mining
Corp of South Africa
iii
A Ala amp Arg Asn Asp ATCC ATP
bp
C CGSC Cys
DNA dNTP(s) DSM
EDTA
9 G-6 P GFAT GIn Glu Gly
hr His
Ile IPTG
kb kD
I LA LB Leu Lys
M Met min ml NAcG-6-P
ORF (s) oriC
ABBREVIATIONS
adenine L alanine ampicillin L-Arginine L-Asparagine L-aspartic acid American Type Culture Collection adenos triphosphate
base pair(s)
cytosine Coli Genetic Stock Centre L-cysteine
deoxyribonucleic acid deoxyribonucleotide triphosphates Deutsche Sammlungvon Mikroorganismen (The German Collection) ethylenediamine tetraacetic acid
pSIS1 construct was further subcloned to plasm ids IS30 IS40 pSlS50
pS183S p81841 and p81 (Fig Some of subclones were extensively mapped
although not all sites are shown in 22 exact positions of the ends of
T jerrooxidans plasmids pTfatpl and pTfatp2 on the cosmid p8I81 were identified
21)
Tfeooxidans
that the cosmid was
unrearranged DNA digests of cosmid pSISI and T jerrooxidans chromosomal DNA were
with pSI The of the bands gave a positive hybridization signal were
identical each of the three different restriction enzyme digests of p8I8l and chromosomal
In order to confirm of pSI81 and to
49
kb kb ~
(A)
11g 50Sts5
middot 2S4
17 bull tU (probe)
- tosect 0S1
(e)
kb
+- 10 kb
- 46 kb
28---+ 26-+
17---
Fig 23 Hybrdization of cosmid pSISI and T jerrooxitians (A TCC 33020) chromosomal
DNA by the KpnI-SalI fragment of pSIS52 (probe) (a) Autoradiographic image of the
restriction digests Lane x contains the probe lane 13 and 5 contain pSISI restricted with BamHl HindIII and Bgffi respectively Lanes 2 4 and 6 contain T errooxitians chromosomal DNA also restricted with same enzymes in similar order (b) The sizes of restricted pSISI
and T jerrooxitians chromosome hybridized by the probe The lanes correspond to those in Fig 23a A DNA digested with Pst served as the molecular weight nlUkermiddot
50
DNA (Fig BamHI digests SHklC at 26 and 2S kb 1 2) HindIII
at 10 kb 3 and 4) and BgnI at 46 (lanes 5 and 6) The signals in
the 181 was because the purified KpnI-San probe from p81 had a small quantity
ofcontaminating vector DNA which has regions ofhomology to the cosmid
vector The observation that the band sizes of hybridizing
for both pS181 and the T ferrooxidans ATCC 33020 same
digest that the region from BgnI site at to HindIII site at 16
kb on -VJjUIU 1 represents unrearranged chromosomal DNA from T ferrooxidans
ATCC there were no hybridization signals in to those predicted
the map with the 2 4 and 6 one can that are no
multiple copies of the probe (a Tn7-like segment) in chromosome of
and Lferrooxidans
of plasmid pSI was found to fall entirely within a Tn7-like trallSDOS()n nrpn
on chromosome 33020 4) A Southern hybridizationUUAcpoundUU
was out to determine whether Tn7-like element is on other
ofT ferrooxidans of T and Lferrooxidans results of this
experiment are shown 24 Lanes D E and F 24 represent strains
19377 DSM and Lferrooxidans DSM 2705 digested to
with BgnI enzyme A hybridization was obtained for
the three strains (lane A) ATCC 19859 (lane B) and UUHUltn
51
(A) AAB CD E F kb
140
115 shy
284 -244 = 199 -
_ I
116 -109 -
08
os -
(8)) A B c o E F
46 kb -----
Fig 24 (a) Autoradiographic image of chromosomal DNA of T ferrooxidans strains ATCC 33020 19859 23270 (lanes A B C) Tthiooxidans strains ATCC 19377 and DSM 504 (D and E) and Lferrooxidans strain DSM 2705 (lane F) all restricted with Bgffi A Pst molecular weight marker was used for sizing (b) Hybridization of the 17 kb KpnI-San piece of p81852 (probe) to the chromosomal DNA of the organisms mentioned 1 Fig 24a The lanes in Fig 24a correspond to those in Fig 24b
(lane C) uuvu (Fig 24) The same size BgllI fragments (46 kb) were
lanes A Band C This result
different countries and grown on two different
they Tn7-like transposon in apparently the same location
no hybridization signal was obtained for T thiooxidans
1 504 or Lferrooxidans DSM 2705 (lanes D E and F of
T thiooxidans have been found to be very closely related based on 1
rRNA et 1992) Since all Tferrooxidans and no Tthiooxidans
~~AA~~ have it implies either T ferrooxidans and
diverged before Tn7-like transposon or Tthiooxidans does not have
an attTn7 attachment the Tn7-like element Though strains
T ferrooxidans were 10catH)uS as apart as the USA and Japan
it is difficult to estimate acquired the Tn7-like transposon as
bacteria get around the world are horizontally transmitted It
remains to be is a general property
of all Lferrooxidans T ferrooxidans strains
harbour this Tn7-like element their
CHAPTER 3
5431 Summary
54Introduction
56Materials and methods
56L Bacterial and plasmid vectors
Media and solutions
56333 DNA
57334 gel electrophoresis
Competent preparation
57336 Transformation of DNA into
58337 Recombinant DNA techniques
58ExonucleaseIII shortening
339 DNA sequencing
633310 Complementation studies
64Results discussion
1 DNA analysis
64Analysis of ORF-l
72343 Glucosamine gene
77344 Complementation of by T ferrooxidans glmS
57cosmid pSIS1 pSlS20
58Plasmidmap of p81816r
Fig Plasmidmap of lS16f
60Fig 34 Restriction digest p81S1Sr and pSIS16f with
63DNA kb BamHI-BamHI
Fig Codon and bias plot of the DNA sequence of pSlS16
Fig 37 Alignment of C-terminal sequences of and Bsubtilis
38 Alignment of amino sequences organisms with high
homology to that of T ferrooxidans 71
Fig Phylogenetic relationship organisms high glmS
sequence homology to Tferrooxidans
31 Summary
A 35 kb BamHI-BamHI p81820 was cloned into pUCBM20 and pUCBM21 to
produce constructs p818 and p81816r respectively This was completely
sequenced both rrelct1()ns and shown to cover the entire gmS (184 kb) Fig 31
and 34 The derived sequence of the T jerrooxidans synthetase was
compared to similar nrvnC ~ other organisms and found to have high sequence
homology was to the glucosamine the best studied
eubacterium EcoU Both constructs p81816f p81 the entire
glmS gene of T jerrooxidans also complemented an Ecoli glmS mutant for growth on medium
lacking N-acetyl glucosamine
32 =-====
Cell wall is vital to every microorganism In most procaryotes this process starts
with amino which are made from rructClsemiddotmiddotn-[)ll()S by the transfer of amide group
to a hexose sugar to form an This reaction is by
glucosamine synthetase the product of the gmS part of the catalysis
to the 40-residue N-terminal glutamine-binding domain (Denisot et al 1991)
participation of Cys 1 to generate a glutamyl thiol ester and nascent ammonia (Buchanan
368-residue C-terminal domain is responsible for the second part of the ClUUU
glucosamine 6-phosphate This been shown to require the ltgtttrltgtt1iltn
of Clpro-R hydrogen of a putative rrulctoseam 6-phosphate to fonn a cis-enolamine
intennediate which upon reprotonation to the gives rise to the product (Golinelli-
Pimpaneau et al 1989)
bacterial enzyme mn two can be separated by limited chymotryptic
proteolysis (Denisot et 199 binding domain encompassing residues 1 to
240 has the same capacity to hydrolyse (and the corresponding p-nitroanilide
derivative) into glutamate as amino acid porI11
binding domain is highly cOllserveu among members of the F-type
ases Enzymes in this include amidophosphophoribosyl et al
1982) asparagine synthetase (Andrulis et al 1987) glucosamine-6-P synmellase (Walker et
aI 1984) and the NodM protein of Rhizobium leguminosarum (Surin and 1988) The
368-residue carboxyl-tenninal domain retains the ability to bind fructose-6-phosphate
The complete DNA sequence of T ferrooxidans glmS a third of the
glmU gene (preceding glmS) sequences downstream of glmS are reported in this
chapter This contains a comparison of the VVA r glmS gene
and Mycobacterium (422 ) An interesting observation was that the T ferrooxidans
glmS gene had comparatively high homology sequences to the Rhizobium leguminosarum and
Rhizobium meliloti M the amino to was 44 and 436
respectively A consensus Dalgarno upstream of the start codon
(ATG) is in 35 No Ecoli a70-type n r~ consensus sequence was
detected in the bp preceding the start codon on intergenic distance
between the two and the absence of any promoter consensus sequence Plumbridge et
al (1993) that the Ecoli glmU and glmS were co-transcribed In the case
the glmU-glmS --n the T errooxidans the to be similar The sequences
homology to six other amidotransferases (Fig 38) which are
(named after the purF-encoded l phosphoribosylpyrophosphate
amidotransferase) and Weng (1987)
The glutamine amide transfer domain of approximately 194 amino acid residues is at the
of protein chain Zalkin and Mei (1989) using site-directed to
the 9 invariant amino acids in the glutamine amide transfer domain of
phosphoribosylpyrophosphated indicated in their a
catalytic triad is involved glutamine amide transfer function of
73
Comparison of the ammo acid sequence alignment of ghtcosamine-6-phosphate
amidotranferases (GFAT) of Rhizobium meliloti (R_m) Rhizobium leguminnosarum (RJ)
Ecoli (E_c) Hinjluenzae Mycobacterium leprae (M_I) Bsubtilis (B_s) and
Scerevisiae (S_c) to that of Tferrooxidans Amino acids are identified by their single
codes The asterisks () represent homologous amino acids of Tferrooxidans glmS to
at least two of the other Consensus ammo acids to all eight organisms are
highlighted (bold and underlined)
1 50 R m i bullbullbullbullbullbull hkps riag s tf bullbullbullbull R~l i bullbullbullbullbullbull hqps rvaep trod bullbullbullbull a
a bullbullbullbullbullbull aa 1r 1 d bullbullbull a a bullbullbullbullbullbull aa inh g bullbullbullbullbullbull sktIv pmilq 1 1g bullbullbull a MCGIVi V D Gli RI IYRGYPSAi AI D 1 gvmar s 1gsaks Ikek a bullbullbullbull qfcny Iversrgi tvq t dgd bullbull ead
51 100 R m hrae 19ktrIk bull bullbull bullbull s t ateR-l tqrae 19rkIk bullbullbull s t atrE-c htIrI qmaqae bull bull h bullbull h gt sv aae in qicI kads stbull bullbull k bullbull i gt T f- Ivsvr aetav bullbullbull r bullbull gq qg c
DL R R G V L AV E L G VGI lTRW E H ntvrrar Isesv1a mv bull sa nIgi rtr gihvfkekr adrvd bullbullbull bullbull nve aka g sy1stfiykqi saketk qnnrdvtv she reqv
101 150 R m ft g skka aevtkqt R 1 ft g ak agaqt E-c v hv hpr krtv aae in sg tf hrI ksrvlq T f- m i h q harah eatt
S E Eamp L A GY l SE TDTEVIAHL ratg eiavv av
qsaIg lqdvk vqv cqrs inkke cky
151 200 bullbull ltk bullbull dgcm hmcve fe a v n bull Iak bullbull dgrrm hmrvk fe st bull bull nweI bullbull kqgtr Iripqr gtvrh 1 s bull bull ewem bullbullbull tds kkvqt gvrh hlvs bull bull hh bullbull at rrvgdr iisg evm
V YR T DLF A A L AV S D PETV AR G M 1 eagvgs lvrrqh tvfana gvrs B s tef rkttIks iInn rvknk 5 c Ihlyntnlqn ~lt klvlees glckschy neitk
74
201 R m ph middot middot middot R 1 ah- middot middot middot middot middot middot E c 1 middot middot middot middot middot middot middot Hae in 1 middot middot middot middot middot T f - c11v middot middotmiddot middot middot middot middot middot M 1 tli middot middot middot middot middot middot middot middot middot B s 11 middot middot middot middot middot S c lvkse kk1kvdfvdv
251 R m middot middot middot middot middot middot middot middot middot middot middot middot middot middot
middot middot middot middot R-1 middot middot middot middot middot middot E c middot middot middot middot middot middot middot middot Hae in middot middot middot middot middot middot middot middot middot middot middot middot T f shy middot middot middot middot middot middot middot middot middot middot middot middot 1M middot middot middot middot middot middot middot middot middot middot middot middot middot middot B s middot middot middot middot middot middot middot middot middot middot middot middot middot middot middot S c feagsqnan1 1piaanefn1
301 R m fndtn wgkt R-1 fneti wgkt E c rf er Hae in srf er T f- rl mlqq
PVTR V YE DGDVA R M 1 ehqaeg qdqavad B s qneyem kmvdd S c khkkl dlhydg
351 R m nhpe v R-1 nhe v E c i nk Hae in k tli T f- lp hr
~ YRHlrM~ ~I EQP AVA M 1 geyl av B-s tpl tdvvrnr S-c pd stf
401 Rm slasa lk R-1 slasca lk E c hql nssr Hae in hq nar-T f f1s hytrq
RVL A SiIS A VG W M 1 ka slakt B s g kqS c lim scatrai
T ferrooxidans (Tf) Scerevisiae (Saee) Mleprae (Myele) and Bsublilis (Baesu)
tr1 ()-ltashyc 8 ~ er (11
-l l
CIgt
~ r (11-CIgt (11
-
$ -0 0shy
a c
omiddot s
S 0 0shyC iii omiddot $
ttl ~ () tI)
I-ltashyt ()
0
~ smiddot (11
81
CHAPTER 4
8341 Summary
8442 Introduction
8643 Materials and methods
431 Bacterial strains and plasmids 86
432 DNA constructs subclones and shortenings 86
864321 Contruct p81852
874322 Construct p81809
874323 Plasmid p8I809 and its shortenings
874324 p8I8II
8844 Results and discussion
441 Analysis of the sequences downstream the glmS gene 88
442 TnsBC homology 96
99443 Homology to the TnsD protein
118444 Analysis of DNA downstream of the region with TnsD homology
LIST OF FIGURES
87Fig 41 Alignment of Tn7-like sequence of T jerrooxidans to Ecoli Tn7
Fig 42 DNA sequences at the proximal end of Tn5468 and Ecoli glmS gene 88
Fig 43 Comparison of the inverted repeats of Tn7 and Tn5468 89
Fig 44 BLAST results of the sequence downtream of T jerrooxidans glmS 90
Fig 45 Alignment of the amino acid sequence of Tn5468 TnsA-like protein 92 to TnsA of Tn7
82
Fig 46 Restriction map of the region of cosmid p8181 with amino acid 95 sequence homology to TnsABC and part of TnsD of Tn7
Fig 47 Restriction map of cosmid p8181Llli with its subclonesmiddot and 96 shortenings
98 Fig 48 BLAST results and DNA sequence of p81852r
100 Fig 49 BLAST results and DNA sequence of p81852f
102 Fig 410 BLAST results and DNA sequence of p81809
Fig 411 Diagrammatic representation of the rearrangement of Tn5468 TnsDshy 106 like protein
107Fig 412 Restriction digests on p818lLlli
108 Fig 413 BLAST results and DNA sequence of p818lOEM
109 Fig 414 BLAST results on joined sequences of p818104 and p818103
111 Fig 415 BLAST results and DNA sequence of p818102
112 Fig 416 BLAST results and nucleotide sequence of p818101
Fig 417 BLAST results and DNA sequence of the combined sequence of 113p818l1f and p81810r
Fig 418 Results of the BLAST search on p81811r and the DNA sequence 117obtained from p81811r
Fig 419 Comparison of the spa operons of Ecali Hinjluenzae and 121 probable structure of T ferraaxidans spa operon
83
CHAPTER FOUR
TN7-LlKE TRANSPOSON OF TFERROOXIDANS
41 Summary
Various constructs and subclones including exonuclease ill shortenings were produced to gain
access to DNA fragments downstream of the T ferrooxidans glmS gene (Chapter 3) and
sequenced Homology searches were performed against sequences in GenBank and EMBL
databases in an attempt to find out how much of a Tn7-like transposon and its antibiotic
markers were present in the T ferrooxidans chromosome (downstream the glmS gene)
Sequences with high homology to the TnsA TnsB TnsC and TnsD proteins of Tn7 were
found covering a region of about 7 kb from the T ferrooxidans glmS gene
Further sequencing (plusmn 45 kb) beyond where TnsD protein homology had been found
revealed no sequences homologous to TnsE protein of Tn7 nor to any of the antibiotic
resistance markers associated with Tn7 However DNA sequences very homologous to Ecoli
ATP-dependentDNA helicase (RecG) protein (EC 361) and guanosine-35-bis (diphosphate)
3-pyrophosphohydrolase (EC 3172) stringent response protein of Ecoli HinJluenzae and
Vibrio sp were found about 15 kb and 4 kb respectively downstream of where homology to
the TnsD protein had been detected
84
42 Transposable insertion sequences in Thiobacillus ferrooxidizns
The presence of two families (family 1 and 2) of repetitive DNA sequences in the genome
of T ferrooxidans has been described previously (Yates and Holmes 1987) One member of
the family was shown to be a 15 kb insertion sequence (IST2) containing open reading
frames (ORFs) (Yates et al 1988) Sequence comparisons have shown that the putative
transposase encoded by IST2 has homology with the proteins encoded by IS256 and ISRm3
present in Staphylococcus aureus and Rhizobium meliloti respectively (Wheatcroft and
Laberge 1991) Restriction enzyme analysis and Southern hybridization of the genome of
T ferrooxidans is consistent with the concept that IST2 can transpose within Tferrooxidans
(Holmes and Haq 1990) Additionally it has been suggested that transposition of family 1
insertion sequences (lST1) might be involved in the phenotypic switching between iron and
sulphur oxidizing modes of growth including the reversible loss of the capacity of
T ferrooxidans to oxidise iron (Schrader and Holmes 1988)
The DNA sequence of IST2 has been determined and exhibits structural features of a typical
insertion sequence such as target-site duplications ORFs and imperfectly matched inverted
repeats (Yates et al 1988) A transposon-like element Tn5467 has been detected in
T ferrooxidans plasmid pTF-FC2 (Rawlings et al 1995) This transposon-like element is
bordered by 38 bp inverted repeat sequences which has sequence identity in 37 of 38 and in
38 of 38 to the tnpA distal and tnpA proximal inverted repeats of Tn21 respectively
Additionally Kusano et al (1991) showed that of the five potential ORFs containing merR
genes in T ferrooxidans strain E-15 ORFs 1 to 3 had significant homology to TnsA from
transposon Tn7
85
Analysis of the sequence at the tenninus of the 35 kb BamHI-BamHI fragment of p81820
revealed an ORF with very high homology to TnsA protein of the transposon Tn7 (Fig 44)
Further studies were carried out to detennine how much of the Tn7 transposition genes were
present in the region further downstream of the T ferroxidans glmS gene Tn7 possesses
trimethoprim streptomycin spectinomycin and streptothricin antibiotic resistance markers
Since T ferrooxidans is not exposed to a hospital environment it was of particular interest to
find out whether similar genes were present in the Tn7-like transposon In the Ecoli
chromosome the Tn7 insertion occurs between the glmS and the pho genes (pstS pstC pstA
pstB and phoU) It was also of interest to detennine whether atp-glm-pst operon order holds
for the T ferrooxidans chromosome It was these questions that motivated the study of this
region of the chromosome
43 OJII
strains and plasm ids used were the same as in Chapter Media and solutions (as
in Chapter 3) can in Appendix Plasmid DNA preparations agarose gel
electrophoresis competent cell preparations transformations and
were all carned out as Chapter 3 Probe preparation Southern blotting hybridization and
detection were as in Chapter 2 The same procedures of nucleotide
DNA sequence described 2 and 3 were
4321 ~lllu~~~
Plasmid p8I852 is a KpnI-SalI subclone p8I820 in the IngtUMnt vector kb) and
was described Chapter 2 where it was used to prepare the probe for Southern hybridization
DNA sequencing was from both (p81852r) and from the Sail sites
(p8I852f)
4322 ==---==
Construct p81809 was made by p8I820 The resulting fragment
(about 42 kb) was then ligated to a Bluescript vector KS+ with the same
enzymes) DNA sequencing was out from end of the construct fA
87
4323 ~mlJtLru1lbJmalpoundsectM~lllgsect
The 40 kb EeoRI-ClaI fragment p8181Llli into Bluescript was called p8181O
Exonuclease III shortening of the fragment was based on the method of Heinikoff (1984) The
vector was protected with and ClaI was as the susceptible for exonuclease III
Another construct p818IOEM was by digesting p81810 and pUCBM20 with MluI and
EeoRI and ligating the approximately 1 kb fragment to the vector
4324 p81811
A 25 ApaI-ClaI digest of p8181Llli was cloned into Bluescript vector KS+ and
sequenced both ends
88
44 Results and discussion
of glmS gene termini (approximately 170
downstream) between
comparison of the nucleotide
coli is shown in Fig 41 This region
site of Tn7 insertion within chromosome of E coli and the imperfect gtpound1
repeat sequences of was marked homology at both the andVI
gene but this homology decreased substantially
beyond the stop codon
amino acid sequence
been associated with duplication at
the insertion at attTn7 by (Lichtenstein and as
CCGGG in and underlined in Figs41
CCGGG and are almost equidistant from their respelt~tle
codons The and the Tn7-like transposon BU- there is
some homology transposons in the regions which includes
repeats
11 inverted
appears to be random thereafter 1) The inverted
repeats of to the inverted repeats of element of
(Fig 43) eight repeats
(four traJrlsposcm was registered with Stanford University
California as
A search on the (GenBank and EMBL) with
of 41 showed high homology to the Tn sA protein 44) Comparison
acid sequence of the TnsA-like protein Tn5468 to the predicted of the
89
Fig 41
Alignment of t DNA sequence of the Tn7-li e
Tferrooxi to E i Tn7 sequence at e of
insertion transcriptional t ion s e of
glmS The ent homology shown low occurs within
the repeats (underl of both the
Tn7-li transposon Tn7 (Fig 43) Homology
becomes before the end of t corresponding
impe s
T V E 10 20 30 40 50 60
Ec Tn 7
Tf7shy(like)
70 80 90 100 110 120
Tf7shy(1
130 140 150 160 170 180 Ec Tn 7 ~~~=-
Tf7shy(like)
90
Fig 42 DNA sequences at the 3 end of the Ecoll glmS and the tnsA proximal of Tn7 to the DNA the 3end of the Tferrooxidans gene and end of Tn7-like (a) DNA sequence determined in the Ecoll strain GD92Tn7 in which Tn been inserted into the glmS transcriptional terminator Walker at ai 1986 Nucleotide 38 onwards are the of the left end of Tn DNA
determined in T strain ATCC 33020 with the of 7-like at the termination site of the glmS
gene Also shown are the 22 bp of the Tn7-like transposon as well as the region where homology the protein begins A good Shine
sequence is shown immediately upstream of what appears to be the TTG initiation codon for the transposon
(a)
_ -35 PLE -10 I tr T~ruR~1 truvmnWCIGACUur ~~GCfuA
100 no 110 110 110
4 M S S bull S I Q I amp I I I I bull I I Q I I 1 I Y I W L ~Tnrcmuamaurmcrmrarr~GGQ1lIGTuaDACf~1lIGcrA
DO 200 amp10 220 DG Zlaquot UO ZIG riO
T Q IT I I 1 I I I I I Y sir r I I T I ILL I D L I ilGIIilJAUlAmrC~GlWllCCCAcarr~GGAWtCmCamp1tlnmcrArClGACTAGNJ
Fig 45 (a) Alignment of the amino acid sequence of Tn5468 (which had homology to TnsA of Tn7 Fig 44) to the amino acid sequence of ORF2 (between the merR genes of Tferrooxidans strain E-1S) ORF2 had been found to have significant homology to Tn sA of Tn7 (Kusano et ai 1991) (b) Alignment of the amino acids translation of Tn5468 (above) to Tn sA of Tn7 (c) Alignment of the amino acid sequence of TnsA of Tn7 to the hypothetical ORF2 protein of Tferrooxidans strain E-1S
(al
Percent Similarity 60000 Percent Identity 44444
Tf Tn5468 1 LARQRYGVDEDRVARFQKEGRGQGRGADYHPWLTIQDVPSQGRSHRLKGI SO 11 1111111 I 1111 1111 I I 111
Fig 49 (a) Results of the BLAST search on the inverted and complemented nucleotide sequence of 1852f (from the Sall site) Results indicate amino acid sequence to the TnsC of Tn7 (protein E is the same as TnsC protein) (b) The nucleotide sequence and open reading frame of p81852f
High Prob producing Segment Pairs Frame Score P(N)
IB255431QQECE7 protein E - Escher +1 176 15e-20 gplX044921lSTN7EUR 1 Tn7 E with +1 176 15e-20 splP058461 ECOLI TRANSPOSON TN7 TRANSPOSITION PR +1 176 64e-20 gplU410111 4 D20248 gene product [Caenorhab +3 59 17e-06
IB255431QQECE7 ical protein E - Escherichia coli transposon Tn7 (fragment)
417 (al BLAST results obtained from combined sequence of (inverted and complemented) and 1810r good sequence to Ecoli and Hinfluenzae DNA RecG (EC 361) was found (b) The combined sequence and open frame which was homologous to RecG
444 Analysis of DNA downstream of region with TnsD homology
The location of the of p81811 ApaI-CLaI construct is shown in Fig 47 The single strand
sequence from the C LaI site was joined to p8181Or and searched using BLAST against the
GenBank and EMBL databases Good homology to Ecoli and Hinjluezae A TP-dependent
DNA helicase recombinase proteins (EC 361) was obtained (Fig 417) The BLAST search
with the sequence from the ApaI end showed high sequence homology to the stringent
response protein guanosine-35-bis (diphosphate) 3-pyrophosphohydrolase (EC 3172) of
Ecoli Hinjluenzae and Scoelicolor (Fig 418) In both Ecoli and Hinjluenzae the two
proteins RecG helicase recombinase and ppGpp stringent response protein constitute part of
the spo operon
445 Spo operon
A brief description will be given on the spo operons of Ecoli and Hinjluenzae which appear
to differ in their arrangements In Ecoli spoT gene encodes guanosine-35-bis
pyrophosphohydrolase (ppGpp) which is synthesized during stringent response to amino acid
starvation It is also known to be responsible for cellular ppGpp degradation (Gentry and
Cashel 1995) The RecG protein is required for normal levels of recombination and DNA
repair RecG protein is a junction specific DNA helicase that acts post-synaptically to drive
branch migration of Holliday junction intermediate made by RecA during the strand
exchange stage of recombination (Whitby and Lloyd 1995)
The spoS (also called rpoZ) encodes the omega subunit of RNA polymerase which is found
associated with core and holoenzyme of RNA polymerase The physiological function of the
omega subunit is unknown Nevertheless it binds stoichiometrically to RNA polymerase
119
Fig 418 BLAST search nucleotide sequence of 1811r I restrict ) inverted and complement high sequence homology to st in s 3 5 1 -bis ( e) 3 I ase (EC 31 7 2) [(ppGpp)shyase] -3-pyropho ase) of Ecoli Hinfluenzae (b) The nucleot and the open frame with n
Add 4 ml 52 mix by inversion at room temp for 5 mins
Add 4 ml 53 Mix by to homogenous suspension
Spin at 15 K 40 mins at 4
remove to fresh tube
Equilibrate column with 2 ml N2
Load supernatant 2 to 4 ml amounts
Wash column 2 X 4 of N3 Elute the DNA the first bed
each) add 07
volumes of isopropanol
Spin at 4 Wash with 70 Ethanol Resuspended pellet in n rv 100 AU of TE and scan
volume of about 8 to 10 drops) To the eluent (two
to
140
SEQUITHERM CYCLE SEQUENCING
Alf-express Cy5 end labelled promer method
only DNA transformed into end- Ecoli
The label is sensitive to light do all with fluorescent lights off
3-5 kb
3-7 kb
7-10 kb
Thaw all reagents from kit at well before use and keep on
1) Label 200 PCR tubes on or little cap flap
(The heated lid removes from the top of the tubes)
2) Add 3 Jtl of termination mixes to labelled tubes
3) On ice with off using 12 ml Eppendorf DNA up to
125 lll with MilliQ water
Add 1 ltl
Add lll of lOX
polymeraseAdd 1 ltl
Mix well Aliquot 38 lll from the eppendorf to Spin down
Push caps on nrnnp1
141
Hybaid thermal Cycler
Program
93 DC for 5 mins 1 cycle
93degC for 30 sees
55 DC for 30 secs
70 DC for 60 secs 30 cycle 93 DC_ 30s 55 DC-30s
70degC for 5 mins 1 cycle
Primer must be min 20 bp long and min 50 GC content if the annealing step is to be
omitted Incubate at 95 DC for 5 mins to denature before running Spin down Load 3 U)
SEQUITHERM CYCLE SEQUENCING
Ordinary method
Use only DNA transformed into end- Ecoli strain
3-5 kb 3J
3-7 kb 44
7-10 kb 6J
Thaw all reagents from kit at RT mix well before use and keep on ice
1) Label 200 PCR tubes on side or little cap flap
(The heated lid removes markings from the top of the tubes)
2) Add 3 AU of termination mixes to labelled tubes
3) On ice using l2 ml Eppendorf tubes make DNA up to 125 41 with MilliQ water
142
Add I Jtl of Primer
Add 25 ttl of lOX sequencing buffer
Add 1 Jtl Sequitherm DNA polymerase
Mix well spin Aliquot 38 ttl from the eppendorf to each termination tube Spin down
Push caps on properly
Hybaid thermal Cycler
Program
93 DC for 5 mins 1 cycle
93 DC for 30 secs
55 DC for 30 secs
70 DC for 60 secs 30 cycles
93 DC_ 30s 55 DC-30s 70 DC for 5 mins 1 cycle
Primer must be minimum of 20 bp long and min 50 GC content if the annealing step is
to be omitted Incubate at 95 DC for 5 mins to denature before running
Spin down Load 3 ttl for short and medium gels 21t1 for long gel runs
143
REFERENCES
144
REFERENCES
Abrahams J P Lutter R Todd R J van Raaij M J Leslie A G W and Walker J E 1993 Inherent asymmetry of the structure of F1-ATPase from bovine heart mitochondria at 65 Aresolution EMBO J 121775-1780
Abrahams J P Leslie A G W Lutter R and Walker 1 E 1994 Structure at 28 A resolution of FeATPase from bovine heart mitochondria Nature 370621-628
Adzumah K and Mizuuchi K 1988 Target immunity of Mu transposition reflects a differential distribution of Mu B protein Cell 53257-266
Akey C W Crepeau R H Dunn S D McCarty R E and Edelstein S J 1983 Electron microscopy of single molecules and crystals of F1-ATPases EMBO J 21409-1415
Allet B 1979 Mu insertion duplicates a 5 bp sequence at the host inserted site Cell 16 123shy129
Amzel L M and Pedersen P L 1983 Proton ATP-ases structure and mechanism Ann Rev Biochem 52801-824
Andersson L Mac Neela J and Wolfenden R 1985 Use of secondary isotope effects and varying pH to investigate mode of binding of inhibitory amino aldehydes by leucine aminopeptidase Biochem 24330-333
Andrews G F Dugan R P and Stevens C J 1992 Combining physical and bacterial treatment for removing pyritic sulfur from coal In Processing and Utilization of High-sulfur coals IV Dugan P R D Quigley and Y Attia (eds) p 515 Elsevier New York
Andrulis I L Chen J and Ray P N 1987 Isolation of human cDNAs for asparagine synthetase and expression in Jansen rat sarcoama cells Mol Cell BioI 72435-2443
Andruszkiewicz R Milewsky S Zieniawa T and Borowski E 1990 Anticandidal properties of N3 -(4-methoxy-fumaroyl)-L-2 3-diamino propanoic acid oligopeptides 1 Med Chern 33132-135
Arciszewska L K Drake D and Craig NL 1989 Transposon Tn7 cis-acting sequences in transposition and transposition immunity J Mol BioI 20735-42
Bachmann B J 1983 Linkage map of Escherichia coli K-12 edition 7 Microbiol Rev 47180-230
145
B Plumbridge 1 A Cochet D Souza J M M M and Calcagno M 1988 Cordinated regulation of amino J
Bacteriol 1754951-4956
0 B Vermoote P V and Le Goffic F 1993 synthetase from Ecoli lUllL- mechanism and inhibition by N3-fumaroyl-2 3-diaminopropionic derivatives Biochem 27 2282-2287
Vermoote P Haumont PY syrlthl~ta~e from Escherichia coli purification site location Biochemistry 26 1940-1948
Badet B Inagaki K Soda K Walsh dependent inhibition of Bacillus stearothermophilus alanine racemase by phosphonate isomers by isomerization to non covalent enzyme-(1-aminoethyl) complexes Biochem 253275-3282
Badet-Denisot M A and Badet of glucoseamine-6shyphosphate synthetase by diethylpyrocarbonates histidine requirement for enzymatic activity Arch Biochem Biophys
Bainton R Gamas P and transposition in vitro proceeds through an excised transposon intermediate (JpnIPtlltpi1 111 breaks in DNA Cell 65805-816
Barry G 1986 Permanent insertion of v~u into the chromosomes of soil bacteria BioTechnology 4446-449
Barth P T Datta N Grinter N S 1976 Transposition a deoxyribonucleic acid sequence trimethoprim and streptomycin resistances from R483 to other replicons 1 Bacteriol 125800-810
Bates C J Adams W and R R 1968 Control of the formation of uri dine diphospho-N-acetyl and glycoprotein synthesis in rat liver J Chern 2411705-17
Benjamin N 1989 Intramolecular transposition of TnlD Cell 59373shy383
Bennet R L and Malamy M resistant mutants of Escgerichia coli and phosphate Commun 40496-503
Benneth1 1978 Bacterial leaching patterns on pyrite crystal J Bacteriol
146
Berg D E Davies 1 Allet B and Rochaix 1 D 1975 Transposition of R factor genes to bacteriophage lambda Proc Natl Acad Sci USA 723268-3632
Berg D E and Drummond M1978 Absence of DNA sequences homologous to transposable element Tn5 (Kan) in the chromosome of Ecoli K-12 J Bcateriol 136419-422
Birkenhager R Hoppert M Deckers-Hebestriet G Mayer F and Altendorf K 1995 The Fo complex of the Ecoli ATP synthase investigation by electron imaging and immunoelectron microscopy Eur J Biochem 23058-67
Bjqgtrbaek C Foersom V and Michelsen O 1990 The transmembrane topology of the a subunit from ATPase in Escherichia coli analysed by PhoA protein fusions FEBS lett 26031-34
Boekemia E J Berden JA and van Heel MG 1986 sructure of mitochondrial F1-ATPase studied by electron microscopy and image processing Biochim Biophys Acta 851353-360
Bolton E Glynn P and OGara F 1984 Site specific transposition of Tn7 into a Rhizobiwn meliloti megaplasmid Mol Gen Genet 193153-157
Boos W 1974 Bacterial transport Annu Rev Biochem 43123-146
Boursaux-Eude C Girons I S and Zuerner R 1995 IS1500 an IS3-like element from Leptospira interrogans Microbiol 1412165-2173
Boyer P D 1993 The binding change mechanism for ATP synthase-some probabilities and possibilities Biochim Biophys Acta 1140215-250
Brachet P Eisen H and Rambach A 1970 Mutations in coliphage lambda affecting the expression of replicative functions 0 and P Mol Gen Genet 108266-276
Brink J Boekemia E 1 and van Bruggen E F 1987 The structure of NADH ubiquitone oxidoreductase fron beef heart mitochondria Crystals containing an octameric arrangement of iron-sulphur protein fragments Eur J Biochem 166287-294
Brock T D and Gustafson J 1976 Ferric iron reduction by sulphur- and iron-oxidizing bacteria Appl Environ Microbiol 32 567-571
Brown L D Dennehy M E and Rawlings D E 1994 The FI genes of the FIFo ATP synthase from the acidophilic bacterium Thiobacillus jerrooxidans complement Escherichia coli FI unc mutants FEMS Microbiol Lett 12219-25
Buchanan 1 1 Adv The Amidotransferases Enzymol 3991-183
Buckley Science
Caruso M Pseudomonas nOVHrn
oxidation of pyrite Applied
1 1982 Interactions Mol Gen Genet
phage 1
Chmara H by Biophysica
synthetase from bacteria antibiotic tetaine Biochimica et
Microbiol Lett 11197-206 controls on the oxidation of refractory Barberton
genetic elements and evolution Nature 263731shyCohen S N 1976 738
Corbet C M and 1 Ingledew 1987 Is oxidation by Thiobacillus ferrooxidans
Couillard D and ~~b 118(5)808-81
Metallurgical residue V UlllLltHIH of metals from sewage sludge J
Cox G B a reassessment of the
F and Hatch L 1986 The mechanism of ATP synthase of the b and a subunits Biochim Acta 84962-69
Craig N L 1995 Unity in Science 270253-254
Craig N and Gamas P 1 Purification and characterization of a transposition protein that binds ATP DNA Nuc Acids Res
1 2873-97 C A 1990 in response to environmental stress Advances in
alUIUH~ on the activity subunit b-specific polyc1onal
G Simoni and Altendorf K 1992 Influence vlJnu~ of the ATP synthase of t-S(nel~lCn
1 BioI Chern 26712364shy
M A Le Goffic F and 1991 Glucosamine- 6P two proteins on limited proteolysis ltVU Biophys 288225-230
148
Dobrogosz W J 1968 _l in Escherichia coli and its to catabolite repression J
Doublet P 1 van Heijenoort and 1993 The murI gene of is an essential gene that encodes klUltUllU~v racemase activity J Bacteriol 1752970-2979
P J van Heijenoort 1992 Identification of the Ecoli which is required for the D-glutamic acid a specific component peptidoglycan 1 Bacteriol
Garren A Garen and Torri ani 1961 Genetic control of repression UCUlllV phosphatase in Ecoli 1 Mol BioI
D J and Boeke 1 D 1990 A 1-1-0- structure is required for Ty1 transposition Genes Develop 4324-330
1982 Transposition of Tn7 occurs at a on Caulobacter crescentus 10SIClmle 1 Bacteriol 151 1056-1 058
H 1990 Molecular IHovUUU)11 -transporting 345-391 In T 12 Bacterial
Press Ltd London
transport and coupling by the synthase insights mechanism of function J Bioenerg 24485-491
1992b Subunit c of FIFo ATP role m transduction Biochim Biophys Acta 1101 v--rJ
Eliopoulos E E Jackson P 1 Keen IN HUIUVI L Thompson P and 1 Structure of a 16 kDa integral AU-UUl that identity to
of the vacuolar H+ -ATPase Protein 15
Fling M 1 C 1985 Nucleotide sequence of encoding the amino glycoside-modifying enzyme 3(9)-O-nucleotidyltransferase Res 137095-7106
Fling M and C l-1UUJIU nucleotide sequence of dihydrofolate rhnrpri by Tn7 Nucleic Acids Res 115
Flores Llchtenstem C P 1990 DNA sequence anlysis of tnsA for the Tn7 transposition Nucleic Acids 18901 911
149
149
Foster D L and Fillingame R H 1982 Stoichiometry of subunits in the H+-ATPase complex of Escherichia coli J BioI Chern 2572009-2015
Fraga D Hermolin J Oldenburg M Miller MJ and Fillingame R H 1994 Arginine 41 of subunit c of Escherichia coli H+-A TP synthase is essential in binding and coupling of F to Fo J BioI Chern 2697532-7537
Friedl P Hoppe J Gunsalus R P Michelsen 0 von Meyenburg K and Schairer H U 1983 Membrane integration and function of the three Fo subunits of the A TP-synthase of Escherichia coli K12 EMBO 1 299-103
Frisa P S and Sonneborn D R 1982 Developmentally regulated interconversion between end product inhibitable and non-inhibitable forms of a first pathway-specific enzyme activity can be mimicked in vitro by dephosphorylation reactions Proc Natl Acad Sci USA 796289-6293
Fujiwara T and Mizuuchi K 1988 Retroviral DNA integration Structure of an integration intermediate Cell 54497-504
Galas D J and Chandler M 1989 Bacterial insertion sequences In Mobile DNA Berg D E and Howe M M (eds) Washington D C American Society for Microbiology pp 109shy162
Gay N J Tybulewicz V L1 Walker JE 1986 Insertion of transposon Tn7 into the Ecoli gmS transcriptional terminator Biochem J 234 111-117
Gentry D R and Cashel M 1995 Cellular localization of the Escherichia coli SpoT protein J Bacteriol 177 3890-3893
Gentry D R Xiao H Burgess R R and Cashel M 1991 The omega subunit of Ecoli K-12 RNA polymerase is not required for stringent RNA control in vivo J Bacteriol 173 3901-3903
Girvin M E and Fillingame R H 1993 Helical structure and folding of subunit c of FIFo ATP synthase IH NMR resonace assignments and NOE analysis Biochemistry 3212167shy12177
Gogol E P Lucken U Bork T and Capaldi R A 1989 Molecular architecture of Escherichia coli F Adenosinetriphosphatase Biochemistry 284709-4716
Gogol E P Aggeler R Sagermann M and Capaldi R A 1989 Cryoelectronic Microscopy of Escherichia coli FI Adenosinetriphosphatase Decorated with Monoclonal Antibodies to Individual Subunits of the complex Biochemistry 284717-4724
150
Heinikoff S 1984
Golinelli-Pimpaneaux B and Badet involvement of Lys603 from Ecoli glucosamoine-6-phosphate synthase substrate fructose-6-phosphate 1 Biochem 201175-182
Gottesman M M and Rosner 1 L of a detenninant of uvu_bullu
resistance by coliphage lambda Sci USA 725041-5045
Grunstein M and Hogness D
Colony hybridization a method for isolation cloned DNAs that contain a 1Jbullu Proc NatL Acad Sci USA 723961-3965
Hauer B and Shapiro 1 A 1984 Control of Tn7 transposition Mol Gen Genet 194
Hedges R W and Jacob Transposition of ampicillin resistance from to other replicons MoL 1-40
Hennolin 1 Gallant 1 psi subunit in the Fo sector of the H+ -ATPase of Ecoli J Bioi
R H 1983 Topology organization and function
Hennolin J and R 1989 Assembly of Fo sector of synthase Interdepenndence of subunit insertion into the membrane 1 2817
van Montagu M Holsters Zambryski P Beuckeleer M Willmitzer and Schell 1 M 1980 interaction of
DNA plant cells Proc Soc Lond 210351-365
P 1972 Insertion mutations control region of Physical characterization of the mutants Mol Gen Genet
115266-276
Holmes applications pI
UI Haq 1990 Adaptation of Thiobacillus vu)nuu for industrial In J Salley R G McCready Wichlacz (ed)
1989 CANMET Ottawa Canada
Holtje J V and U Schwarz 1985 Biosynthesis and growth murein sacculus p 77shy119 InN (ed) Molecular cytology of Escherichia Academic Press Inc New
Acad Sci USA
Heffron E Reubens C and which mediates ampicillin
Translocation of a plasmid DNA sequence nature and specificity of Natl
digestion with exonuclease III creates fl tr breakpoints 1-369
Hernalsteens J M Agrobacterium
Hirsch H the galactose nnnn fJu
151
Holzenburg A Jones P c Franklin T Padi J B and Finbow M E 1993 Evidence for a common structure 1 Biochem 21321-30
Hoppe 1 and Sebald W 1986 Topological pathway of the protons through Fo is provided by amino acid the lipid phase Biochimie (Paris) 68427-434
S Otsubo H Davidson N and 1975 Electron microscope heteroduplex of sequence relations among plasmids identification and mapping of the
insertion sequences IS] and IS2 in F and R plasmids J Bacteriol 22762-775
Inoue c Sugawara K and 1991 regulatory gene in Thiobacillus ferrooxidans is spaced apart from Mol Microbiol 52707-2718
Ish-Horowicz D and Burke and cosmid cloning Nucleic Acids Res 92989-2998
Johnston B Clennell M and D 1995 Structure and function of Tn5467 a Tn2l-like transposon located on T jerrooxidans broad host range plasmid Appl Envir Micro
Kahmann R and Kamp Nucleotide sequences of the attachment bacteriophage Mu DNA Nature 280247-250
Kahn K and Schaefer M R Characterization of trans po son 5469 from cyanobacterium Fremyella diplosiphon J 1777026-7032
Karavaiko G Golovacheva S Pivovarova T A Tzaplina I A and Vartanjan 1988 Thermophilic ltPM Sulfobacillus page 29-41 In Biohydrometallurgyshy87 Norris P and Science and Technology Letters Kew 1
Kennedy J and Humpphreys J D 1976 Microbial cells living immobilised on Nature 261242-244
Koonin 1993 SpoU protein of Escherichia coli belongs to a new family of Nucleic Acids Res 19
Kopecko J and site specific recA mdependtm recombination between bacterial Pt of palindromes at the N ad Acad Sci USA
on L-glutamine D-fructose ud()transtiera~e 1 BioI
152
Krumholz L R Esser U and Simoni RD 1989 Nucleotide sequence of the unc operon of Vibrio alginolyticus Nucleic Acids Res 177993-7994
Kubo K and Craig N 1990 Bacterial transposon Tn7 utilizes two classes of target sites 1 Bacteriol 1722774-2778
Kucharczyk N Denisot M A Le Goffic F and Badet B 1990 Glucosarnine-6-phosphate synthase from Ecoli detennination of the mechanism of inactivation by N3 fumaroyl-L-2-3 diarninoproprionic derivatives Biochemistry 293668-3676
Kusano M Takeshima T Inoue C and Sugawara K 1991 Evidence for two sets of structural genes coding for Ribulose biphosphate carboxylase in Thiobacillus ferrooxidans 1 Bacteriol 1737313-7323
Lane Dl A P Harrison lnr D Stahl B Pace SJ Giovannoni GJ Olsen and N R Pace 1992 Evolutionary relationship among sulphur- and iron-oxidizing eubacteria 1 Bacteriol 174267-278
Lee C H Bhagwhat A and Heffron F 1983 Identification of a transposon TnJ sequence required for transposition immunity Natl Acad Sci USA 806765-6769
Lewis M 1 Chang 1 A and Somoni R D 1990 A topological analysis of subunit a from Escherichia coli F1Fo-ATP synthase predicts eight transmembrane segments 1 BioI Chern 265 10541-10550
Lichtenstein C P and Brenner S 1982 Unique insertion site of Tn7 in the Ecoli chromosome Nature (London) 297601-603
Lichtenstein C P and Brenner S 1981 Site-specific properties of transposition to the Ecoli chromosome Mol Gen Genet 183380-387
Liu X Petersson S and Sandstrom A Mesophilic versus moderate thennophilic bioleaching Biohydrometallurgy Technologies Vol 1 A E Tonna 1 E Wey and Lakshmanan V 1 (ed) pp 29-38
Lizarna H M and Sankey B M 1993 Oxidation of H2S by Thiobacillus thiooxidans is inhibited by substrate and methane BiohydrometaHurgy Technologies Vol II A E Tonna 1 E Wey and C L Brierley (ed) pp 339-364
Lundgren D G and Silver M 1980 Ore leaching by bacteria Ann Rev Microbiol 34263shy268
Makino K Amemura M Shinagawa H Kobayashi A and Nakata A 1985 Sequence of the genes involved in phosphate transport and regulation of the phosphate regulon in Escherichia coli 1 Mol BioI 184231-240
Makino K Shinagawa Amemura M Kimura S Nakata A and Ishihama 1988 Euuv1J of the phosphate regulon of Ecoli Activation of pstS by the PhoB
protein in vitro 1 Mol BioI 20385-95
Makino K Shinagawa H Amemura M Yamada M and 1990 Signal transduction in the phosphate regulon of the Escherichia coli involves phosphotransfer nprlrJp~middotn PhoR and PhoB proteins J Mol BioI 21055
Malamy 1966 Frameshift mutations in the nnlnn of Escherichia coli Cold Harb Symp Quant BioI 31189-201
Malamy M H 1972 Electron microscopy insertions in the lac operon of Escherichia coli MoL Gen
Malamy M H and Bennett R L 1970 mutants of Ecoli and phosphate transport Biochem Biophys Res Commun
Maniatis T Fritsch EF Sambrook J 1982 nn A laboratory manual Cold Spring Harbor Laboratory Press Cold
McKnight G L S L Mudri SH Mathewest R S Marshall P O Sheppard and P J OHara 1990 Molecular cloning synthesis and bacterial expression of human glutaminefructose-6-phosphate UUAUU u J Chem 26725208-25212
Medveczky N and H Rosenburg 1970 phosphate-binding protein of Escherichia Biochim Biophys Acta 211 158-168
Mei B and Zalkin H 1989 A Cysteine-Histidine-Aspartate catalytic triad is involved in Glutamide Amide Transfer Function purF-type Glutamine amodotransferases 1 BioI Chem 264 16613-16619
Mengin-Lecreulx D and J van 1993 Identification of glmU gene encoding Nshyacetylglucosamine in Escherichia coli J Bacteriol 1756150shy6157
Michaelis M J and Criddle M J 1970 Mitochondrial DNA and mutants in Saccharomyces cerevisiae Biochem Genet 5487-495
Michaelis Starlinger P 1969 Two insertions in the jO v
operon having different homologous DNA sequences MoL Gen Genet 10437 377
Miller J H Calos Transposable elements Cell 20579-595
154
Miller J Oldenburg M and Fillingame R 1990 The essential carboxyl group of in subunit c the FIFo ATP synthase can be and H( +)-translocating function retained Proc NatL Acad 874900-4904
Mitcell P 1966 Chemiosmotic coupling in - and phosphorylation BioL Rev 41455-502 (1966)
Mizuuchi K 1984 Mechanism of transposition of bacteriophage Mu Polarity of the strand reaction at the initiation of the transposon Cell 39395-404
C Ph de Donato Berthelin J Surface oxidized species a key factor in the study of bioleaching processes Biohydrometallurgical In A J
Weyand V I Lakshmanan ed 1993 Vol 1 175-184
A Ishino Shinagawa H Makino K and M 1987 Nucleotide of the iap gene responsible for alkaline phosphatase isoenzyme conversion in Ecoli
identification of product 1 1695429-5433
K 1989 The tsr gene-coding prevents thiopeptin from inhibiting ppGpp synthesis in Streptomyces lividans FEMS Microbiol Lett
Ogasawara N and Yoshikawa H 1992 and their organIzatIon in the repnc()n of the bacterial chromosome Mol Microbiol 6(5)629-634
Ohtsubo Davidson N and 1975 microscope heteroduplex studies of sequence relations among plasmids identification and mapping of the insertion sequences 1 and IS2 in F and R plasmids 1 122746-775
Olson G J 1994 Microbial oxidation gold ores and gold bioleaching FEMS un Lett 119 1-6
Orle K A N L 1991 Identification of transposition prroteins by the bacterial transposon [corrected republished originally printed in Gene 1990 Nov 30 96(1) Gene 10425-31
Ouellette M Roy P Homology of ORFs from and from to site specific recombinases 1987 Nucl Acids 10055-10059
Murein synthesis p663-671ln Neidhardt J L Ingraham B Louw M~ M Schaechter H E Umbarger (ed) Escherichia coli and Salmonella
ryphimurium cellular and bioI voL 1 American Society Microbiology Washington
Perlin D S and Senior A Functional and cross-reactivity of antibody to purified subunit b (uncF protein) of Escherichia coli proton-ATPase Arch Biochem Biophys 236603-611
155
Plumbridge J A O Cochet Souza J M Altramirano M M Calcagno M L and Badet B 1993 Coordinated regulation of amino sugar-synthesizing and -degrading enzymes in Escherichia coli K-12 J Bacteriol 175(16)4951-4956
Pretorius I M Rawlings D E and Woods D R 1986 Identification and cloning of Thiobacillus jerrooxidans structural Nif genes in Escherichia coli Gene 4559-65
Qadri M I Flores C c Davis J and Lichtenstein C 1989 Genetic analysis of attTn7 the transposon Tn7 attachment site in Escherichia coli using a novel M13-based transduction assay 1 Mol BioI 20785-98
Radstrom P Skold 0 Swedberg J Roy P H and Sundstrom L 1994 Tn5090 of plasmid R751 which carries integron is related to Tn7 Mu and retroelements J Bacteriol 1763257-3268
Raetz C R H 1987 Structure and biosynthesis of lipid A in Escherichia coli pp 498-503 In F C Niedhardt J L Ingraham K B Low B Magasanik M Schaechter and H E Umbarger (ed) Escherichia coli and Salmonella typhimurium cellular and molecular biology vol 1 American Society for Microbiology Washington DC
Rao N N and Torriani A 1990 Molecular aspects of phosphate transport in Escherichia coli Mol Microbiol 4(7) 1083-1090
Rawlings DE D R Woods and NP Mjoli 1991 The cloning and structre of genes from the autotrophic biomining bacterium Thiobacillusjerrooxidans p 215-237 In PJ Greenaway (ed) Advances in gene technology vol 2 JAI Press London
Richet E and O Raibaud 1989 MalT the regulatory protein of the Escherichia coli maltose system is an A TP-dependent transcriptional activator EMBO 1 8981-987
Rogers M Ekaterrinaki N Nimmo E and Sherratt D 1986 Analysis of Tn7 transposition Mol Gen Genet 205550-556
Ronson C W Nixon B T Albright L M anf Ausubel F M 1987 Rhizobium meliloti ntrA (rpoN) gene is required for diverse metabolic functions J Bacteriol 1692424-2431
Rosenburg H Gerdes R G and Chegwidden K 1977 Two systems for the uptake of phosphate in Ecoli JBacterioL 131505-511
Rosenburg H 1987 In ion transport in Prokaryotes Rosen B P and Silver S (eds) New York Academic Press pp 205-248
Ross D G Swan 1 and N Klecker 1979 Physical structures of TnlO-promoted deletions and inversions role of 1400 base pairs inverted repetitions Cell 16721-731
156
Saedler H and P 19670 mutation in the galactose operon in Ecoli II Physiological characterization MoL Gen Genet 100 190-202
Saedler P 1968 00 and strong polar mutations in the Genet 102353-363
Sambrook J Fritsch Maniatis 1989 Molecular cloning a laboratory manual Cold Harbor Laboratory Cold Spring Harbor New York
Sand W Gehrke T Hallmann Rohde K Sobotke B and Wintzien S 1993 Bioleaching metal sulphides The importance of Leptospirillum ferrooxidans Biohydromemetallurgical Technologies Voll pp 15-25
Sanger Nicklen and Coulson A DNA sequencing with chain-tennination inhibitors Natl Acad USA 745463-5467
Schneider E and Altendorf K All the three subunits are required for an active proton channel (Fo) of Escherichia coli synthase (FIFo) ~-
518
Schneider E and Allendorf K 1987 Bacterial adenosine 5-triphosphate synthase purification and reconstitution complexes and biochemical and functional characterization of subunits Microbio Rev 51477-497
Schrader 1 A and D S Holmes 1988 Phenotypic switching Thiobacillus ferrooxidans J BacterioL 1703915-3023
Scordilis G E H and Lassie 1987 Identification of transposable elements which activates gene expression in Pseudomonas cepacia J Bacteriol 1698-13
Sekine Eisaki N and Ohtsubo E 1996 Identification and characterization of the lS3 molecules generated by breaks 1 BioL Chern 271197-202
Senior A E 1990 The proton-trans locating of Escherichia coli Annu Rev Biophys Chern 194-41
Shapiro 1 and Adhya S 1969 The jLU operon of K-12 n A deletion analysis of the structure polarity 62249-264
J A 1969 Mutations caused by insertion genenc material the galactose operon Escherichia 1 MoL BioI 4093-105
Silvennan M P 1967 Mechanism of bacterial pyrite oxidation 1 Bacteriol 941046-1051
157
C C ElIson and Levinson 1983 Identification of the typel trimethoprim resistance reductase specified by the R-plasmid R43 comparison with procaryotic and eucaryotic dihydrofolate reductase 1 Bacteriol 155 100 1-1008
Smith and Jones P transposition a multigene process Identification of a regulatory product 147915-7927
Sprague Jr Bell R M Cronan 1 A mutant of Ecoli auxotrophic for organic phosphates evidence two defects in phosphate Mol Gen Genet 14371-77
Starlinger and Michaelis 1968 Suppression the sequential aO[)ealame of the galactose enzymes in a transferase amber mutant coli Mol Genet 102367-369
Starlinger 1977 IS elements in microorganisms MicrobioL Immunol
Steffens K Schneider E Herkernhoff B Schmid Altendorf K portion of Escherichia coli A TP synthase resolution of from subunit b J BioI Chern 2625866-5869
Steffens A Deckers-hebestreit G and Altendorf K 1987b The and functional relationship of A TP synthases (FoFI) from Ecoii and the thermophilic bacterium PS3 J Biol 2626334-6338
Strominger J and M S Smith 1959 Uridine diphosphoacetylglucosamine pyrophosphorylase 1 BioI Chern 2341822-1827
Sundstrom Skold 1990 dhfrI trimethoprim resistance gene of can be found at in other genetic surroundings Antimicrob Agents Chemother 34642shy650
Sundstrom L Roy P H and Skold Site-specific of three cassettes in Tn7 J Bacteriol 1733025-3028
Surin B P and Downie JA 1988 of the Rhizobium leguminosarum nodLMN involved efficient host-specific nodulation Mol Microbiol 2 173-183
B P Dixon N and Rosenburg 1986 Purification PhoU protein a OClItnl
regulator of the pho regulon of Ecoli K-1 J Bacteriol 168631
B P D A Jans A L Fimmel Shaw GB Cox Rosenburg Structural gene for the phosphate-repressible phosphate binding of Escherichia coli
own promoter nucleotide of the phoS 1 Bacteriol
158
Suzuki I Chang W and Takeuchi 1994 Oxidation of organic compounds by Thiobacilli Symp Series 55060-67
Takeyama M S Y Noumi T Maeda Ishibashi S and Futai M 1988 Beta subunit of Ecoli amino acid replacement within a conserved sequence G-X-X-X-X-GshyK-TS) v~u binding proteins lett 218222-226
Thomson JA M Hendson and R M 1981 Mutagenesis by insertion of drug resistance Tn7 into a vibrio 11 J Bacteriol
Tichy R Grotenhuis J T c Janssen van Houten R Rulkens and Lettinga 1993 Application of the sulphur cycle bioremediation of soils polluted with heavy metals In Int Conf Contaminated 93 ed F Arendt G J Annokee R Bosman and W J van der Brink Kluwer Academic Publishers Dordrecht pp 1461-1462
G and Mayer An electron approach to the quaternary structure of mitochondrial Eur J Biochem 13237-45
J and Tschape streptothricin resistance transposons Tn1825 and Tn1826 and transposon Tn 7 Plasmidnuu
18246-249
Tso J Y Zalkin H van Cleemput M Yanofsky C and JM 1982 Nucleotide of Escherichia coli and deduced amino glutamine
phosphribosylpyrophosphate am idotransferase J BioI Chern
V Mesyanzhinova I V Koslov I A and Orlova 1984 Structure of studied by electron and image processing Lett 167285-289
P Barber C and transposons Tn5 and Tn7 in Xanthomonas campestris pv campestris MoL Gen 157
Ullrich J and van Putten P Identification of the Gonococcal glmU gene UVUUIJ
the enzyme N-acetylglucosamine 1-Phosphate Uridyltransferase involved in the synthesis UDP-GlcNAc J of Bact 1776902-6909
M 1992 Eight bacterial proteins includin]g UDP-N -acetylglucosamine acyltransferase three vother of Escherichia coli of a six-residue n tVI
theme FEMS MicrobioL 97249-254
van der Steen J J D Doddema J and de Uidoging van zware metalen afvalstromen met behulp van thiobacilli (Removal metals from waste streams
using thiobacilli) Ministry of Housing Physical and Enviromental (VROM) Directoraat-Generaal Milieubeheer Rapport 199210 Hague
Vermoote P 1988 Universite Paris VI Paris Hrllnro
159
Vignais P V Lunard Issartel J P and Dupuis A 1985 Interaction n l1f middotn
oligomycin sensitivity protein (OSCP) beef heart mitochondrial FeATPase 2 Identification of interacting Fl subunits cross-linking Biochem J
Vik S and Dao NN Prediction of transmembrane topology of Fo proteins from Ecoli ATP synthase variational hydrophobic moment analyses Biochim Biophys Acta 1140 199-207
von Meyenburg B B Jcentrgensen J Nielsen and F Hansen 1982 Promoters of the atp operon for the membrane-bound ATP synthase of Ecoli mapped by TnlO insertion mutations MoL Gen Genet 188240-248
von Meyenberg F G Hansen 1980 The origin of replication oriC the Ecoli chromosome near oriC and construction of oriC mutants ICN-UCLA Symp MoLCellBiol 191 159
Waddell S and Craig N 1989 Tn7 transposition recognition of the attTn7 Proc Natl Sci USA 863958-3962
Waddell C S and Craig N L 1988 transposition two transposition pathways directed by five Tn7-encoded genes Develop 21
J E Gay N J Saraste M and A N 1984 DNA sequence the Escherichia coli unc operon Completion of the sequence of a kilobase segment containing
oriC unc and phoS J224799-815
and Collinson 1 1994 the role the stalk in the coupling mechanism of FEBS 34639-43
Walker 1 E Gay N J and Tybulewicz V L 1986 of transposon Tn7 into the Escherichia glmS transcriptional terminator Biochem 1 243 111-1
Wanner Land McSharry 1982 Phosphate-controlled gene in Escherichia coli using Mudl-directed lacZ fusions J Mol BioI 158347-363
Weng M and Zalkin H 1987 Structural role for a region the CTP synthetase glutamide amide transfer domain J Bacteriol 1693023-3028
Wheatcroft R and S and nucleotide sequence of Rhizobiwn meliloti insertion between the transposase encoded by ISRm3 and those encoded by Staphylococcus aureus and Thiobacillus ferrooxidans IST2 J 1732530-2538
160
Whitby M C and Lloyd R 1995 Branch of three-strand recombination intennediates by RecG a possible pathway for securing middotIULl initiated by duplex DNA 1 143303-3310
Wilkens and Capaldi R A Assymmetry and changes in examined by cryoelectronmicroscopy BioI Chern Hoppe-Seyler 37543-51
and Malamy M 1974 The loss of the PhoS periplasmic protein leads to a the specificity a constitutive phosphate transport system in Escherichia coli Biochem Res Commun 60226-233
WiUsky G Rand Malamy M 1980 of two separable inorganic phosphate transport systems in Escherichia coli J BacterioL 144356-365
P J and and C F 1973 enzyme of metabolism and Stadtman R eds) pp 343-363 Academic Press New York
Winterbum P J and Phelps F 197L control of hexosamine biosynthesis by glucosamine synthetase Biochem 1 121711
Wolf-Watz H and Norquist A 1979 Deoxyribonucleic acid and outer membrane protein to outer membrane protein involves a protein J 14043-49
Wolf-Watz H and M 1979 Deoxyribonucleic acid and outer membrane strains for oriC elevated levels deoxyribonucleic acid-binding protein and
11 for specific UU6 of the oriC region to outer membrane J Bacteriol 14050-58
Wolf-Watz H 1984 Affinity of two different chromosome to the outer membrane of Ecoli J Bacteriol 157968-970
Wu C and Te Wu 197 L Isolation and characterization of a glucosamine-requiring mutant of Escherichia 12 defective in glucoamine-6-phosphate synthetase 1 Bacteriol 105455-466
Yamada M Makino Shinagawa Nakata A 1990 Regulation of the phosphate regulon of Escherichia coli properties the pooR mutants and subcellular localization of protein Mol Genet 220366-372
Yates J R and Holmes D S 1987 Two families of repeatcXl DNA sequences Thiobacillus 1 Bacteriol 169 1861-1870
Yates J R R P and D S 1988 an insertion sequence from Thiobacillus errooxidans Proc Natl 857284-7287
161
Youvan D c Elder 1 T Sandlin D E Zsebo K Alder D P Panopoulos N 1 Marrs B L and Hearst 1 E 1982 R-prime site-directed transposon Tn7 mutagenesis of the photosynthetic apparatus in Rhodopseudomonas capsulata 1 Mol BioI 162 17-41
Zalkin H and Mei B 1990 Amino terminal deletions define a glutamide transfer domain in glutamine phosphoribosylpyrophosphate ami do transferase and other purF-type amidotransferases 1 Bacteriol 1723512-3514
Zalkin H and Weng M L 1987 Structural role for a conserved region III the CTP synthetase glutamide amide transfer domain 1 Bacteriol 169 (7)3023-3028
Zhang Y and Fillingame R H 1994 Essential aspartate in subunit c of FIF0 ATP synthase Effect of position 61 substitutions in helix-2 on function of Asp24 in helix-I 1 BioI Chern 2695473-5479
ii
ACKNOWLEDGEMENTS
I am most grateful to my supervisor Prof Doug Rawlings for his excellent supervision
guidance and encouragement throughout the research Without him this work would not
have been accomplished I am also indebted to my colleagues in the Thiobacillus Research
Unit Ant Smith Ros Powles Cliff Dominy Shelly Deane and the rest of my colleagues
in the department who in one way or the other helped me throughout the period of study
I am grateful to all the members of the Department of Microbiology for their assistance
and support during the course of this research Special thanks to Di James Anne Marie
who is always there when you need her and Di DeVillers for her amazing concern to let it
be there when you need it
My friends Kojo Joyce Pee Chateaux Vic Bob Felix Mawanda Moses the list of
which I cannot exhaust you have not been forgotten Thank you for your encouragement
which became the pillar of my strength Last but not the least I acknowledge the financial
support from Foundation for Research and Development of CSIR and the General Mining
Corp of South Africa
iii
A Ala amp Arg Asn Asp ATCC ATP
bp
C CGSC Cys
DNA dNTP(s) DSM
EDTA
9 G-6 P GFAT GIn Glu Gly
hr His
Ile IPTG
kb kD
I LA LB Leu Lys
M Met min ml NAcG-6-P
ORF (s) oriC
ABBREVIATIONS
adenine L alanine ampicillin L-Arginine L-Asparagine L-aspartic acid American Type Culture Collection adenos triphosphate
base pair(s)
cytosine Coli Genetic Stock Centre L-cysteine
deoxyribonucleic acid deoxyribonucleotide triphosphates Deutsche Sammlungvon Mikroorganismen (The German Collection) ethylenediamine tetraacetic acid
pSIS1 construct was further subcloned to plasm ids IS30 IS40 pSlS50
pS183S p81841 and p81 (Fig Some of subclones were extensively mapped
although not all sites are shown in 22 exact positions of the ends of
T jerrooxidans plasmids pTfatpl and pTfatp2 on the cosmid p8I81 were identified
21)
Tfeooxidans
that the cosmid was
unrearranged DNA digests of cosmid pSISI and T jerrooxidans chromosomal DNA were
with pSI The of the bands gave a positive hybridization signal were
identical each of the three different restriction enzyme digests of p8I8l and chromosomal
In order to confirm of pSI81 and to
49
kb kb ~
(A)
11g 50Sts5
middot 2S4
17 bull tU (probe)
- tosect 0S1
(e)
kb
+- 10 kb
- 46 kb
28---+ 26-+
17---
Fig 23 Hybrdization of cosmid pSISI and T jerrooxitians (A TCC 33020) chromosomal
DNA by the KpnI-SalI fragment of pSIS52 (probe) (a) Autoradiographic image of the
restriction digests Lane x contains the probe lane 13 and 5 contain pSISI restricted with BamHl HindIII and Bgffi respectively Lanes 2 4 and 6 contain T errooxitians chromosomal DNA also restricted with same enzymes in similar order (b) The sizes of restricted pSISI
and T jerrooxitians chromosome hybridized by the probe The lanes correspond to those in Fig 23a A DNA digested with Pst served as the molecular weight nlUkermiddot
50
DNA (Fig BamHI digests SHklC at 26 and 2S kb 1 2) HindIII
at 10 kb 3 and 4) and BgnI at 46 (lanes 5 and 6) The signals in
the 181 was because the purified KpnI-San probe from p81 had a small quantity
ofcontaminating vector DNA which has regions ofhomology to the cosmid
vector The observation that the band sizes of hybridizing
for both pS181 and the T ferrooxidans ATCC 33020 same
digest that the region from BgnI site at to HindIII site at 16
kb on -VJjUIU 1 represents unrearranged chromosomal DNA from T ferrooxidans
ATCC there were no hybridization signals in to those predicted
the map with the 2 4 and 6 one can that are no
multiple copies of the probe (a Tn7-like segment) in chromosome of
and Lferrooxidans
of plasmid pSI was found to fall entirely within a Tn7-like trallSDOS()n nrpn
on chromosome 33020 4) A Southern hybridizationUUAcpoundUU
was out to determine whether Tn7-like element is on other
ofT ferrooxidans of T and Lferrooxidans results of this
experiment are shown 24 Lanes D E and F 24 represent strains
19377 DSM and Lferrooxidans DSM 2705 digested to
with BgnI enzyme A hybridization was obtained for
the three strains (lane A) ATCC 19859 (lane B) and UUHUltn
51
(A) AAB CD E F kb
140
115 shy
284 -244 = 199 -
_ I
116 -109 -
08
os -
(8)) A B c o E F
46 kb -----
Fig 24 (a) Autoradiographic image of chromosomal DNA of T ferrooxidans strains ATCC 33020 19859 23270 (lanes A B C) Tthiooxidans strains ATCC 19377 and DSM 504 (D and E) and Lferrooxidans strain DSM 2705 (lane F) all restricted with Bgffi A Pst molecular weight marker was used for sizing (b) Hybridization of the 17 kb KpnI-San piece of p81852 (probe) to the chromosomal DNA of the organisms mentioned 1 Fig 24a The lanes in Fig 24a correspond to those in Fig 24b
(lane C) uuvu (Fig 24) The same size BgllI fragments (46 kb) were
lanes A Band C This result
different countries and grown on two different
they Tn7-like transposon in apparently the same location
no hybridization signal was obtained for T thiooxidans
1 504 or Lferrooxidans DSM 2705 (lanes D E and F of
T thiooxidans have been found to be very closely related based on 1
rRNA et 1992) Since all Tferrooxidans and no Tthiooxidans
~~AA~~ have it implies either T ferrooxidans and
diverged before Tn7-like transposon or Tthiooxidans does not have
an attTn7 attachment the Tn7-like element Though strains
T ferrooxidans were 10catH)uS as apart as the USA and Japan
it is difficult to estimate acquired the Tn7-like transposon as
bacteria get around the world are horizontally transmitted It
remains to be is a general property
of all Lferrooxidans T ferrooxidans strains
harbour this Tn7-like element their
CHAPTER 3
5431 Summary
54Introduction
56Materials and methods
56L Bacterial and plasmid vectors
Media and solutions
56333 DNA
57334 gel electrophoresis
Competent preparation
57336 Transformation of DNA into
58337 Recombinant DNA techniques
58ExonucleaseIII shortening
339 DNA sequencing
633310 Complementation studies
64Results discussion
1 DNA analysis
64Analysis of ORF-l
72343 Glucosamine gene
77344 Complementation of by T ferrooxidans glmS
57cosmid pSIS1 pSlS20
58Plasmidmap of p81816r
Fig Plasmidmap of lS16f
60Fig 34 Restriction digest p81S1Sr and pSIS16f with
63DNA kb BamHI-BamHI
Fig Codon and bias plot of the DNA sequence of pSlS16
Fig 37 Alignment of C-terminal sequences of and Bsubtilis
38 Alignment of amino sequences organisms with high
homology to that of T ferrooxidans 71
Fig Phylogenetic relationship organisms high glmS
sequence homology to Tferrooxidans
31 Summary
A 35 kb BamHI-BamHI p81820 was cloned into pUCBM20 and pUCBM21 to
produce constructs p818 and p81816r respectively This was completely
sequenced both rrelct1()ns and shown to cover the entire gmS (184 kb) Fig 31
and 34 The derived sequence of the T jerrooxidans synthetase was
compared to similar nrvnC ~ other organisms and found to have high sequence
homology was to the glucosamine the best studied
eubacterium EcoU Both constructs p81816f p81 the entire
glmS gene of T jerrooxidans also complemented an Ecoli glmS mutant for growth on medium
lacking N-acetyl glucosamine
32 =-====
Cell wall is vital to every microorganism In most procaryotes this process starts
with amino which are made from rructClsemiddotmiddotn-[)ll()S by the transfer of amide group
to a hexose sugar to form an This reaction is by
glucosamine synthetase the product of the gmS part of the catalysis
to the 40-residue N-terminal glutamine-binding domain (Denisot et al 1991)
participation of Cys 1 to generate a glutamyl thiol ester and nascent ammonia (Buchanan
368-residue C-terminal domain is responsible for the second part of the ClUUU
glucosamine 6-phosphate This been shown to require the ltgtttrltgtt1iltn
of Clpro-R hydrogen of a putative rrulctoseam 6-phosphate to fonn a cis-enolamine
intennediate which upon reprotonation to the gives rise to the product (Golinelli-
Pimpaneau et al 1989)
bacterial enzyme mn two can be separated by limited chymotryptic
proteolysis (Denisot et 199 binding domain encompassing residues 1 to
240 has the same capacity to hydrolyse (and the corresponding p-nitroanilide
derivative) into glutamate as amino acid porI11
binding domain is highly cOllserveu among members of the F-type
ases Enzymes in this include amidophosphophoribosyl et al
1982) asparagine synthetase (Andrulis et al 1987) glucosamine-6-P synmellase (Walker et
aI 1984) and the NodM protein of Rhizobium leguminosarum (Surin and 1988) The
368-residue carboxyl-tenninal domain retains the ability to bind fructose-6-phosphate
The complete DNA sequence of T ferrooxidans glmS a third of the
glmU gene (preceding glmS) sequences downstream of glmS are reported in this
chapter This contains a comparison of the VVA r glmS gene
and Mycobacterium (422 ) An interesting observation was that the T ferrooxidans
glmS gene had comparatively high homology sequences to the Rhizobium leguminosarum and
Rhizobium meliloti M the amino to was 44 and 436
respectively A consensus Dalgarno upstream of the start codon
(ATG) is in 35 No Ecoli a70-type n r~ consensus sequence was
detected in the bp preceding the start codon on intergenic distance
between the two and the absence of any promoter consensus sequence Plumbridge et
al (1993) that the Ecoli glmU and glmS were co-transcribed In the case
the glmU-glmS --n the T errooxidans the to be similar The sequences
homology to six other amidotransferases (Fig 38) which are
(named after the purF-encoded l phosphoribosylpyrophosphate
amidotransferase) and Weng (1987)
The glutamine amide transfer domain of approximately 194 amino acid residues is at the
of protein chain Zalkin and Mei (1989) using site-directed to
the 9 invariant amino acids in the glutamine amide transfer domain of
phosphoribosylpyrophosphated indicated in their a
catalytic triad is involved glutamine amide transfer function of
73
Comparison of the ammo acid sequence alignment of ghtcosamine-6-phosphate
amidotranferases (GFAT) of Rhizobium meliloti (R_m) Rhizobium leguminnosarum (RJ)
Ecoli (E_c) Hinjluenzae Mycobacterium leprae (M_I) Bsubtilis (B_s) and
Scerevisiae (S_c) to that of Tferrooxidans Amino acids are identified by their single
codes The asterisks () represent homologous amino acids of Tferrooxidans glmS to
at least two of the other Consensus ammo acids to all eight organisms are
highlighted (bold and underlined)
1 50 R m i bullbullbullbullbullbull hkps riag s tf bullbullbullbull R~l i bullbullbullbullbullbull hqps rvaep trod bullbullbullbull a
a bullbullbullbullbullbull aa 1r 1 d bullbullbull a a bullbullbullbullbullbull aa inh g bullbullbullbullbullbull sktIv pmilq 1 1g bullbullbull a MCGIVi V D Gli RI IYRGYPSAi AI D 1 gvmar s 1gsaks Ikek a bullbullbullbull qfcny Iversrgi tvq t dgd bullbull ead
51 100 R m hrae 19ktrIk bull bullbull bullbull s t ateR-l tqrae 19rkIk bullbullbull s t atrE-c htIrI qmaqae bull bull h bullbull h gt sv aae in qicI kads stbull bullbull k bullbull i gt T f- Ivsvr aetav bullbullbull r bullbull gq qg c
DL R R G V L AV E L G VGI lTRW E H ntvrrar Isesv1a mv bull sa nIgi rtr gihvfkekr adrvd bullbullbull bullbull nve aka g sy1stfiykqi saketk qnnrdvtv she reqv
101 150 R m ft g skka aevtkqt R 1 ft g ak agaqt E-c v hv hpr krtv aae in sg tf hrI ksrvlq T f- m i h q harah eatt
S E Eamp L A GY l SE TDTEVIAHL ratg eiavv av
qsaIg lqdvk vqv cqrs inkke cky
151 200 bullbull ltk bullbull dgcm hmcve fe a v n bull Iak bullbull dgrrm hmrvk fe st bull bull nweI bullbull kqgtr Iripqr gtvrh 1 s bull bull ewem bullbullbull tds kkvqt gvrh hlvs bull bull hh bullbull at rrvgdr iisg evm
V YR T DLF A A L AV S D PETV AR G M 1 eagvgs lvrrqh tvfana gvrs B s tef rkttIks iInn rvknk 5 c Ihlyntnlqn ~lt klvlees glckschy neitk
74
201 R m ph middot middot middot R 1 ah- middot middot middot middot middot middot E c 1 middot middot middot middot middot middot middot Hae in 1 middot middot middot middot middot T f - c11v middot middotmiddot middot middot middot middot middot M 1 tli middot middot middot middot middot middot middot middot middot B s 11 middot middot middot middot middot S c lvkse kk1kvdfvdv
251 R m middot middot middot middot middot middot middot middot middot middot middot middot middot middot
middot middot middot middot R-1 middot middot middot middot middot middot E c middot middot middot middot middot middot middot middot Hae in middot middot middot middot middot middot middot middot middot middot middot middot T f shy middot middot middot middot middot middot middot middot middot middot middot middot 1M middot middot middot middot middot middot middot middot middot middot middot middot middot middot B s middot middot middot middot middot middot middot middot middot middot middot middot middot middot middot S c feagsqnan1 1piaanefn1
301 R m fndtn wgkt R-1 fneti wgkt E c rf er Hae in srf er T f- rl mlqq
PVTR V YE DGDVA R M 1 ehqaeg qdqavad B s qneyem kmvdd S c khkkl dlhydg
351 R m nhpe v R-1 nhe v E c i nk Hae in k tli T f- lp hr
~ YRHlrM~ ~I EQP AVA M 1 geyl av B-s tpl tdvvrnr S-c pd stf
401 Rm slasa lk R-1 slasca lk E c hql nssr Hae in hq nar-T f f1s hytrq
RVL A SiIS A VG W M 1 ka slakt B s g kqS c lim scatrai
T ferrooxidans (Tf) Scerevisiae (Saee) Mleprae (Myele) and Bsublilis (Baesu)
tr1 ()-ltashyc 8 ~ er (11
-l l
CIgt
~ r (11-CIgt (11
-
$ -0 0shy
a c
omiddot s
S 0 0shyC iii omiddot $
ttl ~ () tI)
I-ltashyt ()
0
~ smiddot (11
81
CHAPTER 4
8341 Summary
8442 Introduction
8643 Materials and methods
431 Bacterial strains and plasmids 86
432 DNA constructs subclones and shortenings 86
864321 Contruct p81852
874322 Construct p81809
874323 Plasmid p8I809 and its shortenings
874324 p8I8II
8844 Results and discussion
441 Analysis of the sequences downstream the glmS gene 88
442 TnsBC homology 96
99443 Homology to the TnsD protein
118444 Analysis of DNA downstream of the region with TnsD homology
LIST OF FIGURES
87Fig 41 Alignment of Tn7-like sequence of T jerrooxidans to Ecoli Tn7
Fig 42 DNA sequences at the proximal end of Tn5468 and Ecoli glmS gene 88
Fig 43 Comparison of the inverted repeats of Tn7 and Tn5468 89
Fig 44 BLAST results of the sequence downtream of T jerrooxidans glmS 90
Fig 45 Alignment of the amino acid sequence of Tn5468 TnsA-like protein 92 to TnsA of Tn7
82
Fig 46 Restriction map of the region of cosmid p8181 with amino acid 95 sequence homology to TnsABC and part of TnsD of Tn7
Fig 47 Restriction map of cosmid p8181Llli with its subclonesmiddot and 96 shortenings
98 Fig 48 BLAST results and DNA sequence of p81852r
100 Fig 49 BLAST results and DNA sequence of p81852f
102 Fig 410 BLAST results and DNA sequence of p81809
Fig 411 Diagrammatic representation of the rearrangement of Tn5468 TnsDshy 106 like protein
107Fig 412 Restriction digests on p818lLlli
108 Fig 413 BLAST results and DNA sequence of p818lOEM
109 Fig 414 BLAST results on joined sequences of p818104 and p818103
111 Fig 415 BLAST results and DNA sequence of p818102
112 Fig 416 BLAST results and nucleotide sequence of p818101
Fig 417 BLAST results and DNA sequence of the combined sequence of 113p818l1f and p81810r
Fig 418 Results of the BLAST search on p81811r and the DNA sequence 117obtained from p81811r
Fig 419 Comparison of the spa operons of Ecali Hinjluenzae and 121 probable structure of T ferraaxidans spa operon
83
CHAPTER FOUR
TN7-LlKE TRANSPOSON OF TFERROOXIDANS
41 Summary
Various constructs and subclones including exonuclease ill shortenings were produced to gain
access to DNA fragments downstream of the T ferrooxidans glmS gene (Chapter 3) and
sequenced Homology searches were performed against sequences in GenBank and EMBL
databases in an attempt to find out how much of a Tn7-like transposon and its antibiotic
markers were present in the T ferrooxidans chromosome (downstream the glmS gene)
Sequences with high homology to the TnsA TnsB TnsC and TnsD proteins of Tn7 were
found covering a region of about 7 kb from the T ferrooxidans glmS gene
Further sequencing (plusmn 45 kb) beyond where TnsD protein homology had been found
revealed no sequences homologous to TnsE protein of Tn7 nor to any of the antibiotic
resistance markers associated with Tn7 However DNA sequences very homologous to Ecoli
ATP-dependentDNA helicase (RecG) protein (EC 361) and guanosine-35-bis (diphosphate)
3-pyrophosphohydrolase (EC 3172) stringent response protein of Ecoli HinJluenzae and
Vibrio sp were found about 15 kb and 4 kb respectively downstream of where homology to
the TnsD protein had been detected
84
42 Transposable insertion sequences in Thiobacillus ferrooxidizns
The presence of two families (family 1 and 2) of repetitive DNA sequences in the genome
of T ferrooxidans has been described previously (Yates and Holmes 1987) One member of
the family was shown to be a 15 kb insertion sequence (IST2) containing open reading
frames (ORFs) (Yates et al 1988) Sequence comparisons have shown that the putative
transposase encoded by IST2 has homology with the proteins encoded by IS256 and ISRm3
present in Staphylococcus aureus and Rhizobium meliloti respectively (Wheatcroft and
Laberge 1991) Restriction enzyme analysis and Southern hybridization of the genome of
T ferrooxidans is consistent with the concept that IST2 can transpose within Tferrooxidans
(Holmes and Haq 1990) Additionally it has been suggested that transposition of family 1
insertion sequences (lST1) might be involved in the phenotypic switching between iron and
sulphur oxidizing modes of growth including the reversible loss of the capacity of
T ferrooxidans to oxidise iron (Schrader and Holmes 1988)
The DNA sequence of IST2 has been determined and exhibits structural features of a typical
insertion sequence such as target-site duplications ORFs and imperfectly matched inverted
repeats (Yates et al 1988) A transposon-like element Tn5467 has been detected in
T ferrooxidans plasmid pTF-FC2 (Rawlings et al 1995) This transposon-like element is
bordered by 38 bp inverted repeat sequences which has sequence identity in 37 of 38 and in
38 of 38 to the tnpA distal and tnpA proximal inverted repeats of Tn21 respectively
Additionally Kusano et al (1991) showed that of the five potential ORFs containing merR
genes in T ferrooxidans strain E-15 ORFs 1 to 3 had significant homology to TnsA from
transposon Tn7
85
Analysis of the sequence at the tenninus of the 35 kb BamHI-BamHI fragment of p81820
revealed an ORF with very high homology to TnsA protein of the transposon Tn7 (Fig 44)
Further studies were carried out to detennine how much of the Tn7 transposition genes were
present in the region further downstream of the T ferroxidans glmS gene Tn7 possesses
trimethoprim streptomycin spectinomycin and streptothricin antibiotic resistance markers
Since T ferrooxidans is not exposed to a hospital environment it was of particular interest to
find out whether similar genes were present in the Tn7-like transposon In the Ecoli
chromosome the Tn7 insertion occurs between the glmS and the pho genes (pstS pstC pstA
pstB and phoU) It was also of interest to detennine whether atp-glm-pst operon order holds
for the T ferrooxidans chromosome It was these questions that motivated the study of this
region of the chromosome
43 OJII
strains and plasm ids used were the same as in Chapter Media and solutions (as
in Chapter 3) can in Appendix Plasmid DNA preparations agarose gel
electrophoresis competent cell preparations transformations and
were all carned out as Chapter 3 Probe preparation Southern blotting hybridization and
detection were as in Chapter 2 The same procedures of nucleotide
DNA sequence described 2 and 3 were
4321 ~lllu~~~
Plasmid p8I852 is a KpnI-SalI subclone p8I820 in the IngtUMnt vector kb) and
was described Chapter 2 where it was used to prepare the probe for Southern hybridization
DNA sequencing was from both (p81852r) and from the Sail sites
(p8I852f)
4322 ==---==
Construct p81809 was made by p8I820 The resulting fragment
(about 42 kb) was then ligated to a Bluescript vector KS+ with the same
enzymes) DNA sequencing was out from end of the construct fA
87
4323 ~mlJtLru1lbJmalpoundsectM~lllgsect
The 40 kb EeoRI-ClaI fragment p8181Llli into Bluescript was called p8181O
Exonuclease III shortening of the fragment was based on the method of Heinikoff (1984) The
vector was protected with and ClaI was as the susceptible for exonuclease III
Another construct p818IOEM was by digesting p81810 and pUCBM20 with MluI and
EeoRI and ligating the approximately 1 kb fragment to the vector
4324 p81811
A 25 ApaI-ClaI digest of p8181Llli was cloned into Bluescript vector KS+ and
sequenced both ends
88
44 Results and discussion
of glmS gene termini (approximately 170
downstream) between
comparison of the nucleotide
coli is shown in Fig 41 This region
site of Tn7 insertion within chromosome of E coli and the imperfect gtpound1
repeat sequences of was marked homology at both the andVI
gene but this homology decreased substantially
beyond the stop codon
amino acid sequence
been associated with duplication at
the insertion at attTn7 by (Lichtenstein and as
CCGGG in and underlined in Figs41
CCGGG and are almost equidistant from their respelt~tle
codons The and the Tn7-like transposon BU- there is
some homology transposons in the regions which includes
repeats
11 inverted
appears to be random thereafter 1) The inverted
repeats of to the inverted repeats of element of
(Fig 43) eight repeats
(four traJrlsposcm was registered with Stanford University
California as
A search on the (GenBank and EMBL) with
of 41 showed high homology to the Tn sA protein 44) Comparison
acid sequence of the TnsA-like protein Tn5468 to the predicted of the
89
Fig 41
Alignment of t DNA sequence of the Tn7-li e
Tferrooxi to E i Tn7 sequence at e of
insertion transcriptional t ion s e of
glmS The ent homology shown low occurs within
the repeats (underl of both the
Tn7-li transposon Tn7 (Fig 43) Homology
becomes before the end of t corresponding
impe s
T V E 10 20 30 40 50 60
Ec Tn 7
Tf7shy(like)
70 80 90 100 110 120
Tf7shy(1
130 140 150 160 170 180 Ec Tn 7 ~~~=-
Tf7shy(like)
90
Fig 42 DNA sequences at the 3 end of the Ecoll glmS and the tnsA proximal of Tn7 to the DNA the 3end of the Tferrooxidans gene and end of Tn7-like (a) DNA sequence determined in the Ecoll strain GD92Tn7 in which Tn been inserted into the glmS transcriptional terminator Walker at ai 1986 Nucleotide 38 onwards are the of the left end of Tn DNA
determined in T strain ATCC 33020 with the of 7-like at the termination site of the glmS
gene Also shown are the 22 bp of the Tn7-like transposon as well as the region where homology the protein begins A good Shine
sequence is shown immediately upstream of what appears to be the TTG initiation codon for the transposon
(a)
_ -35 PLE -10 I tr T~ruR~1 truvmnWCIGACUur ~~GCfuA
100 no 110 110 110
4 M S S bull S I Q I amp I I I I bull I I Q I I 1 I Y I W L ~Tnrcmuamaurmcrmrarr~GGQ1lIGTuaDACf~1lIGcrA
DO 200 amp10 220 DG Zlaquot UO ZIG riO
T Q IT I I 1 I I I I I Y sir r I I T I ILL I D L I ilGIIilJAUlAmrC~GlWllCCCAcarr~GGAWtCmCamp1tlnmcrArClGACTAGNJ
Fig 45 (a) Alignment of the amino acid sequence of Tn5468 (which had homology to TnsA of Tn7 Fig 44) to the amino acid sequence of ORF2 (between the merR genes of Tferrooxidans strain E-1S) ORF2 had been found to have significant homology to Tn sA of Tn7 (Kusano et ai 1991) (b) Alignment of the amino acids translation of Tn5468 (above) to Tn sA of Tn7 (c) Alignment of the amino acid sequence of TnsA of Tn7 to the hypothetical ORF2 protein of Tferrooxidans strain E-1S
(al
Percent Similarity 60000 Percent Identity 44444
Tf Tn5468 1 LARQRYGVDEDRVARFQKEGRGQGRGADYHPWLTIQDVPSQGRSHRLKGI SO 11 1111111 I 1111 1111 I I 111
Fig 49 (a) Results of the BLAST search on the inverted and complemented nucleotide sequence of 1852f (from the Sall site) Results indicate amino acid sequence to the TnsC of Tn7 (protein E is the same as TnsC protein) (b) The nucleotide sequence and open reading frame of p81852f
High Prob producing Segment Pairs Frame Score P(N)
IB255431QQECE7 protein E - Escher +1 176 15e-20 gplX044921lSTN7EUR 1 Tn7 E with +1 176 15e-20 splP058461 ECOLI TRANSPOSON TN7 TRANSPOSITION PR +1 176 64e-20 gplU410111 4 D20248 gene product [Caenorhab +3 59 17e-06
IB255431QQECE7 ical protein E - Escherichia coli transposon Tn7 (fragment)
417 (al BLAST results obtained from combined sequence of (inverted and complemented) and 1810r good sequence to Ecoli and Hinfluenzae DNA RecG (EC 361) was found (b) The combined sequence and open frame which was homologous to RecG
444 Analysis of DNA downstream of region with TnsD homology
The location of the of p81811 ApaI-CLaI construct is shown in Fig 47 The single strand
sequence from the C LaI site was joined to p8181Or and searched using BLAST against the
GenBank and EMBL databases Good homology to Ecoli and Hinjluezae A TP-dependent
DNA helicase recombinase proteins (EC 361) was obtained (Fig 417) The BLAST search
with the sequence from the ApaI end showed high sequence homology to the stringent
response protein guanosine-35-bis (diphosphate) 3-pyrophosphohydrolase (EC 3172) of
Ecoli Hinjluenzae and Scoelicolor (Fig 418) In both Ecoli and Hinjluenzae the two
proteins RecG helicase recombinase and ppGpp stringent response protein constitute part of
the spo operon
445 Spo operon
A brief description will be given on the spo operons of Ecoli and Hinjluenzae which appear
to differ in their arrangements In Ecoli spoT gene encodes guanosine-35-bis
pyrophosphohydrolase (ppGpp) which is synthesized during stringent response to amino acid
starvation It is also known to be responsible for cellular ppGpp degradation (Gentry and
Cashel 1995) The RecG protein is required for normal levels of recombination and DNA
repair RecG protein is a junction specific DNA helicase that acts post-synaptically to drive
branch migration of Holliday junction intermediate made by RecA during the strand
exchange stage of recombination (Whitby and Lloyd 1995)
The spoS (also called rpoZ) encodes the omega subunit of RNA polymerase which is found
associated with core and holoenzyme of RNA polymerase The physiological function of the
omega subunit is unknown Nevertheless it binds stoichiometrically to RNA polymerase
119
Fig 418 BLAST search nucleotide sequence of 1811r I restrict ) inverted and complement high sequence homology to st in s 3 5 1 -bis ( e) 3 I ase (EC 31 7 2) [(ppGpp)shyase] -3-pyropho ase) of Ecoli Hinfluenzae (b) The nucleot and the open frame with n
Add 4 ml 52 mix by inversion at room temp for 5 mins
Add 4 ml 53 Mix by to homogenous suspension
Spin at 15 K 40 mins at 4
remove to fresh tube
Equilibrate column with 2 ml N2
Load supernatant 2 to 4 ml amounts
Wash column 2 X 4 of N3 Elute the DNA the first bed
each) add 07
volumes of isopropanol
Spin at 4 Wash with 70 Ethanol Resuspended pellet in n rv 100 AU of TE and scan
volume of about 8 to 10 drops) To the eluent (two
to
140
SEQUITHERM CYCLE SEQUENCING
Alf-express Cy5 end labelled promer method
only DNA transformed into end- Ecoli
The label is sensitive to light do all with fluorescent lights off
3-5 kb
3-7 kb
7-10 kb
Thaw all reagents from kit at well before use and keep on
1) Label 200 PCR tubes on or little cap flap
(The heated lid removes from the top of the tubes)
2) Add 3 Jtl of termination mixes to labelled tubes
3) On ice with off using 12 ml Eppendorf DNA up to
125 lll with MilliQ water
Add 1 ltl
Add lll of lOX
polymeraseAdd 1 ltl
Mix well Aliquot 38 lll from the eppendorf to Spin down
Push caps on nrnnp1
141
Hybaid thermal Cycler
Program
93 DC for 5 mins 1 cycle
93degC for 30 sees
55 DC for 30 secs
70 DC for 60 secs 30 cycle 93 DC_ 30s 55 DC-30s
70degC for 5 mins 1 cycle
Primer must be min 20 bp long and min 50 GC content if the annealing step is to be
omitted Incubate at 95 DC for 5 mins to denature before running Spin down Load 3 U)
SEQUITHERM CYCLE SEQUENCING
Ordinary method
Use only DNA transformed into end- Ecoli strain
3-5 kb 3J
3-7 kb 44
7-10 kb 6J
Thaw all reagents from kit at RT mix well before use and keep on ice
1) Label 200 PCR tubes on side or little cap flap
(The heated lid removes markings from the top of the tubes)
2) Add 3 AU of termination mixes to labelled tubes
3) On ice using l2 ml Eppendorf tubes make DNA up to 125 41 with MilliQ water
142
Add I Jtl of Primer
Add 25 ttl of lOX sequencing buffer
Add 1 Jtl Sequitherm DNA polymerase
Mix well spin Aliquot 38 ttl from the eppendorf to each termination tube Spin down
Push caps on properly
Hybaid thermal Cycler
Program
93 DC for 5 mins 1 cycle
93 DC for 30 secs
55 DC for 30 secs
70 DC for 60 secs 30 cycles
93 DC_ 30s 55 DC-30s 70 DC for 5 mins 1 cycle
Primer must be minimum of 20 bp long and min 50 GC content if the annealing step is
to be omitted Incubate at 95 DC for 5 mins to denature before running
Spin down Load 3 ttl for short and medium gels 21t1 for long gel runs
143
REFERENCES
144
REFERENCES
Abrahams J P Lutter R Todd R J van Raaij M J Leslie A G W and Walker J E 1993 Inherent asymmetry of the structure of F1-ATPase from bovine heart mitochondria at 65 Aresolution EMBO J 121775-1780
Abrahams J P Leslie A G W Lutter R and Walker 1 E 1994 Structure at 28 A resolution of FeATPase from bovine heart mitochondria Nature 370621-628
Adzumah K and Mizuuchi K 1988 Target immunity of Mu transposition reflects a differential distribution of Mu B protein Cell 53257-266
Akey C W Crepeau R H Dunn S D McCarty R E and Edelstein S J 1983 Electron microscopy of single molecules and crystals of F1-ATPases EMBO J 21409-1415
Allet B 1979 Mu insertion duplicates a 5 bp sequence at the host inserted site Cell 16 123shy129
Amzel L M and Pedersen P L 1983 Proton ATP-ases structure and mechanism Ann Rev Biochem 52801-824
Andersson L Mac Neela J and Wolfenden R 1985 Use of secondary isotope effects and varying pH to investigate mode of binding of inhibitory amino aldehydes by leucine aminopeptidase Biochem 24330-333
Andrews G F Dugan R P and Stevens C J 1992 Combining physical and bacterial treatment for removing pyritic sulfur from coal In Processing and Utilization of High-sulfur coals IV Dugan P R D Quigley and Y Attia (eds) p 515 Elsevier New York
Andrulis I L Chen J and Ray P N 1987 Isolation of human cDNAs for asparagine synthetase and expression in Jansen rat sarcoama cells Mol Cell BioI 72435-2443
Andruszkiewicz R Milewsky S Zieniawa T and Borowski E 1990 Anticandidal properties of N3 -(4-methoxy-fumaroyl)-L-2 3-diamino propanoic acid oligopeptides 1 Med Chern 33132-135
Arciszewska L K Drake D and Craig NL 1989 Transposon Tn7 cis-acting sequences in transposition and transposition immunity J Mol BioI 20735-42
Bachmann B J 1983 Linkage map of Escherichia coli K-12 edition 7 Microbiol Rev 47180-230
145
B Plumbridge 1 A Cochet D Souza J M M M and Calcagno M 1988 Cordinated regulation of amino J
Bacteriol 1754951-4956
0 B Vermoote P V and Le Goffic F 1993 synthetase from Ecoli lUllL- mechanism and inhibition by N3-fumaroyl-2 3-diaminopropionic derivatives Biochem 27 2282-2287
Vermoote P Haumont PY syrlthl~ta~e from Escherichia coli purification site location Biochemistry 26 1940-1948
Badet B Inagaki K Soda K Walsh dependent inhibition of Bacillus stearothermophilus alanine racemase by phosphonate isomers by isomerization to non covalent enzyme-(1-aminoethyl) complexes Biochem 253275-3282
Badet-Denisot M A and Badet of glucoseamine-6shyphosphate synthetase by diethylpyrocarbonates histidine requirement for enzymatic activity Arch Biochem Biophys
Bainton R Gamas P and transposition in vitro proceeds through an excised transposon intermediate (JpnIPtlltpi1 111 breaks in DNA Cell 65805-816
Barry G 1986 Permanent insertion of v~u into the chromosomes of soil bacteria BioTechnology 4446-449
Barth P T Datta N Grinter N S 1976 Transposition a deoxyribonucleic acid sequence trimethoprim and streptomycin resistances from R483 to other replicons 1 Bacteriol 125800-810
Bates C J Adams W and R R 1968 Control of the formation of uri dine diphospho-N-acetyl and glycoprotein synthesis in rat liver J Chern 2411705-17
Benjamin N 1989 Intramolecular transposition of TnlD Cell 59373shy383
Bennet R L and Malamy M resistant mutants of Escgerichia coli and phosphate Commun 40496-503
Benneth1 1978 Bacterial leaching patterns on pyrite crystal J Bacteriol
146
Berg D E Davies 1 Allet B and Rochaix 1 D 1975 Transposition of R factor genes to bacteriophage lambda Proc Natl Acad Sci USA 723268-3632
Berg D E and Drummond M1978 Absence of DNA sequences homologous to transposable element Tn5 (Kan) in the chromosome of Ecoli K-12 J Bcateriol 136419-422
Birkenhager R Hoppert M Deckers-Hebestriet G Mayer F and Altendorf K 1995 The Fo complex of the Ecoli ATP synthase investigation by electron imaging and immunoelectron microscopy Eur J Biochem 23058-67
Bjqgtrbaek C Foersom V and Michelsen O 1990 The transmembrane topology of the a subunit from ATPase in Escherichia coli analysed by PhoA protein fusions FEBS lett 26031-34
Boekemia E J Berden JA and van Heel MG 1986 sructure of mitochondrial F1-ATPase studied by electron microscopy and image processing Biochim Biophys Acta 851353-360
Bolton E Glynn P and OGara F 1984 Site specific transposition of Tn7 into a Rhizobiwn meliloti megaplasmid Mol Gen Genet 193153-157
Boos W 1974 Bacterial transport Annu Rev Biochem 43123-146
Boursaux-Eude C Girons I S and Zuerner R 1995 IS1500 an IS3-like element from Leptospira interrogans Microbiol 1412165-2173
Boyer P D 1993 The binding change mechanism for ATP synthase-some probabilities and possibilities Biochim Biophys Acta 1140215-250
Brachet P Eisen H and Rambach A 1970 Mutations in coliphage lambda affecting the expression of replicative functions 0 and P Mol Gen Genet 108266-276
Brink J Boekemia E 1 and van Bruggen E F 1987 The structure of NADH ubiquitone oxidoreductase fron beef heart mitochondria Crystals containing an octameric arrangement of iron-sulphur protein fragments Eur J Biochem 166287-294
Brock T D and Gustafson J 1976 Ferric iron reduction by sulphur- and iron-oxidizing bacteria Appl Environ Microbiol 32 567-571
Brown L D Dennehy M E and Rawlings D E 1994 The FI genes of the FIFo ATP synthase from the acidophilic bacterium Thiobacillus jerrooxidans complement Escherichia coli FI unc mutants FEMS Microbiol Lett 12219-25
Buchanan 1 1 Adv The Amidotransferases Enzymol 3991-183
Buckley Science
Caruso M Pseudomonas nOVHrn
oxidation of pyrite Applied
1 1982 Interactions Mol Gen Genet
phage 1
Chmara H by Biophysica
synthetase from bacteria antibiotic tetaine Biochimica et
Microbiol Lett 11197-206 controls on the oxidation of refractory Barberton
genetic elements and evolution Nature 263731shyCohen S N 1976 738
Corbet C M and 1 Ingledew 1987 Is oxidation by Thiobacillus ferrooxidans
Couillard D and ~~b 118(5)808-81
Metallurgical residue V UlllLltHIH of metals from sewage sludge J
Cox G B a reassessment of the
F and Hatch L 1986 The mechanism of ATP synthase of the b and a subunits Biochim Acta 84962-69
Craig N L 1995 Unity in Science 270253-254
Craig N and Gamas P 1 Purification and characterization of a transposition protein that binds ATP DNA Nuc Acids Res
1 2873-97 C A 1990 in response to environmental stress Advances in
alUIUH~ on the activity subunit b-specific polyc1onal
G Simoni and Altendorf K 1992 Influence vlJnu~ of the ATP synthase of t-S(nel~lCn
1 BioI Chern 26712364shy
M A Le Goffic F and 1991 Glucosamine- 6P two proteins on limited proteolysis ltVU Biophys 288225-230
148
Dobrogosz W J 1968 _l in Escherichia coli and its to catabolite repression J
Doublet P 1 van Heijenoort and 1993 The murI gene of is an essential gene that encodes klUltUllU~v racemase activity J Bacteriol 1752970-2979
P J van Heijenoort 1992 Identification of the Ecoli which is required for the D-glutamic acid a specific component peptidoglycan 1 Bacteriol
Garren A Garen and Torri ani 1961 Genetic control of repression UCUlllV phosphatase in Ecoli 1 Mol BioI
D J and Boeke 1 D 1990 A 1-1-0- structure is required for Ty1 transposition Genes Develop 4324-330
1982 Transposition of Tn7 occurs at a on Caulobacter crescentus 10SIClmle 1 Bacteriol 151 1056-1 058
H 1990 Molecular IHovUUU)11 -transporting 345-391 In T 12 Bacterial
Press Ltd London
transport and coupling by the synthase insights mechanism of function J Bioenerg 24485-491
1992b Subunit c of FIFo ATP role m transduction Biochim Biophys Acta 1101 v--rJ
Eliopoulos E E Jackson P 1 Keen IN HUIUVI L Thompson P and 1 Structure of a 16 kDa integral AU-UUl that identity to
of the vacuolar H+ -ATPase Protein 15
Fling M 1 C 1985 Nucleotide sequence of encoding the amino glycoside-modifying enzyme 3(9)-O-nucleotidyltransferase Res 137095-7106
Fling M and C l-1UUJIU nucleotide sequence of dihydrofolate rhnrpri by Tn7 Nucleic Acids Res 115
Flores Llchtenstem C P 1990 DNA sequence anlysis of tnsA for the Tn7 transposition Nucleic Acids 18901 911
149
149
Foster D L and Fillingame R H 1982 Stoichiometry of subunits in the H+-ATPase complex of Escherichia coli J BioI Chern 2572009-2015
Fraga D Hermolin J Oldenburg M Miller MJ and Fillingame R H 1994 Arginine 41 of subunit c of Escherichia coli H+-A TP synthase is essential in binding and coupling of F to Fo J BioI Chern 2697532-7537
Friedl P Hoppe J Gunsalus R P Michelsen 0 von Meyenburg K and Schairer H U 1983 Membrane integration and function of the three Fo subunits of the A TP-synthase of Escherichia coli K12 EMBO 1 299-103
Frisa P S and Sonneborn D R 1982 Developmentally regulated interconversion between end product inhibitable and non-inhibitable forms of a first pathway-specific enzyme activity can be mimicked in vitro by dephosphorylation reactions Proc Natl Acad Sci USA 796289-6293
Fujiwara T and Mizuuchi K 1988 Retroviral DNA integration Structure of an integration intermediate Cell 54497-504
Galas D J and Chandler M 1989 Bacterial insertion sequences In Mobile DNA Berg D E and Howe M M (eds) Washington D C American Society for Microbiology pp 109shy162
Gay N J Tybulewicz V L1 Walker JE 1986 Insertion of transposon Tn7 into the Ecoli gmS transcriptional terminator Biochem J 234 111-117
Gentry D R and Cashel M 1995 Cellular localization of the Escherichia coli SpoT protein J Bacteriol 177 3890-3893
Gentry D R Xiao H Burgess R R and Cashel M 1991 The omega subunit of Ecoli K-12 RNA polymerase is not required for stringent RNA control in vivo J Bacteriol 173 3901-3903
Girvin M E and Fillingame R H 1993 Helical structure and folding of subunit c of FIFo ATP synthase IH NMR resonace assignments and NOE analysis Biochemistry 3212167shy12177
Gogol E P Lucken U Bork T and Capaldi R A 1989 Molecular architecture of Escherichia coli F Adenosinetriphosphatase Biochemistry 284709-4716
Gogol E P Aggeler R Sagermann M and Capaldi R A 1989 Cryoelectronic Microscopy of Escherichia coli FI Adenosinetriphosphatase Decorated with Monoclonal Antibodies to Individual Subunits of the complex Biochemistry 284717-4724
150
Heinikoff S 1984
Golinelli-Pimpaneaux B and Badet involvement of Lys603 from Ecoli glucosamoine-6-phosphate synthase substrate fructose-6-phosphate 1 Biochem 201175-182
Gottesman M M and Rosner 1 L of a detenninant of uvu_bullu
resistance by coliphage lambda Sci USA 725041-5045
Grunstein M and Hogness D
Colony hybridization a method for isolation cloned DNAs that contain a 1Jbullu Proc NatL Acad Sci USA 723961-3965
Hauer B and Shapiro 1 A 1984 Control of Tn7 transposition Mol Gen Genet 194
Hedges R W and Jacob Transposition of ampicillin resistance from to other replicons MoL 1-40
Hennolin 1 Gallant 1 psi subunit in the Fo sector of the H+ -ATPase of Ecoli J Bioi
R H 1983 Topology organization and function
Hennolin J and R 1989 Assembly of Fo sector of synthase Interdepenndence of subunit insertion into the membrane 1 2817
van Montagu M Holsters Zambryski P Beuckeleer M Willmitzer and Schell 1 M 1980 interaction of
DNA plant cells Proc Soc Lond 210351-365
P 1972 Insertion mutations control region of Physical characterization of the mutants Mol Gen Genet
115266-276
Holmes applications pI
UI Haq 1990 Adaptation of Thiobacillus vu)nuu for industrial In J Salley R G McCready Wichlacz (ed)
1989 CANMET Ottawa Canada
Holtje J V and U Schwarz 1985 Biosynthesis and growth murein sacculus p 77shy119 InN (ed) Molecular cytology of Escherichia Academic Press Inc New
Acad Sci USA
Heffron E Reubens C and which mediates ampicillin
Translocation of a plasmid DNA sequence nature and specificity of Natl
digestion with exonuclease III creates fl tr breakpoints 1-369
Hernalsteens J M Agrobacterium
Hirsch H the galactose nnnn fJu
151
Holzenburg A Jones P c Franklin T Padi J B and Finbow M E 1993 Evidence for a common structure 1 Biochem 21321-30
Hoppe 1 and Sebald W 1986 Topological pathway of the protons through Fo is provided by amino acid the lipid phase Biochimie (Paris) 68427-434
S Otsubo H Davidson N and 1975 Electron microscope heteroduplex of sequence relations among plasmids identification and mapping of the
insertion sequences IS] and IS2 in F and R plasmids J Bacteriol 22762-775
Inoue c Sugawara K and 1991 regulatory gene in Thiobacillus ferrooxidans is spaced apart from Mol Microbiol 52707-2718
Ish-Horowicz D and Burke and cosmid cloning Nucleic Acids Res 92989-2998
Johnston B Clennell M and D 1995 Structure and function of Tn5467 a Tn2l-like transposon located on T jerrooxidans broad host range plasmid Appl Envir Micro
Kahmann R and Kamp Nucleotide sequences of the attachment bacteriophage Mu DNA Nature 280247-250
Kahn K and Schaefer M R Characterization of trans po son 5469 from cyanobacterium Fremyella diplosiphon J 1777026-7032
Karavaiko G Golovacheva S Pivovarova T A Tzaplina I A and Vartanjan 1988 Thermophilic ltPM Sulfobacillus page 29-41 In Biohydrometallurgyshy87 Norris P and Science and Technology Letters Kew 1
Kennedy J and Humpphreys J D 1976 Microbial cells living immobilised on Nature 261242-244
Koonin 1993 SpoU protein of Escherichia coli belongs to a new family of Nucleic Acids Res 19
Kopecko J and site specific recA mdependtm recombination between bacterial Pt of palindromes at the N ad Acad Sci USA
on L-glutamine D-fructose ud()transtiera~e 1 BioI
152
Krumholz L R Esser U and Simoni RD 1989 Nucleotide sequence of the unc operon of Vibrio alginolyticus Nucleic Acids Res 177993-7994
Kubo K and Craig N 1990 Bacterial transposon Tn7 utilizes two classes of target sites 1 Bacteriol 1722774-2778
Kucharczyk N Denisot M A Le Goffic F and Badet B 1990 Glucosarnine-6-phosphate synthase from Ecoli detennination of the mechanism of inactivation by N3 fumaroyl-L-2-3 diarninoproprionic derivatives Biochemistry 293668-3676
Kusano M Takeshima T Inoue C and Sugawara K 1991 Evidence for two sets of structural genes coding for Ribulose biphosphate carboxylase in Thiobacillus ferrooxidans 1 Bacteriol 1737313-7323
Lane Dl A P Harrison lnr D Stahl B Pace SJ Giovannoni GJ Olsen and N R Pace 1992 Evolutionary relationship among sulphur- and iron-oxidizing eubacteria 1 Bacteriol 174267-278
Lee C H Bhagwhat A and Heffron F 1983 Identification of a transposon TnJ sequence required for transposition immunity Natl Acad Sci USA 806765-6769
Lewis M 1 Chang 1 A and Somoni R D 1990 A topological analysis of subunit a from Escherichia coli F1Fo-ATP synthase predicts eight transmembrane segments 1 BioI Chern 265 10541-10550
Lichtenstein C P and Brenner S 1982 Unique insertion site of Tn7 in the Ecoli chromosome Nature (London) 297601-603
Lichtenstein C P and Brenner S 1981 Site-specific properties of transposition to the Ecoli chromosome Mol Gen Genet 183380-387
Liu X Petersson S and Sandstrom A Mesophilic versus moderate thennophilic bioleaching Biohydrometallurgy Technologies Vol 1 A E Tonna 1 E Wey and Lakshmanan V 1 (ed) pp 29-38
Lizarna H M and Sankey B M 1993 Oxidation of H2S by Thiobacillus thiooxidans is inhibited by substrate and methane BiohydrometaHurgy Technologies Vol II A E Tonna 1 E Wey and C L Brierley (ed) pp 339-364
Lundgren D G and Silver M 1980 Ore leaching by bacteria Ann Rev Microbiol 34263shy268
Makino K Amemura M Shinagawa H Kobayashi A and Nakata A 1985 Sequence of the genes involved in phosphate transport and regulation of the phosphate regulon in Escherichia coli 1 Mol BioI 184231-240
Makino K Shinagawa Amemura M Kimura S Nakata A and Ishihama 1988 Euuv1J of the phosphate regulon of Ecoli Activation of pstS by the PhoB
protein in vitro 1 Mol BioI 20385-95
Makino K Shinagawa H Amemura M Yamada M and 1990 Signal transduction in the phosphate regulon of the Escherichia coli involves phosphotransfer nprlrJp~middotn PhoR and PhoB proteins J Mol BioI 21055
Malamy 1966 Frameshift mutations in the nnlnn of Escherichia coli Cold Harb Symp Quant BioI 31189-201
Malamy M H 1972 Electron microscopy insertions in the lac operon of Escherichia coli MoL Gen
Malamy M H and Bennett R L 1970 mutants of Ecoli and phosphate transport Biochem Biophys Res Commun
Maniatis T Fritsch EF Sambrook J 1982 nn A laboratory manual Cold Spring Harbor Laboratory Press Cold
McKnight G L S L Mudri SH Mathewest R S Marshall P O Sheppard and P J OHara 1990 Molecular cloning synthesis and bacterial expression of human glutaminefructose-6-phosphate UUAUU u J Chem 26725208-25212
Medveczky N and H Rosenburg 1970 phosphate-binding protein of Escherichia Biochim Biophys Acta 211 158-168
Mei B and Zalkin H 1989 A Cysteine-Histidine-Aspartate catalytic triad is involved in Glutamide Amide Transfer Function purF-type Glutamine amodotransferases 1 BioI Chem 264 16613-16619
Mengin-Lecreulx D and J van 1993 Identification of glmU gene encoding Nshyacetylglucosamine in Escherichia coli J Bacteriol 1756150shy6157
Michaelis M J and Criddle M J 1970 Mitochondrial DNA and mutants in Saccharomyces cerevisiae Biochem Genet 5487-495
Michaelis Starlinger P 1969 Two insertions in the jO v
operon having different homologous DNA sequences MoL Gen Genet 10437 377
Miller J H Calos Transposable elements Cell 20579-595
154
Miller J Oldenburg M and Fillingame R 1990 The essential carboxyl group of in subunit c the FIFo ATP synthase can be and H( +)-translocating function retained Proc NatL Acad 874900-4904
Mitcell P 1966 Chemiosmotic coupling in - and phosphorylation BioL Rev 41455-502 (1966)
Mizuuchi K 1984 Mechanism of transposition of bacteriophage Mu Polarity of the strand reaction at the initiation of the transposon Cell 39395-404
C Ph de Donato Berthelin J Surface oxidized species a key factor in the study of bioleaching processes Biohydrometallurgical In A J
Weyand V I Lakshmanan ed 1993 Vol 1 175-184
A Ishino Shinagawa H Makino K and M 1987 Nucleotide of the iap gene responsible for alkaline phosphatase isoenzyme conversion in Ecoli
identification of product 1 1695429-5433
K 1989 The tsr gene-coding prevents thiopeptin from inhibiting ppGpp synthesis in Streptomyces lividans FEMS Microbiol Lett
Ogasawara N and Yoshikawa H 1992 and their organIzatIon in the repnc()n of the bacterial chromosome Mol Microbiol 6(5)629-634
Ohtsubo Davidson N and 1975 microscope heteroduplex studies of sequence relations among plasmids identification and mapping of the insertion sequences 1 and IS2 in F and R plasmids 1 122746-775
Olson G J 1994 Microbial oxidation gold ores and gold bioleaching FEMS un Lett 119 1-6
Orle K A N L 1991 Identification of transposition prroteins by the bacterial transposon [corrected republished originally printed in Gene 1990 Nov 30 96(1) Gene 10425-31
Ouellette M Roy P Homology of ORFs from and from to site specific recombinases 1987 Nucl Acids 10055-10059
Murein synthesis p663-671ln Neidhardt J L Ingraham B Louw M~ M Schaechter H E Umbarger (ed) Escherichia coli and Salmonella
ryphimurium cellular and bioI voL 1 American Society Microbiology Washington
Perlin D S and Senior A Functional and cross-reactivity of antibody to purified subunit b (uncF protein) of Escherichia coli proton-ATPase Arch Biochem Biophys 236603-611
155
Plumbridge J A O Cochet Souza J M Altramirano M M Calcagno M L and Badet B 1993 Coordinated regulation of amino sugar-synthesizing and -degrading enzymes in Escherichia coli K-12 J Bacteriol 175(16)4951-4956
Pretorius I M Rawlings D E and Woods D R 1986 Identification and cloning of Thiobacillus jerrooxidans structural Nif genes in Escherichia coli Gene 4559-65
Qadri M I Flores C c Davis J and Lichtenstein C 1989 Genetic analysis of attTn7 the transposon Tn7 attachment site in Escherichia coli using a novel M13-based transduction assay 1 Mol BioI 20785-98
Radstrom P Skold 0 Swedberg J Roy P H and Sundstrom L 1994 Tn5090 of plasmid R751 which carries integron is related to Tn7 Mu and retroelements J Bacteriol 1763257-3268
Raetz C R H 1987 Structure and biosynthesis of lipid A in Escherichia coli pp 498-503 In F C Niedhardt J L Ingraham K B Low B Magasanik M Schaechter and H E Umbarger (ed) Escherichia coli and Salmonella typhimurium cellular and molecular biology vol 1 American Society for Microbiology Washington DC
Rao N N and Torriani A 1990 Molecular aspects of phosphate transport in Escherichia coli Mol Microbiol 4(7) 1083-1090
Rawlings DE D R Woods and NP Mjoli 1991 The cloning and structre of genes from the autotrophic biomining bacterium Thiobacillusjerrooxidans p 215-237 In PJ Greenaway (ed) Advances in gene technology vol 2 JAI Press London
Richet E and O Raibaud 1989 MalT the regulatory protein of the Escherichia coli maltose system is an A TP-dependent transcriptional activator EMBO 1 8981-987
Rogers M Ekaterrinaki N Nimmo E and Sherratt D 1986 Analysis of Tn7 transposition Mol Gen Genet 205550-556
Ronson C W Nixon B T Albright L M anf Ausubel F M 1987 Rhizobium meliloti ntrA (rpoN) gene is required for diverse metabolic functions J Bacteriol 1692424-2431
Rosenburg H Gerdes R G and Chegwidden K 1977 Two systems for the uptake of phosphate in Ecoli JBacterioL 131505-511
Rosenburg H 1987 In ion transport in Prokaryotes Rosen B P and Silver S (eds) New York Academic Press pp 205-248
Ross D G Swan 1 and N Klecker 1979 Physical structures of TnlO-promoted deletions and inversions role of 1400 base pairs inverted repetitions Cell 16721-731
156
Saedler H and P 19670 mutation in the galactose operon in Ecoli II Physiological characterization MoL Gen Genet 100 190-202
Saedler P 1968 00 and strong polar mutations in the Genet 102353-363
Sambrook J Fritsch Maniatis 1989 Molecular cloning a laboratory manual Cold Harbor Laboratory Cold Spring Harbor New York
Sand W Gehrke T Hallmann Rohde K Sobotke B and Wintzien S 1993 Bioleaching metal sulphides The importance of Leptospirillum ferrooxidans Biohydromemetallurgical Technologies Voll pp 15-25
Sanger Nicklen and Coulson A DNA sequencing with chain-tennination inhibitors Natl Acad USA 745463-5467
Schneider E and Altendorf K All the three subunits are required for an active proton channel (Fo) of Escherichia coli synthase (FIFo) ~-
518
Schneider E and Allendorf K 1987 Bacterial adenosine 5-triphosphate synthase purification and reconstitution complexes and biochemical and functional characterization of subunits Microbio Rev 51477-497
Schrader 1 A and D S Holmes 1988 Phenotypic switching Thiobacillus ferrooxidans J BacterioL 1703915-3023
Scordilis G E H and Lassie 1987 Identification of transposable elements which activates gene expression in Pseudomonas cepacia J Bacteriol 1698-13
Sekine Eisaki N and Ohtsubo E 1996 Identification and characterization of the lS3 molecules generated by breaks 1 BioL Chern 271197-202
Senior A E 1990 The proton-trans locating of Escherichia coli Annu Rev Biophys Chern 194-41
Shapiro 1 and Adhya S 1969 The jLU operon of K-12 n A deletion analysis of the structure polarity 62249-264
J A 1969 Mutations caused by insertion genenc material the galactose operon Escherichia 1 MoL BioI 4093-105
Silvennan M P 1967 Mechanism of bacterial pyrite oxidation 1 Bacteriol 941046-1051
157
C C ElIson and Levinson 1983 Identification of the typel trimethoprim resistance reductase specified by the R-plasmid R43 comparison with procaryotic and eucaryotic dihydrofolate reductase 1 Bacteriol 155 100 1-1008
Smith and Jones P transposition a multigene process Identification of a regulatory product 147915-7927
Sprague Jr Bell R M Cronan 1 A mutant of Ecoli auxotrophic for organic phosphates evidence two defects in phosphate Mol Gen Genet 14371-77
Starlinger and Michaelis 1968 Suppression the sequential aO[)ealame of the galactose enzymes in a transferase amber mutant coli Mol Genet 102367-369
Starlinger 1977 IS elements in microorganisms MicrobioL Immunol
Steffens K Schneider E Herkernhoff B Schmid Altendorf K portion of Escherichia coli A TP synthase resolution of from subunit b J BioI Chern 2625866-5869
Steffens A Deckers-hebestreit G and Altendorf K 1987b The and functional relationship of A TP synthases (FoFI) from Ecoii and the thermophilic bacterium PS3 J Biol 2626334-6338
Strominger J and M S Smith 1959 Uridine diphosphoacetylglucosamine pyrophosphorylase 1 BioI Chern 2341822-1827
Sundstrom Skold 1990 dhfrI trimethoprim resistance gene of can be found at in other genetic surroundings Antimicrob Agents Chemother 34642shy650
Sundstrom L Roy P H and Skold Site-specific of three cassettes in Tn7 J Bacteriol 1733025-3028
Surin B P and Downie JA 1988 of the Rhizobium leguminosarum nodLMN involved efficient host-specific nodulation Mol Microbiol 2 173-183
B P Dixon N and Rosenburg 1986 Purification PhoU protein a OClItnl
regulator of the pho regulon of Ecoli K-1 J Bacteriol 168631
B P D A Jans A L Fimmel Shaw GB Cox Rosenburg Structural gene for the phosphate-repressible phosphate binding of Escherichia coli
own promoter nucleotide of the phoS 1 Bacteriol
158
Suzuki I Chang W and Takeuchi 1994 Oxidation of organic compounds by Thiobacilli Symp Series 55060-67
Takeyama M S Y Noumi T Maeda Ishibashi S and Futai M 1988 Beta subunit of Ecoli amino acid replacement within a conserved sequence G-X-X-X-X-GshyK-TS) v~u binding proteins lett 218222-226
Thomson JA M Hendson and R M 1981 Mutagenesis by insertion of drug resistance Tn7 into a vibrio 11 J Bacteriol
Tichy R Grotenhuis J T c Janssen van Houten R Rulkens and Lettinga 1993 Application of the sulphur cycle bioremediation of soils polluted with heavy metals In Int Conf Contaminated 93 ed F Arendt G J Annokee R Bosman and W J van der Brink Kluwer Academic Publishers Dordrecht pp 1461-1462
G and Mayer An electron approach to the quaternary structure of mitochondrial Eur J Biochem 13237-45
J and Tschape streptothricin resistance transposons Tn1825 and Tn1826 and transposon Tn 7 Plasmidnuu
18246-249
Tso J Y Zalkin H van Cleemput M Yanofsky C and JM 1982 Nucleotide of Escherichia coli and deduced amino glutamine
phosphribosylpyrophosphate am idotransferase J BioI Chern
V Mesyanzhinova I V Koslov I A and Orlova 1984 Structure of studied by electron and image processing Lett 167285-289
P Barber C and transposons Tn5 and Tn7 in Xanthomonas campestris pv campestris MoL Gen 157
Ullrich J and van Putten P Identification of the Gonococcal glmU gene UVUUIJ
the enzyme N-acetylglucosamine 1-Phosphate Uridyltransferase involved in the synthesis UDP-GlcNAc J of Bact 1776902-6909
M 1992 Eight bacterial proteins includin]g UDP-N -acetylglucosamine acyltransferase three vother of Escherichia coli of a six-residue n tVI
theme FEMS MicrobioL 97249-254
van der Steen J J D Doddema J and de Uidoging van zware metalen afvalstromen met behulp van thiobacilli (Removal metals from waste streams
using thiobacilli) Ministry of Housing Physical and Enviromental (VROM) Directoraat-Generaal Milieubeheer Rapport 199210 Hague
Vermoote P 1988 Universite Paris VI Paris Hrllnro
159
Vignais P V Lunard Issartel J P and Dupuis A 1985 Interaction n l1f middotn
oligomycin sensitivity protein (OSCP) beef heart mitochondrial FeATPase 2 Identification of interacting Fl subunits cross-linking Biochem J
Vik S and Dao NN Prediction of transmembrane topology of Fo proteins from Ecoli ATP synthase variational hydrophobic moment analyses Biochim Biophys Acta 1140 199-207
von Meyenburg B B Jcentrgensen J Nielsen and F Hansen 1982 Promoters of the atp operon for the membrane-bound ATP synthase of Ecoli mapped by TnlO insertion mutations MoL Gen Genet 188240-248
von Meyenberg F G Hansen 1980 The origin of replication oriC the Ecoli chromosome near oriC and construction of oriC mutants ICN-UCLA Symp MoLCellBiol 191 159
Waddell S and Craig N 1989 Tn7 transposition recognition of the attTn7 Proc Natl Sci USA 863958-3962
Waddell C S and Craig N L 1988 transposition two transposition pathways directed by five Tn7-encoded genes Develop 21
J E Gay N J Saraste M and A N 1984 DNA sequence the Escherichia coli unc operon Completion of the sequence of a kilobase segment containing
oriC unc and phoS J224799-815
and Collinson 1 1994 the role the stalk in the coupling mechanism of FEBS 34639-43
Walker 1 E Gay N J and Tybulewicz V L 1986 of transposon Tn7 into the Escherichia glmS transcriptional terminator Biochem 1 243 111-1
Wanner Land McSharry 1982 Phosphate-controlled gene in Escherichia coli using Mudl-directed lacZ fusions J Mol BioI 158347-363
Weng M and Zalkin H 1987 Structural role for a region the CTP synthetase glutamide amide transfer domain J Bacteriol 1693023-3028
Wheatcroft R and S and nucleotide sequence of Rhizobiwn meliloti insertion between the transposase encoded by ISRm3 and those encoded by Staphylococcus aureus and Thiobacillus ferrooxidans IST2 J 1732530-2538
160
Whitby M C and Lloyd R 1995 Branch of three-strand recombination intennediates by RecG a possible pathway for securing middotIULl initiated by duplex DNA 1 143303-3310
Wilkens and Capaldi R A Assymmetry and changes in examined by cryoelectronmicroscopy BioI Chern Hoppe-Seyler 37543-51
and Malamy M 1974 The loss of the PhoS periplasmic protein leads to a the specificity a constitutive phosphate transport system in Escherichia coli Biochem Res Commun 60226-233
WiUsky G Rand Malamy M 1980 of two separable inorganic phosphate transport systems in Escherichia coli J BacterioL 144356-365
P J and and C F 1973 enzyme of metabolism and Stadtman R eds) pp 343-363 Academic Press New York
Winterbum P J and Phelps F 197L control of hexosamine biosynthesis by glucosamine synthetase Biochem 1 121711
Wolf-Watz H and Norquist A 1979 Deoxyribonucleic acid and outer membrane protein to outer membrane protein involves a protein J 14043-49
Wolf-Watz H and M 1979 Deoxyribonucleic acid and outer membrane strains for oriC elevated levels deoxyribonucleic acid-binding protein and
11 for specific UU6 of the oriC region to outer membrane J Bacteriol 14050-58
Wolf-Watz H 1984 Affinity of two different chromosome to the outer membrane of Ecoli J Bacteriol 157968-970
Wu C and Te Wu 197 L Isolation and characterization of a glucosamine-requiring mutant of Escherichia 12 defective in glucoamine-6-phosphate synthetase 1 Bacteriol 105455-466
Yamada M Makino Shinagawa Nakata A 1990 Regulation of the phosphate regulon of Escherichia coli properties the pooR mutants and subcellular localization of protein Mol Genet 220366-372
Yates J R and Holmes D S 1987 Two families of repeatcXl DNA sequences Thiobacillus 1 Bacteriol 169 1861-1870
Yates J R R P and D S 1988 an insertion sequence from Thiobacillus errooxidans Proc Natl 857284-7287
161
Youvan D c Elder 1 T Sandlin D E Zsebo K Alder D P Panopoulos N 1 Marrs B L and Hearst 1 E 1982 R-prime site-directed transposon Tn7 mutagenesis of the photosynthetic apparatus in Rhodopseudomonas capsulata 1 Mol BioI 162 17-41
Zalkin H and Mei B 1990 Amino terminal deletions define a glutamide transfer domain in glutamine phosphoribosylpyrophosphate ami do transferase and other purF-type amidotransferases 1 Bacteriol 1723512-3514
Zalkin H and Weng M L 1987 Structural role for a conserved region III the CTP synthetase glutamide amide transfer domain 1 Bacteriol 169 (7)3023-3028
Zhang Y and Fillingame R H 1994 Essential aspartate in subunit c of FIF0 ATP synthase Effect of position 61 substitutions in helix-2 on function of Asp24 in helix-I 1 BioI Chern 2695473-5479
iii
A Ala amp Arg Asn Asp ATCC ATP
bp
C CGSC Cys
DNA dNTP(s) DSM
EDTA
9 G-6 P GFAT GIn Glu Gly
hr His
Ile IPTG
kb kD
I LA LB Leu Lys
M Met min ml NAcG-6-P
ORF (s) oriC
ABBREVIATIONS
adenine L alanine ampicillin L-Arginine L-Asparagine L-aspartic acid American Type Culture Collection adenos triphosphate
base pair(s)
cytosine Coli Genetic Stock Centre L-cysteine
deoxyribonucleic acid deoxyribonucleotide triphosphates Deutsche Sammlungvon Mikroorganismen (The German Collection) ethylenediamine tetraacetic acid
pSIS1 construct was further subcloned to plasm ids IS30 IS40 pSlS50
pS183S p81841 and p81 (Fig Some of subclones were extensively mapped
although not all sites are shown in 22 exact positions of the ends of
T jerrooxidans plasmids pTfatpl and pTfatp2 on the cosmid p8I81 were identified
21)
Tfeooxidans
that the cosmid was
unrearranged DNA digests of cosmid pSISI and T jerrooxidans chromosomal DNA were
with pSI The of the bands gave a positive hybridization signal were
identical each of the three different restriction enzyme digests of p8I8l and chromosomal
In order to confirm of pSI81 and to
49
kb kb ~
(A)
11g 50Sts5
middot 2S4
17 bull tU (probe)
- tosect 0S1
(e)
kb
+- 10 kb
- 46 kb
28---+ 26-+
17---
Fig 23 Hybrdization of cosmid pSISI and T jerrooxitians (A TCC 33020) chromosomal
DNA by the KpnI-SalI fragment of pSIS52 (probe) (a) Autoradiographic image of the
restriction digests Lane x contains the probe lane 13 and 5 contain pSISI restricted with BamHl HindIII and Bgffi respectively Lanes 2 4 and 6 contain T errooxitians chromosomal DNA also restricted with same enzymes in similar order (b) The sizes of restricted pSISI
and T jerrooxitians chromosome hybridized by the probe The lanes correspond to those in Fig 23a A DNA digested with Pst served as the molecular weight nlUkermiddot
50
DNA (Fig BamHI digests SHklC at 26 and 2S kb 1 2) HindIII
at 10 kb 3 and 4) and BgnI at 46 (lanes 5 and 6) The signals in
the 181 was because the purified KpnI-San probe from p81 had a small quantity
ofcontaminating vector DNA which has regions ofhomology to the cosmid
vector The observation that the band sizes of hybridizing
for both pS181 and the T ferrooxidans ATCC 33020 same
digest that the region from BgnI site at to HindIII site at 16
kb on -VJjUIU 1 represents unrearranged chromosomal DNA from T ferrooxidans
ATCC there were no hybridization signals in to those predicted
the map with the 2 4 and 6 one can that are no
multiple copies of the probe (a Tn7-like segment) in chromosome of
and Lferrooxidans
of plasmid pSI was found to fall entirely within a Tn7-like trallSDOS()n nrpn
on chromosome 33020 4) A Southern hybridizationUUAcpoundUU
was out to determine whether Tn7-like element is on other
ofT ferrooxidans of T and Lferrooxidans results of this
experiment are shown 24 Lanes D E and F 24 represent strains
19377 DSM and Lferrooxidans DSM 2705 digested to
with BgnI enzyme A hybridization was obtained for
the three strains (lane A) ATCC 19859 (lane B) and UUHUltn
51
(A) AAB CD E F kb
140
115 shy
284 -244 = 199 -
_ I
116 -109 -
08
os -
(8)) A B c o E F
46 kb -----
Fig 24 (a) Autoradiographic image of chromosomal DNA of T ferrooxidans strains ATCC 33020 19859 23270 (lanes A B C) Tthiooxidans strains ATCC 19377 and DSM 504 (D and E) and Lferrooxidans strain DSM 2705 (lane F) all restricted with Bgffi A Pst molecular weight marker was used for sizing (b) Hybridization of the 17 kb KpnI-San piece of p81852 (probe) to the chromosomal DNA of the organisms mentioned 1 Fig 24a The lanes in Fig 24a correspond to those in Fig 24b
(lane C) uuvu (Fig 24) The same size BgllI fragments (46 kb) were
lanes A Band C This result
different countries and grown on two different
they Tn7-like transposon in apparently the same location
no hybridization signal was obtained for T thiooxidans
1 504 or Lferrooxidans DSM 2705 (lanes D E and F of
T thiooxidans have been found to be very closely related based on 1
rRNA et 1992) Since all Tferrooxidans and no Tthiooxidans
~~AA~~ have it implies either T ferrooxidans and
diverged before Tn7-like transposon or Tthiooxidans does not have
an attTn7 attachment the Tn7-like element Though strains
T ferrooxidans were 10catH)uS as apart as the USA and Japan
it is difficult to estimate acquired the Tn7-like transposon as
bacteria get around the world are horizontally transmitted It
remains to be is a general property
of all Lferrooxidans T ferrooxidans strains
harbour this Tn7-like element their
CHAPTER 3
5431 Summary
54Introduction
56Materials and methods
56L Bacterial and plasmid vectors
Media and solutions
56333 DNA
57334 gel electrophoresis
Competent preparation
57336 Transformation of DNA into
58337 Recombinant DNA techniques
58ExonucleaseIII shortening
339 DNA sequencing
633310 Complementation studies
64Results discussion
1 DNA analysis
64Analysis of ORF-l
72343 Glucosamine gene
77344 Complementation of by T ferrooxidans glmS
57cosmid pSIS1 pSlS20
58Plasmidmap of p81816r
Fig Plasmidmap of lS16f
60Fig 34 Restriction digest p81S1Sr and pSIS16f with
63DNA kb BamHI-BamHI
Fig Codon and bias plot of the DNA sequence of pSlS16
Fig 37 Alignment of C-terminal sequences of and Bsubtilis
38 Alignment of amino sequences organisms with high
homology to that of T ferrooxidans 71
Fig Phylogenetic relationship organisms high glmS
sequence homology to Tferrooxidans
31 Summary
A 35 kb BamHI-BamHI p81820 was cloned into pUCBM20 and pUCBM21 to
produce constructs p818 and p81816r respectively This was completely
sequenced both rrelct1()ns and shown to cover the entire gmS (184 kb) Fig 31
and 34 The derived sequence of the T jerrooxidans synthetase was
compared to similar nrvnC ~ other organisms and found to have high sequence
homology was to the glucosamine the best studied
eubacterium EcoU Both constructs p81816f p81 the entire
glmS gene of T jerrooxidans also complemented an Ecoli glmS mutant for growth on medium
lacking N-acetyl glucosamine
32 =-====
Cell wall is vital to every microorganism In most procaryotes this process starts
with amino which are made from rructClsemiddotmiddotn-[)ll()S by the transfer of amide group
to a hexose sugar to form an This reaction is by
glucosamine synthetase the product of the gmS part of the catalysis
to the 40-residue N-terminal glutamine-binding domain (Denisot et al 1991)
participation of Cys 1 to generate a glutamyl thiol ester and nascent ammonia (Buchanan
368-residue C-terminal domain is responsible for the second part of the ClUUU
glucosamine 6-phosphate This been shown to require the ltgtttrltgtt1iltn
of Clpro-R hydrogen of a putative rrulctoseam 6-phosphate to fonn a cis-enolamine
intennediate which upon reprotonation to the gives rise to the product (Golinelli-
Pimpaneau et al 1989)
bacterial enzyme mn two can be separated by limited chymotryptic
proteolysis (Denisot et 199 binding domain encompassing residues 1 to
240 has the same capacity to hydrolyse (and the corresponding p-nitroanilide
derivative) into glutamate as amino acid porI11
binding domain is highly cOllserveu among members of the F-type
ases Enzymes in this include amidophosphophoribosyl et al
1982) asparagine synthetase (Andrulis et al 1987) glucosamine-6-P synmellase (Walker et
aI 1984) and the NodM protein of Rhizobium leguminosarum (Surin and 1988) The
368-residue carboxyl-tenninal domain retains the ability to bind fructose-6-phosphate
The complete DNA sequence of T ferrooxidans glmS a third of the
glmU gene (preceding glmS) sequences downstream of glmS are reported in this
chapter This contains a comparison of the VVA r glmS gene
and Mycobacterium (422 ) An interesting observation was that the T ferrooxidans
glmS gene had comparatively high homology sequences to the Rhizobium leguminosarum and
Rhizobium meliloti M the amino to was 44 and 436
respectively A consensus Dalgarno upstream of the start codon
(ATG) is in 35 No Ecoli a70-type n r~ consensus sequence was
detected in the bp preceding the start codon on intergenic distance
between the two and the absence of any promoter consensus sequence Plumbridge et
al (1993) that the Ecoli glmU and glmS were co-transcribed In the case
the glmU-glmS --n the T errooxidans the to be similar The sequences
homology to six other amidotransferases (Fig 38) which are
(named after the purF-encoded l phosphoribosylpyrophosphate
amidotransferase) and Weng (1987)
The glutamine amide transfer domain of approximately 194 amino acid residues is at the
of protein chain Zalkin and Mei (1989) using site-directed to
the 9 invariant amino acids in the glutamine amide transfer domain of
phosphoribosylpyrophosphated indicated in their a
catalytic triad is involved glutamine amide transfer function of
73
Comparison of the ammo acid sequence alignment of ghtcosamine-6-phosphate
amidotranferases (GFAT) of Rhizobium meliloti (R_m) Rhizobium leguminnosarum (RJ)
Ecoli (E_c) Hinjluenzae Mycobacterium leprae (M_I) Bsubtilis (B_s) and
Scerevisiae (S_c) to that of Tferrooxidans Amino acids are identified by their single
codes The asterisks () represent homologous amino acids of Tferrooxidans glmS to
at least two of the other Consensus ammo acids to all eight organisms are
highlighted (bold and underlined)
1 50 R m i bullbullbullbullbullbull hkps riag s tf bullbullbullbull R~l i bullbullbullbullbullbull hqps rvaep trod bullbullbullbull a
a bullbullbullbullbullbull aa 1r 1 d bullbullbull a a bullbullbullbullbullbull aa inh g bullbullbullbullbullbull sktIv pmilq 1 1g bullbullbull a MCGIVi V D Gli RI IYRGYPSAi AI D 1 gvmar s 1gsaks Ikek a bullbullbullbull qfcny Iversrgi tvq t dgd bullbull ead
51 100 R m hrae 19ktrIk bull bullbull bullbull s t ateR-l tqrae 19rkIk bullbullbull s t atrE-c htIrI qmaqae bull bull h bullbull h gt sv aae in qicI kads stbull bullbull k bullbull i gt T f- Ivsvr aetav bullbullbull r bullbull gq qg c
DL R R G V L AV E L G VGI lTRW E H ntvrrar Isesv1a mv bull sa nIgi rtr gihvfkekr adrvd bullbullbull bullbull nve aka g sy1stfiykqi saketk qnnrdvtv she reqv
101 150 R m ft g skka aevtkqt R 1 ft g ak agaqt E-c v hv hpr krtv aae in sg tf hrI ksrvlq T f- m i h q harah eatt
S E Eamp L A GY l SE TDTEVIAHL ratg eiavv av
qsaIg lqdvk vqv cqrs inkke cky
151 200 bullbull ltk bullbull dgcm hmcve fe a v n bull Iak bullbull dgrrm hmrvk fe st bull bull nweI bullbull kqgtr Iripqr gtvrh 1 s bull bull ewem bullbullbull tds kkvqt gvrh hlvs bull bull hh bullbull at rrvgdr iisg evm
V YR T DLF A A L AV S D PETV AR G M 1 eagvgs lvrrqh tvfana gvrs B s tef rkttIks iInn rvknk 5 c Ihlyntnlqn ~lt klvlees glckschy neitk
74
201 R m ph middot middot middot R 1 ah- middot middot middot middot middot middot E c 1 middot middot middot middot middot middot middot Hae in 1 middot middot middot middot middot T f - c11v middot middotmiddot middot middot middot middot middot M 1 tli middot middot middot middot middot middot middot middot middot B s 11 middot middot middot middot middot S c lvkse kk1kvdfvdv
251 R m middot middot middot middot middot middot middot middot middot middot middot middot middot middot
middot middot middot middot R-1 middot middot middot middot middot middot E c middot middot middot middot middot middot middot middot Hae in middot middot middot middot middot middot middot middot middot middot middot middot T f shy middot middot middot middot middot middot middot middot middot middot middot middot 1M middot middot middot middot middot middot middot middot middot middot middot middot middot middot B s middot middot middot middot middot middot middot middot middot middot middot middot middot middot middot S c feagsqnan1 1piaanefn1
301 R m fndtn wgkt R-1 fneti wgkt E c rf er Hae in srf er T f- rl mlqq
PVTR V YE DGDVA R M 1 ehqaeg qdqavad B s qneyem kmvdd S c khkkl dlhydg
351 R m nhpe v R-1 nhe v E c i nk Hae in k tli T f- lp hr
~ YRHlrM~ ~I EQP AVA M 1 geyl av B-s tpl tdvvrnr S-c pd stf
401 Rm slasa lk R-1 slasca lk E c hql nssr Hae in hq nar-T f f1s hytrq
RVL A SiIS A VG W M 1 ka slakt B s g kqS c lim scatrai
T ferrooxidans (Tf) Scerevisiae (Saee) Mleprae (Myele) and Bsublilis (Baesu)
tr1 ()-ltashyc 8 ~ er (11
-l l
CIgt
~ r (11-CIgt (11
-
$ -0 0shy
a c
omiddot s
S 0 0shyC iii omiddot $
ttl ~ () tI)
I-ltashyt ()
0
~ smiddot (11
81
CHAPTER 4
8341 Summary
8442 Introduction
8643 Materials and methods
431 Bacterial strains and plasmids 86
432 DNA constructs subclones and shortenings 86
864321 Contruct p81852
874322 Construct p81809
874323 Plasmid p8I809 and its shortenings
874324 p8I8II
8844 Results and discussion
441 Analysis of the sequences downstream the glmS gene 88
442 TnsBC homology 96
99443 Homology to the TnsD protein
118444 Analysis of DNA downstream of the region with TnsD homology
LIST OF FIGURES
87Fig 41 Alignment of Tn7-like sequence of T jerrooxidans to Ecoli Tn7
Fig 42 DNA sequences at the proximal end of Tn5468 and Ecoli glmS gene 88
Fig 43 Comparison of the inverted repeats of Tn7 and Tn5468 89
Fig 44 BLAST results of the sequence downtream of T jerrooxidans glmS 90
Fig 45 Alignment of the amino acid sequence of Tn5468 TnsA-like protein 92 to TnsA of Tn7
82
Fig 46 Restriction map of the region of cosmid p8181 with amino acid 95 sequence homology to TnsABC and part of TnsD of Tn7
Fig 47 Restriction map of cosmid p8181Llli with its subclonesmiddot and 96 shortenings
98 Fig 48 BLAST results and DNA sequence of p81852r
100 Fig 49 BLAST results and DNA sequence of p81852f
102 Fig 410 BLAST results and DNA sequence of p81809
Fig 411 Diagrammatic representation of the rearrangement of Tn5468 TnsDshy 106 like protein
107Fig 412 Restriction digests on p818lLlli
108 Fig 413 BLAST results and DNA sequence of p818lOEM
109 Fig 414 BLAST results on joined sequences of p818104 and p818103
111 Fig 415 BLAST results and DNA sequence of p818102
112 Fig 416 BLAST results and nucleotide sequence of p818101
Fig 417 BLAST results and DNA sequence of the combined sequence of 113p818l1f and p81810r
Fig 418 Results of the BLAST search on p81811r and the DNA sequence 117obtained from p81811r
Fig 419 Comparison of the spa operons of Ecali Hinjluenzae and 121 probable structure of T ferraaxidans spa operon
83
CHAPTER FOUR
TN7-LlKE TRANSPOSON OF TFERROOXIDANS
41 Summary
Various constructs and subclones including exonuclease ill shortenings were produced to gain
access to DNA fragments downstream of the T ferrooxidans glmS gene (Chapter 3) and
sequenced Homology searches were performed against sequences in GenBank and EMBL
databases in an attempt to find out how much of a Tn7-like transposon and its antibiotic
markers were present in the T ferrooxidans chromosome (downstream the glmS gene)
Sequences with high homology to the TnsA TnsB TnsC and TnsD proteins of Tn7 were
found covering a region of about 7 kb from the T ferrooxidans glmS gene
Further sequencing (plusmn 45 kb) beyond where TnsD protein homology had been found
revealed no sequences homologous to TnsE protein of Tn7 nor to any of the antibiotic
resistance markers associated with Tn7 However DNA sequences very homologous to Ecoli
ATP-dependentDNA helicase (RecG) protein (EC 361) and guanosine-35-bis (diphosphate)
3-pyrophosphohydrolase (EC 3172) stringent response protein of Ecoli HinJluenzae and
Vibrio sp were found about 15 kb and 4 kb respectively downstream of where homology to
the TnsD protein had been detected
84
42 Transposable insertion sequences in Thiobacillus ferrooxidizns
The presence of two families (family 1 and 2) of repetitive DNA sequences in the genome
of T ferrooxidans has been described previously (Yates and Holmes 1987) One member of
the family was shown to be a 15 kb insertion sequence (IST2) containing open reading
frames (ORFs) (Yates et al 1988) Sequence comparisons have shown that the putative
transposase encoded by IST2 has homology with the proteins encoded by IS256 and ISRm3
present in Staphylococcus aureus and Rhizobium meliloti respectively (Wheatcroft and
Laberge 1991) Restriction enzyme analysis and Southern hybridization of the genome of
T ferrooxidans is consistent with the concept that IST2 can transpose within Tferrooxidans
(Holmes and Haq 1990) Additionally it has been suggested that transposition of family 1
insertion sequences (lST1) might be involved in the phenotypic switching between iron and
sulphur oxidizing modes of growth including the reversible loss of the capacity of
T ferrooxidans to oxidise iron (Schrader and Holmes 1988)
The DNA sequence of IST2 has been determined and exhibits structural features of a typical
insertion sequence such as target-site duplications ORFs and imperfectly matched inverted
repeats (Yates et al 1988) A transposon-like element Tn5467 has been detected in
T ferrooxidans plasmid pTF-FC2 (Rawlings et al 1995) This transposon-like element is
bordered by 38 bp inverted repeat sequences which has sequence identity in 37 of 38 and in
38 of 38 to the tnpA distal and tnpA proximal inverted repeats of Tn21 respectively
Additionally Kusano et al (1991) showed that of the five potential ORFs containing merR
genes in T ferrooxidans strain E-15 ORFs 1 to 3 had significant homology to TnsA from
transposon Tn7
85
Analysis of the sequence at the tenninus of the 35 kb BamHI-BamHI fragment of p81820
revealed an ORF with very high homology to TnsA protein of the transposon Tn7 (Fig 44)
Further studies were carried out to detennine how much of the Tn7 transposition genes were
present in the region further downstream of the T ferroxidans glmS gene Tn7 possesses
trimethoprim streptomycin spectinomycin and streptothricin antibiotic resistance markers
Since T ferrooxidans is not exposed to a hospital environment it was of particular interest to
find out whether similar genes were present in the Tn7-like transposon In the Ecoli
chromosome the Tn7 insertion occurs between the glmS and the pho genes (pstS pstC pstA
pstB and phoU) It was also of interest to detennine whether atp-glm-pst operon order holds
for the T ferrooxidans chromosome It was these questions that motivated the study of this
region of the chromosome
43 OJII
strains and plasm ids used were the same as in Chapter Media and solutions (as
in Chapter 3) can in Appendix Plasmid DNA preparations agarose gel
electrophoresis competent cell preparations transformations and
were all carned out as Chapter 3 Probe preparation Southern blotting hybridization and
detection were as in Chapter 2 The same procedures of nucleotide
DNA sequence described 2 and 3 were
4321 ~lllu~~~
Plasmid p8I852 is a KpnI-SalI subclone p8I820 in the IngtUMnt vector kb) and
was described Chapter 2 where it was used to prepare the probe for Southern hybridization
DNA sequencing was from both (p81852r) and from the Sail sites
(p8I852f)
4322 ==---==
Construct p81809 was made by p8I820 The resulting fragment
(about 42 kb) was then ligated to a Bluescript vector KS+ with the same
enzymes) DNA sequencing was out from end of the construct fA
87
4323 ~mlJtLru1lbJmalpoundsectM~lllgsect
The 40 kb EeoRI-ClaI fragment p8181Llli into Bluescript was called p8181O
Exonuclease III shortening of the fragment was based on the method of Heinikoff (1984) The
vector was protected with and ClaI was as the susceptible for exonuclease III
Another construct p818IOEM was by digesting p81810 and pUCBM20 with MluI and
EeoRI and ligating the approximately 1 kb fragment to the vector
4324 p81811
A 25 ApaI-ClaI digest of p8181Llli was cloned into Bluescript vector KS+ and
sequenced both ends
88
44 Results and discussion
of glmS gene termini (approximately 170
downstream) between
comparison of the nucleotide
coli is shown in Fig 41 This region
site of Tn7 insertion within chromosome of E coli and the imperfect gtpound1
repeat sequences of was marked homology at both the andVI
gene but this homology decreased substantially
beyond the stop codon
amino acid sequence
been associated with duplication at
the insertion at attTn7 by (Lichtenstein and as
CCGGG in and underlined in Figs41
CCGGG and are almost equidistant from their respelt~tle
codons The and the Tn7-like transposon BU- there is
some homology transposons in the regions which includes
repeats
11 inverted
appears to be random thereafter 1) The inverted
repeats of to the inverted repeats of element of
(Fig 43) eight repeats
(four traJrlsposcm was registered with Stanford University
California as
A search on the (GenBank and EMBL) with
of 41 showed high homology to the Tn sA protein 44) Comparison
acid sequence of the TnsA-like protein Tn5468 to the predicted of the
89
Fig 41
Alignment of t DNA sequence of the Tn7-li e
Tferrooxi to E i Tn7 sequence at e of
insertion transcriptional t ion s e of
glmS The ent homology shown low occurs within
the repeats (underl of both the
Tn7-li transposon Tn7 (Fig 43) Homology
becomes before the end of t corresponding
impe s
T V E 10 20 30 40 50 60
Ec Tn 7
Tf7shy(like)
70 80 90 100 110 120
Tf7shy(1
130 140 150 160 170 180 Ec Tn 7 ~~~=-
Tf7shy(like)
90
Fig 42 DNA sequences at the 3 end of the Ecoll glmS and the tnsA proximal of Tn7 to the DNA the 3end of the Tferrooxidans gene and end of Tn7-like (a) DNA sequence determined in the Ecoll strain GD92Tn7 in which Tn been inserted into the glmS transcriptional terminator Walker at ai 1986 Nucleotide 38 onwards are the of the left end of Tn DNA
determined in T strain ATCC 33020 with the of 7-like at the termination site of the glmS
gene Also shown are the 22 bp of the Tn7-like transposon as well as the region where homology the protein begins A good Shine
sequence is shown immediately upstream of what appears to be the TTG initiation codon for the transposon
(a)
_ -35 PLE -10 I tr T~ruR~1 truvmnWCIGACUur ~~GCfuA
100 no 110 110 110
4 M S S bull S I Q I amp I I I I bull I I Q I I 1 I Y I W L ~Tnrcmuamaurmcrmrarr~GGQ1lIGTuaDACf~1lIGcrA
DO 200 amp10 220 DG Zlaquot UO ZIG riO
T Q IT I I 1 I I I I I Y sir r I I T I ILL I D L I ilGIIilJAUlAmrC~GlWllCCCAcarr~GGAWtCmCamp1tlnmcrArClGACTAGNJ
Fig 45 (a) Alignment of the amino acid sequence of Tn5468 (which had homology to TnsA of Tn7 Fig 44) to the amino acid sequence of ORF2 (between the merR genes of Tferrooxidans strain E-1S) ORF2 had been found to have significant homology to Tn sA of Tn7 (Kusano et ai 1991) (b) Alignment of the amino acids translation of Tn5468 (above) to Tn sA of Tn7 (c) Alignment of the amino acid sequence of TnsA of Tn7 to the hypothetical ORF2 protein of Tferrooxidans strain E-1S
(al
Percent Similarity 60000 Percent Identity 44444
Tf Tn5468 1 LARQRYGVDEDRVARFQKEGRGQGRGADYHPWLTIQDVPSQGRSHRLKGI SO 11 1111111 I 1111 1111 I I 111
Fig 49 (a) Results of the BLAST search on the inverted and complemented nucleotide sequence of 1852f (from the Sall site) Results indicate amino acid sequence to the TnsC of Tn7 (protein E is the same as TnsC protein) (b) The nucleotide sequence and open reading frame of p81852f
High Prob producing Segment Pairs Frame Score P(N)
IB255431QQECE7 protein E - Escher +1 176 15e-20 gplX044921lSTN7EUR 1 Tn7 E with +1 176 15e-20 splP058461 ECOLI TRANSPOSON TN7 TRANSPOSITION PR +1 176 64e-20 gplU410111 4 D20248 gene product [Caenorhab +3 59 17e-06
IB255431QQECE7 ical protein E - Escherichia coli transposon Tn7 (fragment)
417 (al BLAST results obtained from combined sequence of (inverted and complemented) and 1810r good sequence to Ecoli and Hinfluenzae DNA RecG (EC 361) was found (b) The combined sequence and open frame which was homologous to RecG
444 Analysis of DNA downstream of region with TnsD homology
The location of the of p81811 ApaI-CLaI construct is shown in Fig 47 The single strand
sequence from the C LaI site was joined to p8181Or and searched using BLAST against the
GenBank and EMBL databases Good homology to Ecoli and Hinjluezae A TP-dependent
DNA helicase recombinase proteins (EC 361) was obtained (Fig 417) The BLAST search
with the sequence from the ApaI end showed high sequence homology to the stringent
response protein guanosine-35-bis (diphosphate) 3-pyrophosphohydrolase (EC 3172) of
Ecoli Hinjluenzae and Scoelicolor (Fig 418) In both Ecoli and Hinjluenzae the two
proteins RecG helicase recombinase and ppGpp stringent response protein constitute part of
the spo operon
445 Spo operon
A brief description will be given on the spo operons of Ecoli and Hinjluenzae which appear
to differ in their arrangements In Ecoli spoT gene encodes guanosine-35-bis
pyrophosphohydrolase (ppGpp) which is synthesized during stringent response to amino acid
starvation It is also known to be responsible for cellular ppGpp degradation (Gentry and
Cashel 1995) The RecG protein is required for normal levels of recombination and DNA
repair RecG protein is a junction specific DNA helicase that acts post-synaptically to drive
branch migration of Holliday junction intermediate made by RecA during the strand
exchange stage of recombination (Whitby and Lloyd 1995)
The spoS (also called rpoZ) encodes the omega subunit of RNA polymerase which is found
associated with core and holoenzyme of RNA polymerase The physiological function of the
omega subunit is unknown Nevertheless it binds stoichiometrically to RNA polymerase
119
Fig 418 BLAST search nucleotide sequence of 1811r I restrict ) inverted and complement high sequence homology to st in s 3 5 1 -bis ( e) 3 I ase (EC 31 7 2) [(ppGpp)shyase] -3-pyropho ase) of Ecoli Hinfluenzae (b) The nucleot and the open frame with n
Add 4 ml 52 mix by inversion at room temp for 5 mins
Add 4 ml 53 Mix by to homogenous suspension
Spin at 15 K 40 mins at 4
remove to fresh tube
Equilibrate column with 2 ml N2
Load supernatant 2 to 4 ml amounts
Wash column 2 X 4 of N3 Elute the DNA the first bed
each) add 07
volumes of isopropanol
Spin at 4 Wash with 70 Ethanol Resuspended pellet in n rv 100 AU of TE and scan
volume of about 8 to 10 drops) To the eluent (two
to
140
SEQUITHERM CYCLE SEQUENCING
Alf-express Cy5 end labelled promer method
only DNA transformed into end- Ecoli
The label is sensitive to light do all with fluorescent lights off
3-5 kb
3-7 kb
7-10 kb
Thaw all reagents from kit at well before use and keep on
1) Label 200 PCR tubes on or little cap flap
(The heated lid removes from the top of the tubes)
2) Add 3 Jtl of termination mixes to labelled tubes
3) On ice with off using 12 ml Eppendorf DNA up to
125 lll with MilliQ water
Add 1 ltl
Add lll of lOX
polymeraseAdd 1 ltl
Mix well Aliquot 38 lll from the eppendorf to Spin down
Push caps on nrnnp1
141
Hybaid thermal Cycler
Program
93 DC for 5 mins 1 cycle
93degC for 30 sees
55 DC for 30 secs
70 DC for 60 secs 30 cycle 93 DC_ 30s 55 DC-30s
70degC for 5 mins 1 cycle
Primer must be min 20 bp long and min 50 GC content if the annealing step is to be
omitted Incubate at 95 DC for 5 mins to denature before running Spin down Load 3 U)
SEQUITHERM CYCLE SEQUENCING
Ordinary method
Use only DNA transformed into end- Ecoli strain
3-5 kb 3J
3-7 kb 44
7-10 kb 6J
Thaw all reagents from kit at RT mix well before use and keep on ice
1) Label 200 PCR tubes on side or little cap flap
(The heated lid removes markings from the top of the tubes)
2) Add 3 AU of termination mixes to labelled tubes
3) On ice using l2 ml Eppendorf tubes make DNA up to 125 41 with MilliQ water
142
Add I Jtl of Primer
Add 25 ttl of lOX sequencing buffer
Add 1 Jtl Sequitherm DNA polymerase
Mix well spin Aliquot 38 ttl from the eppendorf to each termination tube Spin down
Push caps on properly
Hybaid thermal Cycler
Program
93 DC for 5 mins 1 cycle
93 DC for 30 secs
55 DC for 30 secs
70 DC for 60 secs 30 cycles
93 DC_ 30s 55 DC-30s 70 DC for 5 mins 1 cycle
Primer must be minimum of 20 bp long and min 50 GC content if the annealing step is
to be omitted Incubate at 95 DC for 5 mins to denature before running
Spin down Load 3 ttl for short and medium gels 21t1 for long gel runs
143
REFERENCES
144
REFERENCES
Abrahams J P Lutter R Todd R J van Raaij M J Leslie A G W and Walker J E 1993 Inherent asymmetry of the structure of F1-ATPase from bovine heart mitochondria at 65 Aresolution EMBO J 121775-1780
Abrahams J P Leslie A G W Lutter R and Walker 1 E 1994 Structure at 28 A resolution of FeATPase from bovine heart mitochondria Nature 370621-628
Adzumah K and Mizuuchi K 1988 Target immunity of Mu transposition reflects a differential distribution of Mu B protein Cell 53257-266
Akey C W Crepeau R H Dunn S D McCarty R E and Edelstein S J 1983 Electron microscopy of single molecules and crystals of F1-ATPases EMBO J 21409-1415
Allet B 1979 Mu insertion duplicates a 5 bp sequence at the host inserted site Cell 16 123shy129
Amzel L M and Pedersen P L 1983 Proton ATP-ases structure and mechanism Ann Rev Biochem 52801-824
Andersson L Mac Neela J and Wolfenden R 1985 Use of secondary isotope effects and varying pH to investigate mode of binding of inhibitory amino aldehydes by leucine aminopeptidase Biochem 24330-333
Andrews G F Dugan R P and Stevens C J 1992 Combining physical and bacterial treatment for removing pyritic sulfur from coal In Processing and Utilization of High-sulfur coals IV Dugan P R D Quigley and Y Attia (eds) p 515 Elsevier New York
Andrulis I L Chen J and Ray P N 1987 Isolation of human cDNAs for asparagine synthetase and expression in Jansen rat sarcoama cells Mol Cell BioI 72435-2443
Andruszkiewicz R Milewsky S Zieniawa T and Borowski E 1990 Anticandidal properties of N3 -(4-methoxy-fumaroyl)-L-2 3-diamino propanoic acid oligopeptides 1 Med Chern 33132-135
Arciszewska L K Drake D and Craig NL 1989 Transposon Tn7 cis-acting sequences in transposition and transposition immunity J Mol BioI 20735-42
Bachmann B J 1983 Linkage map of Escherichia coli K-12 edition 7 Microbiol Rev 47180-230
145
B Plumbridge 1 A Cochet D Souza J M M M and Calcagno M 1988 Cordinated regulation of amino J
Bacteriol 1754951-4956
0 B Vermoote P V and Le Goffic F 1993 synthetase from Ecoli lUllL- mechanism and inhibition by N3-fumaroyl-2 3-diaminopropionic derivatives Biochem 27 2282-2287
Vermoote P Haumont PY syrlthl~ta~e from Escherichia coli purification site location Biochemistry 26 1940-1948
Badet B Inagaki K Soda K Walsh dependent inhibition of Bacillus stearothermophilus alanine racemase by phosphonate isomers by isomerization to non covalent enzyme-(1-aminoethyl) complexes Biochem 253275-3282
Badet-Denisot M A and Badet of glucoseamine-6shyphosphate synthetase by diethylpyrocarbonates histidine requirement for enzymatic activity Arch Biochem Biophys
Bainton R Gamas P and transposition in vitro proceeds through an excised transposon intermediate (JpnIPtlltpi1 111 breaks in DNA Cell 65805-816
Barry G 1986 Permanent insertion of v~u into the chromosomes of soil bacteria BioTechnology 4446-449
Barth P T Datta N Grinter N S 1976 Transposition a deoxyribonucleic acid sequence trimethoprim and streptomycin resistances from R483 to other replicons 1 Bacteriol 125800-810
Bates C J Adams W and R R 1968 Control of the formation of uri dine diphospho-N-acetyl and glycoprotein synthesis in rat liver J Chern 2411705-17
Benjamin N 1989 Intramolecular transposition of TnlD Cell 59373shy383
Bennet R L and Malamy M resistant mutants of Escgerichia coli and phosphate Commun 40496-503
Benneth1 1978 Bacterial leaching patterns on pyrite crystal J Bacteriol
146
Berg D E Davies 1 Allet B and Rochaix 1 D 1975 Transposition of R factor genes to bacteriophage lambda Proc Natl Acad Sci USA 723268-3632
Berg D E and Drummond M1978 Absence of DNA sequences homologous to transposable element Tn5 (Kan) in the chromosome of Ecoli K-12 J Bcateriol 136419-422
Birkenhager R Hoppert M Deckers-Hebestriet G Mayer F and Altendorf K 1995 The Fo complex of the Ecoli ATP synthase investigation by electron imaging and immunoelectron microscopy Eur J Biochem 23058-67
Bjqgtrbaek C Foersom V and Michelsen O 1990 The transmembrane topology of the a subunit from ATPase in Escherichia coli analysed by PhoA protein fusions FEBS lett 26031-34
Boekemia E J Berden JA and van Heel MG 1986 sructure of mitochondrial F1-ATPase studied by electron microscopy and image processing Biochim Biophys Acta 851353-360
Bolton E Glynn P and OGara F 1984 Site specific transposition of Tn7 into a Rhizobiwn meliloti megaplasmid Mol Gen Genet 193153-157
Boos W 1974 Bacterial transport Annu Rev Biochem 43123-146
Boursaux-Eude C Girons I S and Zuerner R 1995 IS1500 an IS3-like element from Leptospira interrogans Microbiol 1412165-2173
Boyer P D 1993 The binding change mechanism for ATP synthase-some probabilities and possibilities Biochim Biophys Acta 1140215-250
Brachet P Eisen H and Rambach A 1970 Mutations in coliphage lambda affecting the expression of replicative functions 0 and P Mol Gen Genet 108266-276
Brink J Boekemia E 1 and van Bruggen E F 1987 The structure of NADH ubiquitone oxidoreductase fron beef heart mitochondria Crystals containing an octameric arrangement of iron-sulphur protein fragments Eur J Biochem 166287-294
Brock T D and Gustafson J 1976 Ferric iron reduction by sulphur- and iron-oxidizing bacteria Appl Environ Microbiol 32 567-571
Brown L D Dennehy M E and Rawlings D E 1994 The FI genes of the FIFo ATP synthase from the acidophilic bacterium Thiobacillus jerrooxidans complement Escherichia coli FI unc mutants FEMS Microbiol Lett 12219-25
Buchanan 1 1 Adv The Amidotransferases Enzymol 3991-183
Buckley Science
Caruso M Pseudomonas nOVHrn
oxidation of pyrite Applied
1 1982 Interactions Mol Gen Genet
phage 1
Chmara H by Biophysica
synthetase from bacteria antibiotic tetaine Biochimica et
Microbiol Lett 11197-206 controls on the oxidation of refractory Barberton
genetic elements and evolution Nature 263731shyCohen S N 1976 738
Corbet C M and 1 Ingledew 1987 Is oxidation by Thiobacillus ferrooxidans
Couillard D and ~~b 118(5)808-81
Metallurgical residue V UlllLltHIH of metals from sewage sludge J
Cox G B a reassessment of the
F and Hatch L 1986 The mechanism of ATP synthase of the b and a subunits Biochim Acta 84962-69
Craig N L 1995 Unity in Science 270253-254
Craig N and Gamas P 1 Purification and characterization of a transposition protein that binds ATP DNA Nuc Acids Res
1 2873-97 C A 1990 in response to environmental stress Advances in
alUIUH~ on the activity subunit b-specific polyc1onal
G Simoni and Altendorf K 1992 Influence vlJnu~ of the ATP synthase of t-S(nel~lCn
1 BioI Chern 26712364shy
M A Le Goffic F and 1991 Glucosamine- 6P two proteins on limited proteolysis ltVU Biophys 288225-230
148
Dobrogosz W J 1968 _l in Escherichia coli and its to catabolite repression J
Doublet P 1 van Heijenoort and 1993 The murI gene of is an essential gene that encodes klUltUllU~v racemase activity J Bacteriol 1752970-2979
P J van Heijenoort 1992 Identification of the Ecoli which is required for the D-glutamic acid a specific component peptidoglycan 1 Bacteriol
Garren A Garen and Torri ani 1961 Genetic control of repression UCUlllV phosphatase in Ecoli 1 Mol BioI
D J and Boeke 1 D 1990 A 1-1-0- structure is required for Ty1 transposition Genes Develop 4324-330
1982 Transposition of Tn7 occurs at a on Caulobacter crescentus 10SIClmle 1 Bacteriol 151 1056-1 058
H 1990 Molecular IHovUUU)11 -transporting 345-391 In T 12 Bacterial
Press Ltd London
transport and coupling by the synthase insights mechanism of function J Bioenerg 24485-491
1992b Subunit c of FIFo ATP role m transduction Biochim Biophys Acta 1101 v--rJ
Eliopoulos E E Jackson P 1 Keen IN HUIUVI L Thompson P and 1 Structure of a 16 kDa integral AU-UUl that identity to
of the vacuolar H+ -ATPase Protein 15
Fling M 1 C 1985 Nucleotide sequence of encoding the amino glycoside-modifying enzyme 3(9)-O-nucleotidyltransferase Res 137095-7106
Fling M and C l-1UUJIU nucleotide sequence of dihydrofolate rhnrpri by Tn7 Nucleic Acids Res 115
Flores Llchtenstem C P 1990 DNA sequence anlysis of tnsA for the Tn7 transposition Nucleic Acids 18901 911
149
149
Foster D L and Fillingame R H 1982 Stoichiometry of subunits in the H+-ATPase complex of Escherichia coli J BioI Chern 2572009-2015
Fraga D Hermolin J Oldenburg M Miller MJ and Fillingame R H 1994 Arginine 41 of subunit c of Escherichia coli H+-A TP synthase is essential in binding and coupling of F to Fo J BioI Chern 2697532-7537
Friedl P Hoppe J Gunsalus R P Michelsen 0 von Meyenburg K and Schairer H U 1983 Membrane integration and function of the three Fo subunits of the A TP-synthase of Escherichia coli K12 EMBO 1 299-103
Frisa P S and Sonneborn D R 1982 Developmentally regulated interconversion between end product inhibitable and non-inhibitable forms of a first pathway-specific enzyme activity can be mimicked in vitro by dephosphorylation reactions Proc Natl Acad Sci USA 796289-6293
Fujiwara T and Mizuuchi K 1988 Retroviral DNA integration Structure of an integration intermediate Cell 54497-504
Galas D J and Chandler M 1989 Bacterial insertion sequences In Mobile DNA Berg D E and Howe M M (eds) Washington D C American Society for Microbiology pp 109shy162
Gay N J Tybulewicz V L1 Walker JE 1986 Insertion of transposon Tn7 into the Ecoli gmS transcriptional terminator Biochem J 234 111-117
Gentry D R and Cashel M 1995 Cellular localization of the Escherichia coli SpoT protein J Bacteriol 177 3890-3893
Gentry D R Xiao H Burgess R R and Cashel M 1991 The omega subunit of Ecoli K-12 RNA polymerase is not required for stringent RNA control in vivo J Bacteriol 173 3901-3903
Girvin M E and Fillingame R H 1993 Helical structure and folding of subunit c of FIFo ATP synthase IH NMR resonace assignments and NOE analysis Biochemistry 3212167shy12177
Gogol E P Lucken U Bork T and Capaldi R A 1989 Molecular architecture of Escherichia coli F Adenosinetriphosphatase Biochemistry 284709-4716
Gogol E P Aggeler R Sagermann M and Capaldi R A 1989 Cryoelectronic Microscopy of Escherichia coli FI Adenosinetriphosphatase Decorated with Monoclonal Antibodies to Individual Subunits of the complex Biochemistry 284717-4724
150
Heinikoff S 1984
Golinelli-Pimpaneaux B and Badet involvement of Lys603 from Ecoli glucosamoine-6-phosphate synthase substrate fructose-6-phosphate 1 Biochem 201175-182
Gottesman M M and Rosner 1 L of a detenninant of uvu_bullu
resistance by coliphage lambda Sci USA 725041-5045
Grunstein M and Hogness D
Colony hybridization a method for isolation cloned DNAs that contain a 1Jbullu Proc NatL Acad Sci USA 723961-3965
Hauer B and Shapiro 1 A 1984 Control of Tn7 transposition Mol Gen Genet 194
Hedges R W and Jacob Transposition of ampicillin resistance from to other replicons MoL 1-40
Hennolin 1 Gallant 1 psi subunit in the Fo sector of the H+ -ATPase of Ecoli J Bioi
R H 1983 Topology organization and function
Hennolin J and R 1989 Assembly of Fo sector of synthase Interdepenndence of subunit insertion into the membrane 1 2817
van Montagu M Holsters Zambryski P Beuckeleer M Willmitzer and Schell 1 M 1980 interaction of
DNA plant cells Proc Soc Lond 210351-365
P 1972 Insertion mutations control region of Physical characterization of the mutants Mol Gen Genet
115266-276
Holmes applications pI
UI Haq 1990 Adaptation of Thiobacillus vu)nuu for industrial In J Salley R G McCready Wichlacz (ed)
1989 CANMET Ottawa Canada
Holtje J V and U Schwarz 1985 Biosynthesis and growth murein sacculus p 77shy119 InN (ed) Molecular cytology of Escherichia Academic Press Inc New
Acad Sci USA
Heffron E Reubens C and which mediates ampicillin
Translocation of a plasmid DNA sequence nature and specificity of Natl
digestion with exonuclease III creates fl tr breakpoints 1-369
Hernalsteens J M Agrobacterium
Hirsch H the galactose nnnn fJu
151
Holzenburg A Jones P c Franklin T Padi J B and Finbow M E 1993 Evidence for a common structure 1 Biochem 21321-30
Hoppe 1 and Sebald W 1986 Topological pathway of the protons through Fo is provided by amino acid the lipid phase Biochimie (Paris) 68427-434
S Otsubo H Davidson N and 1975 Electron microscope heteroduplex of sequence relations among plasmids identification and mapping of the
insertion sequences IS] and IS2 in F and R plasmids J Bacteriol 22762-775
Inoue c Sugawara K and 1991 regulatory gene in Thiobacillus ferrooxidans is spaced apart from Mol Microbiol 52707-2718
Ish-Horowicz D and Burke and cosmid cloning Nucleic Acids Res 92989-2998
Johnston B Clennell M and D 1995 Structure and function of Tn5467 a Tn2l-like transposon located on T jerrooxidans broad host range plasmid Appl Envir Micro
Kahmann R and Kamp Nucleotide sequences of the attachment bacteriophage Mu DNA Nature 280247-250
Kahn K and Schaefer M R Characterization of trans po son 5469 from cyanobacterium Fremyella diplosiphon J 1777026-7032
Karavaiko G Golovacheva S Pivovarova T A Tzaplina I A and Vartanjan 1988 Thermophilic ltPM Sulfobacillus page 29-41 In Biohydrometallurgyshy87 Norris P and Science and Technology Letters Kew 1
Kennedy J and Humpphreys J D 1976 Microbial cells living immobilised on Nature 261242-244
Koonin 1993 SpoU protein of Escherichia coli belongs to a new family of Nucleic Acids Res 19
Kopecko J and site specific recA mdependtm recombination between bacterial Pt of palindromes at the N ad Acad Sci USA
on L-glutamine D-fructose ud()transtiera~e 1 BioI
152
Krumholz L R Esser U and Simoni RD 1989 Nucleotide sequence of the unc operon of Vibrio alginolyticus Nucleic Acids Res 177993-7994
Kubo K and Craig N 1990 Bacterial transposon Tn7 utilizes two classes of target sites 1 Bacteriol 1722774-2778
Kucharczyk N Denisot M A Le Goffic F and Badet B 1990 Glucosarnine-6-phosphate synthase from Ecoli detennination of the mechanism of inactivation by N3 fumaroyl-L-2-3 diarninoproprionic derivatives Biochemistry 293668-3676
Kusano M Takeshima T Inoue C and Sugawara K 1991 Evidence for two sets of structural genes coding for Ribulose biphosphate carboxylase in Thiobacillus ferrooxidans 1 Bacteriol 1737313-7323
Lane Dl A P Harrison lnr D Stahl B Pace SJ Giovannoni GJ Olsen and N R Pace 1992 Evolutionary relationship among sulphur- and iron-oxidizing eubacteria 1 Bacteriol 174267-278
Lee C H Bhagwhat A and Heffron F 1983 Identification of a transposon TnJ sequence required for transposition immunity Natl Acad Sci USA 806765-6769
Lewis M 1 Chang 1 A and Somoni R D 1990 A topological analysis of subunit a from Escherichia coli F1Fo-ATP synthase predicts eight transmembrane segments 1 BioI Chern 265 10541-10550
Lichtenstein C P and Brenner S 1982 Unique insertion site of Tn7 in the Ecoli chromosome Nature (London) 297601-603
Lichtenstein C P and Brenner S 1981 Site-specific properties of transposition to the Ecoli chromosome Mol Gen Genet 183380-387
Liu X Petersson S and Sandstrom A Mesophilic versus moderate thennophilic bioleaching Biohydrometallurgy Technologies Vol 1 A E Tonna 1 E Wey and Lakshmanan V 1 (ed) pp 29-38
Lizarna H M and Sankey B M 1993 Oxidation of H2S by Thiobacillus thiooxidans is inhibited by substrate and methane BiohydrometaHurgy Technologies Vol II A E Tonna 1 E Wey and C L Brierley (ed) pp 339-364
Lundgren D G and Silver M 1980 Ore leaching by bacteria Ann Rev Microbiol 34263shy268
Makino K Amemura M Shinagawa H Kobayashi A and Nakata A 1985 Sequence of the genes involved in phosphate transport and regulation of the phosphate regulon in Escherichia coli 1 Mol BioI 184231-240
Makino K Shinagawa Amemura M Kimura S Nakata A and Ishihama 1988 Euuv1J of the phosphate regulon of Ecoli Activation of pstS by the PhoB
protein in vitro 1 Mol BioI 20385-95
Makino K Shinagawa H Amemura M Yamada M and 1990 Signal transduction in the phosphate regulon of the Escherichia coli involves phosphotransfer nprlrJp~middotn PhoR and PhoB proteins J Mol BioI 21055
Malamy 1966 Frameshift mutations in the nnlnn of Escherichia coli Cold Harb Symp Quant BioI 31189-201
Malamy M H 1972 Electron microscopy insertions in the lac operon of Escherichia coli MoL Gen
Malamy M H and Bennett R L 1970 mutants of Ecoli and phosphate transport Biochem Biophys Res Commun
Maniatis T Fritsch EF Sambrook J 1982 nn A laboratory manual Cold Spring Harbor Laboratory Press Cold
McKnight G L S L Mudri SH Mathewest R S Marshall P O Sheppard and P J OHara 1990 Molecular cloning synthesis and bacterial expression of human glutaminefructose-6-phosphate UUAUU u J Chem 26725208-25212
Medveczky N and H Rosenburg 1970 phosphate-binding protein of Escherichia Biochim Biophys Acta 211 158-168
Mei B and Zalkin H 1989 A Cysteine-Histidine-Aspartate catalytic triad is involved in Glutamide Amide Transfer Function purF-type Glutamine amodotransferases 1 BioI Chem 264 16613-16619
Mengin-Lecreulx D and J van 1993 Identification of glmU gene encoding Nshyacetylglucosamine in Escherichia coli J Bacteriol 1756150shy6157
Michaelis M J and Criddle M J 1970 Mitochondrial DNA and mutants in Saccharomyces cerevisiae Biochem Genet 5487-495
Michaelis Starlinger P 1969 Two insertions in the jO v
operon having different homologous DNA sequences MoL Gen Genet 10437 377
Miller J H Calos Transposable elements Cell 20579-595
154
Miller J Oldenburg M and Fillingame R 1990 The essential carboxyl group of in subunit c the FIFo ATP synthase can be and H( +)-translocating function retained Proc NatL Acad 874900-4904
Mitcell P 1966 Chemiosmotic coupling in - and phosphorylation BioL Rev 41455-502 (1966)
Mizuuchi K 1984 Mechanism of transposition of bacteriophage Mu Polarity of the strand reaction at the initiation of the transposon Cell 39395-404
C Ph de Donato Berthelin J Surface oxidized species a key factor in the study of bioleaching processes Biohydrometallurgical In A J
Weyand V I Lakshmanan ed 1993 Vol 1 175-184
A Ishino Shinagawa H Makino K and M 1987 Nucleotide of the iap gene responsible for alkaline phosphatase isoenzyme conversion in Ecoli
identification of product 1 1695429-5433
K 1989 The tsr gene-coding prevents thiopeptin from inhibiting ppGpp synthesis in Streptomyces lividans FEMS Microbiol Lett
Ogasawara N and Yoshikawa H 1992 and their organIzatIon in the repnc()n of the bacterial chromosome Mol Microbiol 6(5)629-634
Ohtsubo Davidson N and 1975 microscope heteroduplex studies of sequence relations among plasmids identification and mapping of the insertion sequences 1 and IS2 in F and R plasmids 1 122746-775
Olson G J 1994 Microbial oxidation gold ores and gold bioleaching FEMS un Lett 119 1-6
Orle K A N L 1991 Identification of transposition prroteins by the bacterial transposon [corrected republished originally printed in Gene 1990 Nov 30 96(1) Gene 10425-31
Ouellette M Roy P Homology of ORFs from and from to site specific recombinases 1987 Nucl Acids 10055-10059
Murein synthesis p663-671ln Neidhardt J L Ingraham B Louw M~ M Schaechter H E Umbarger (ed) Escherichia coli and Salmonella
ryphimurium cellular and bioI voL 1 American Society Microbiology Washington
Perlin D S and Senior A Functional and cross-reactivity of antibody to purified subunit b (uncF protein) of Escherichia coli proton-ATPase Arch Biochem Biophys 236603-611
155
Plumbridge J A O Cochet Souza J M Altramirano M M Calcagno M L and Badet B 1993 Coordinated regulation of amino sugar-synthesizing and -degrading enzymes in Escherichia coli K-12 J Bacteriol 175(16)4951-4956
Pretorius I M Rawlings D E and Woods D R 1986 Identification and cloning of Thiobacillus jerrooxidans structural Nif genes in Escherichia coli Gene 4559-65
Qadri M I Flores C c Davis J and Lichtenstein C 1989 Genetic analysis of attTn7 the transposon Tn7 attachment site in Escherichia coli using a novel M13-based transduction assay 1 Mol BioI 20785-98
Radstrom P Skold 0 Swedberg J Roy P H and Sundstrom L 1994 Tn5090 of plasmid R751 which carries integron is related to Tn7 Mu and retroelements J Bacteriol 1763257-3268
Raetz C R H 1987 Structure and biosynthesis of lipid A in Escherichia coli pp 498-503 In F C Niedhardt J L Ingraham K B Low B Magasanik M Schaechter and H E Umbarger (ed) Escherichia coli and Salmonella typhimurium cellular and molecular biology vol 1 American Society for Microbiology Washington DC
Rao N N and Torriani A 1990 Molecular aspects of phosphate transport in Escherichia coli Mol Microbiol 4(7) 1083-1090
Rawlings DE D R Woods and NP Mjoli 1991 The cloning and structre of genes from the autotrophic biomining bacterium Thiobacillusjerrooxidans p 215-237 In PJ Greenaway (ed) Advances in gene technology vol 2 JAI Press London
Richet E and O Raibaud 1989 MalT the regulatory protein of the Escherichia coli maltose system is an A TP-dependent transcriptional activator EMBO 1 8981-987
Rogers M Ekaterrinaki N Nimmo E and Sherratt D 1986 Analysis of Tn7 transposition Mol Gen Genet 205550-556
Ronson C W Nixon B T Albright L M anf Ausubel F M 1987 Rhizobium meliloti ntrA (rpoN) gene is required for diverse metabolic functions J Bacteriol 1692424-2431
Rosenburg H Gerdes R G and Chegwidden K 1977 Two systems for the uptake of phosphate in Ecoli JBacterioL 131505-511
Rosenburg H 1987 In ion transport in Prokaryotes Rosen B P and Silver S (eds) New York Academic Press pp 205-248
Ross D G Swan 1 and N Klecker 1979 Physical structures of TnlO-promoted deletions and inversions role of 1400 base pairs inverted repetitions Cell 16721-731
156
Saedler H and P 19670 mutation in the galactose operon in Ecoli II Physiological characterization MoL Gen Genet 100 190-202
Saedler P 1968 00 and strong polar mutations in the Genet 102353-363
Sambrook J Fritsch Maniatis 1989 Molecular cloning a laboratory manual Cold Harbor Laboratory Cold Spring Harbor New York
Sand W Gehrke T Hallmann Rohde K Sobotke B and Wintzien S 1993 Bioleaching metal sulphides The importance of Leptospirillum ferrooxidans Biohydromemetallurgical Technologies Voll pp 15-25
Sanger Nicklen and Coulson A DNA sequencing with chain-tennination inhibitors Natl Acad USA 745463-5467
Schneider E and Altendorf K All the three subunits are required for an active proton channel (Fo) of Escherichia coli synthase (FIFo) ~-
518
Schneider E and Allendorf K 1987 Bacterial adenosine 5-triphosphate synthase purification and reconstitution complexes and biochemical and functional characterization of subunits Microbio Rev 51477-497
Schrader 1 A and D S Holmes 1988 Phenotypic switching Thiobacillus ferrooxidans J BacterioL 1703915-3023
Scordilis G E H and Lassie 1987 Identification of transposable elements which activates gene expression in Pseudomonas cepacia J Bacteriol 1698-13
Sekine Eisaki N and Ohtsubo E 1996 Identification and characterization of the lS3 molecules generated by breaks 1 BioL Chern 271197-202
Senior A E 1990 The proton-trans locating of Escherichia coli Annu Rev Biophys Chern 194-41
Shapiro 1 and Adhya S 1969 The jLU operon of K-12 n A deletion analysis of the structure polarity 62249-264
J A 1969 Mutations caused by insertion genenc material the galactose operon Escherichia 1 MoL BioI 4093-105
Silvennan M P 1967 Mechanism of bacterial pyrite oxidation 1 Bacteriol 941046-1051
157
C C ElIson and Levinson 1983 Identification of the typel trimethoprim resistance reductase specified by the R-plasmid R43 comparison with procaryotic and eucaryotic dihydrofolate reductase 1 Bacteriol 155 100 1-1008
Smith and Jones P transposition a multigene process Identification of a regulatory product 147915-7927
Sprague Jr Bell R M Cronan 1 A mutant of Ecoli auxotrophic for organic phosphates evidence two defects in phosphate Mol Gen Genet 14371-77
Starlinger and Michaelis 1968 Suppression the sequential aO[)ealame of the galactose enzymes in a transferase amber mutant coli Mol Genet 102367-369
Starlinger 1977 IS elements in microorganisms MicrobioL Immunol
Steffens K Schneider E Herkernhoff B Schmid Altendorf K portion of Escherichia coli A TP synthase resolution of from subunit b J BioI Chern 2625866-5869
Steffens A Deckers-hebestreit G and Altendorf K 1987b The and functional relationship of A TP synthases (FoFI) from Ecoii and the thermophilic bacterium PS3 J Biol 2626334-6338
Strominger J and M S Smith 1959 Uridine diphosphoacetylglucosamine pyrophosphorylase 1 BioI Chern 2341822-1827
Sundstrom Skold 1990 dhfrI trimethoprim resistance gene of can be found at in other genetic surroundings Antimicrob Agents Chemother 34642shy650
Sundstrom L Roy P H and Skold Site-specific of three cassettes in Tn7 J Bacteriol 1733025-3028
Surin B P and Downie JA 1988 of the Rhizobium leguminosarum nodLMN involved efficient host-specific nodulation Mol Microbiol 2 173-183
B P Dixon N and Rosenburg 1986 Purification PhoU protein a OClItnl
regulator of the pho regulon of Ecoli K-1 J Bacteriol 168631
B P D A Jans A L Fimmel Shaw GB Cox Rosenburg Structural gene for the phosphate-repressible phosphate binding of Escherichia coli
own promoter nucleotide of the phoS 1 Bacteriol
158
Suzuki I Chang W and Takeuchi 1994 Oxidation of organic compounds by Thiobacilli Symp Series 55060-67
Takeyama M S Y Noumi T Maeda Ishibashi S and Futai M 1988 Beta subunit of Ecoli amino acid replacement within a conserved sequence G-X-X-X-X-GshyK-TS) v~u binding proteins lett 218222-226
Thomson JA M Hendson and R M 1981 Mutagenesis by insertion of drug resistance Tn7 into a vibrio 11 J Bacteriol
Tichy R Grotenhuis J T c Janssen van Houten R Rulkens and Lettinga 1993 Application of the sulphur cycle bioremediation of soils polluted with heavy metals In Int Conf Contaminated 93 ed F Arendt G J Annokee R Bosman and W J van der Brink Kluwer Academic Publishers Dordrecht pp 1461-1462
G and Mayer An electron approach to the quaternary structure of mitochondrial Eur J Biochem 13237-45
J and Tschape streptothricin resistance transposons Tn1825 and Tn1826 and transposon Tn 7 Plasmidnuu
18246-249
Tso J Y Zalkin H van Cleemput M Yanofsky C and JM 1982 Nucleotide of Escherichia coli and deduced amino glutamine
phosphribosylpyrophosphate am idotransferase J BioI Chern
V Mesyanzhinova I V Koslov I A and Orlova 1984 Structure of studied by electron and image processing Lett 167285-289
P Barber C and transposons Tn5 and Tn7 in Xanthomonas campestris pv campestris MoL Gen 157
Ullrich J and van Putten P Identification of the Gonococcal glmU gene UVUUIJ
the enzyme N-acetylglucosamine 1-Phosphate Uridyltransferase involved in the synthesis UDP-GlcNAc J of Bact 1776902-6909
M 1992 Eight bacterial proteins includin]g UDP-N -acetylglucosamine acyltransferase three vother of Escherichia coli of a six-residue n tVI
theme FEMS MicrobioL 97249-254
van der Steen J J D Doddema J and de Uidoging van zware metalen afvalstromen met behulp van thiobacilli (Removal metals from waste streams
using thiobacilli) Ministry of Housing Physical and Enviromental (VROM) Directoraat-Generaal Milieubeheer Rapport 199210 Hague
Vermoote P 1988 Universite Paris VI Paris Hrllnro
159
Vignais P V Lunard Issartel J P and Dupuis A 1985 Interaction n l1f middotn
oligomycin sensitivity protein (OSCP) beef heart mitochondrial FeATPase 2 Identification of interacting Fl subunits cross-linking Biochem J
Vik S and Dao NN Prediction of transmembrane topology of Fo proteins from Ecoli ATP synthase variational hydrophobic moment analyses Biochim Biophys Acta 1140 199-207
von Meyenburg B B Jcentrgensen J Nielsen and F Hansen 1982 Promoters of the atp operon for the membrane-bound ATP synthase of Ecoli mapped by TnlO insertion mutations MoL Gen Genet 188240-248
von Meyenberg F G Hansen 1980 The origin of replication oriC the Ecoli chromosome near oriC and construction of oriC mutants ICN-UCLA Symp MoLCellBiol 191 159
Waddell S and Craig N 1989 Tn7 transposition recognition of the attTn7 Proc Natl Sci USA 863958-3962
Waddell C S and Craig N L 1988 transposition two transposition pathways directed by five Tn7-encoded genes Develop 21
J E Gay N J Saraste M and A N 1984 DNA sequence the Escherichia coli unc operon Completion of the sequence of a kilobase segment containing
oriC unc and phoS J224799-815
and Collinson 1 1994 the role the stalk in the coupling mechanism of FEBS 34639-43
Walker 1 E Gay N J and Tybulewicz V L 1986 of transposon Tn7 into the Escherichia glmS transcriptional terminator Biochem 1 243 111-1
Wanner Land McSharry 1982 Phosphate-controlled gene in Escherichia coli using Mudl-directed lacZ fusions J Mol BioI 158347-363
Weng M and Zalkin H 1987 Structural role for a region the CTP synthetase glutamide amide transfer domain J Bacteriol 1693023-3028
Wheatcroft R and S and nucleotide sequence of Rhizobiwn meliloti insertion between the transposase encoded by ISRm3 and those encoded by Staphylococcus aureus and Thiobacillus ferrooxidans IST2 J 1732530-2538
160
Whitby M C and Lloyd R 1995 Branch of three-strand recombination intennediates by RecG a possible pathway for securing middotIULl initiated by duplex DNA 1 143303-3310
Wilkens and Capaldi R A Assymmetry and changes in examined by cryoelectronmicroscopy BioI Chern Hoppe-Seyler 37543-51
and Malamy M 1974 The loss of the PhoS periplasmic protein leads to a the specificity a constitutive phosphate transport system in Escherichia coli Biochem Res Commun 60226-233
WiUsky G Rand Malamy M 1980 of two separable inorganic phosphate transport systems in Escherichia coli J BacterioL 144356-365
P J and and C F 1973 enzyme of metabolism and Stadtman R eds) pp 343-363 Academic Press New York
Winterbum P J and Phelps F 197L control of hexosamine biosynthesis by glucosamine synthetase Biochem 1 121711
Wolf-Watz H and Norquist A 1979 Deoxyribonucleic acid and outer membrane protein to outer membrane protein involves a protein J 14043-49
Wolf-Watz H and M 1979 Deoxyribonucleic acid and outer membrane strains for oriC elevated levels deoxyribonucleic acid-binding protein and
11 for specific UU6 of the oriC region to outer membrane J Bacteriol 14050-58
Wolf-Watz H 1984 Affinity of two different chromosome to the outer membrane of Ecoli J Bacteriol 157968-970
Wu C and Te Wu 197 L Isolation and characterization of a glucosamine-requiring mutant of Escherichia 12 defective in glucoamine-6-phosphate synthetase 1 Bacteriol 105455-466
Yamada M Makino Shinagawa Nakata A 1990 Regulation of the phosphate regulon of Escherichia coli properties the pooR mutants and subcellular localization of protein Mol Genet 220366-372
Yates J R and Holmes D S 1987 Two families of repeatcXl DNA sequences Thiobacillus 1 Bacteriol 169 1861-1870
Yates J R R P and D S 1988 an insertion sequence from Thiobacillus errooxidans Proc Natl 857284-7287
161
Youvan D c Elder 1 T Sandlin D E Zsebo K Alder D P Panopoulos N 1 Marrs B L and Hearst 1 E 1982 R-prime site-directed transposon Tn7 mutagenesis of the photosynthetic apparatus in Rhodopseudomonas capsulata 1 Mol BioI 162 17-41
Zalkin H and Mei B 1990 Amino terminal deletions define a glutamide transfer domain in glutamine phosphoribosylpyrophosphate ami do transferase and other purF-type amidotransferases 1 Bacteriol 1723512-3514
Zalkin H and Weng M L 1987 Structural role for a conserved region III the CTP synthetase glutamide amide transfer domain 1 Bacteriol 169 (7)3023-3028
Zhang Y and Fillingame R H 1994 Essential aspartate in subunit c of FIF0 ATP synthase Effect of position 61 substitutions in helix-2 on function of Asp24 in helix-I 1 BioI Chern 2695473-5479
pSIS1 construct was further subcloned to plasm ids IS30 IS40 pSlS50
pS183S p81841 and p81 (Fig Some of subclones were extensively mapped
although not all sites are shown in 22 exact positions of the ends of
T jerrooxidans plasmids pTfatpl and pTfatp2 on the cosmid p8I81 were identified
21)
Tfeooxidans
that the cosmid was
unrearranged DNA digests of cosmid pSISI and T jerrooxidans chromosomal DNA were
with pSI The of the bands gave a positive hybridization signal were
identical each of the three different restriction enzyme digests of p8I8l and chromosomal
In order to confirm of pSI81 and to
49
kb kb ~
(A)
11g 50Sts5
middot 2S4
17 bull tU (probe)
- tosect 0S1
(e)
kb
+- 10 kb
- 46 kb
28---+ 26-+
17---
Fig 23 Hybrdization of cosmid pSISI and T jerrooxitians (A TCC 33020) chromosomal
DNA by the KpnI-SalI fragment of pSIS52 (probe) (a) Autoradiographic image of the
restriction digests Lane x contains the probe lane 13 and 5 contain pSISI restricted with BamHl HindIII and Bgffi respectively Lanes 2 4 and 6 contain T errooxitians chromosomal DNA also restricted with same enzymes in similar order (b) The sizes of restricted pSISI
and T jerrooxitians chromosome hybridized by the probe The lanes correspond to those in Fig 23a A DNA digested with Pst served as the molecular weight nlUkermiddot
50
DNA (Fig BamHI digests SHklC at 26 and 2S kb 1 2) HindIII
at 10 kb 3 and 4) and BgnI at 46 (lanes 5 and 6) The signals in
the 181 was because the purified KpnI-San probe from p81 had a small quantity
ofcontaminating vector DNA which has regions ofhomology to the cosmid
vector The observation that the band sizes of hybridizing
for both pS181 and the T ferrooxidans ATCC 33020 same
digest that the region from BgnI site at to HindIII site at 16
kb on -VJjUIU 1 represents unrearranged chromosomal DNA from T ferrooxidans
ATCC there were no hybridization signals in to those predicted
the map with the 2 4 and 6 one can that are no
multiple copies of the probe (a Tn7-like segment) in chromosome of
and Lferrooxidans
of plasmid pSI was found to fall entirely within a Tn7-like trallSDOS()n nrpn
on chromosome 33020 4) A Southern hybridizationUUAcpoundUU
was out to determine whether Tn7-like element is on other
ofT ferrooxidans of T and Lferrooxidans results of this
experiment are shown 24 Lanes D E and F 24 represent strains
19377 DSM and Lferrooxidans DSM 2705 digested to
with BgnI enzyme A hybridization was obtained for
the three strains (lane A) ATCC 19859 (lane B) and UUHUltn
51
(A) AAB CD E F kb
140
115 shy
284 -244 = 199 -
_ I
116 -109 -
08
os -
(8)) A B c o E F
46 kb -----
Fig 24 (a) Autoradiographic image of chromosomal DNA of T ferrooxidans strains ATCC 33020 19859 23270 (lanes A B C) Tthiooxidans strains ATCC 19377 and DSM 504 (D and E) and Lferrooxidans strain DSM 2705 (lane F) all restricted with Bgffi A Pst molecular weight marker was used for sizing (b) Hybridization of the 17 kb KpnI-San piece of p81852 (probe) to the chromosomal DNA of the organisms mentioned 1 Fig 24a The lanes in Fig 24a correspond to those in Fig 24b
(lane C) uuvu (Fig 24) The same size BgllI fragments (46 kb) were
lanes A Band C This result
different countries and grown on two different
they Tn7-like transposon in apparently the same location
no hybridization signal was obtained for T thiooxidans
1 504 or Lferrooxidans DSM 2705 (lanes D E and F of
T thiooxidans have been found to be very closely related based on 1
rRNA et 1992) Since all Tferrooxidans and no Tthiooxidans
~~AA~~ have it implies either T ferrooxidans and
diverged before Tn7-like transposon or Tthiooxidans does not have
an attTn7 attachment the Tn7-like element Though strains
T ferrooxidans were 10catH)uS as apart as the USA and Japan
it is difficult to estimate acquired the Tn7-like transposon as
bacteria get around the world are horizontally transmitted It
remains to be is a general property
of all Lferrooxidans T ferrooxidans strains
harbour this Tn7-like element their
CHAPTER 3
5431 Summary
54Introduction
56Materials and methods
56L Bacterial and plasmid vectors
Media and solutions
56333 DNA
57334 gel electrophoresis
Competent preparation
57336 Transformation of DNA into
58337 Recombinant DNA techniques
58ExonucleaseIII shortening
339 DNA sequencing
633310 Complementation studies
64Results discussion
1 DNA analysis
64Analysis of ORF-l
72343 Glucosamine gene
77344 Complementation of by T ferrooxidans glmS
57cosmid pSIS1 pSlS20
58Plasmidmap of p81816r
Fig Plasmidmap of lS16f
60Fig 34 Restriction digest p81S1Sr and pSIS16f with
63DNA kb BamHI-BamHI
Fig Codon and bias plot of the DNA sequence of pSlS16
Fig 37 Alignment of C-terminal sequences of and Bsubtilis
38 Alignment of amino sequences organisms with high
homology to that of T ferrooxidans 71
Fig Phylogenetic relationship organisms high glmS
sequence homology to Tferrooxidans
31 Summary
A 35 kb BamHI-BamHI p81820 was cloned into pUCBM20 and pUCBM21 to
produce constructs p818 and p81816r respectively This was completely
sequenced both rrelct1()ns and shown to cover the entire gmS (184 kb) Fig 31
and 34 The derived sequence of the T jerrooxidans synthetase was
compared to similar nrvnC ~ other organisms and found to have high sequence
homology was to the glucosamine the best studied
eubacterium EcoU Both constructs p81816f p81 the entire
glmS gene of T jerrooxidans also complemented an Ecoli glmS mutant for growth on medium
lacking N-acetyl glucosamine
32 =-====
Cell wall is vital to every microorganism In most procaryotes this process starts
with amino which are made from rructClsemiddotmiddotn-[)ll()S by the transfer of amide group
to a hexose sugar to form an This reaction is by
glucosamine synthetase the product of the gmS part of the catalysis
to the 40-residue N-terminal glutamine-binding domain (Denisot et al 1991)
participation of Cys 1 to generate a glutamyl thiol ester and nascent ammonia (Buchanan
368-residue C-terminal domain is responsible for the second part of the ClUUU
glucosamine 6-phosphate This been shown to require the ltgtttrltgtt1iltn
of Clpro-R hydrogen of a putative rrulctoseam 6-phosphate to fonn a cis-enolamine
intennediate which upon reprotonation to the gives rise to the product (Golinelli-
Pimpaneau et al 1989)
bacterial enzyme mn two can be separated by limited chymotryptic
proteolysis (Denisot et 199 binding domain encompassing residues 1 to
240 has the same capacity to hydrolyse (and the corresponding p-nitroanilide
derivative) into glutamate as amino acid porI11
binding domain is highly cOllserveu among members of the F-type
ases Enzymes in this include amidophosphophoribosyl et al
1982) asparagine synthetase (Andrulis et al 1987) glucosamine-6-P synmellase (Walker et
aI 1984) and the NodM protein of Rhizobium leguminosarum (Surin and 1988) The
368-residue carboxyl-tenninal domain retains the ability to bind fructose-6-phosphate
The complete DNA sequence of T ferrooxidans glmS a third of the
glmU gene (preceding glmS) sequences downstream of glmS are reported in this
chapter This contains a comparison of the VVA r glmS gene
and Mycobacterium (422 ) An interesting observation was that the T ferrooxidans
glmS gene had comparatively high homology sequences to the Rhizobium leguminosarum and
Rhizobium meliloti M the amino to was 44 and 436
respectively A consensus Dalgarno upstream of the start codon
(ATG) is in 35 No Ecoli a70-type n r~ consensus sequence was
detected in the bp preceding the start codon on intergenic distance
between the two and the absence of any promoter consensus sequence Plumbridge et
al (1993) that the Ecoli glmU and glmS were co-transcribed In the case
the glmU-glmS --n the T errooxidans the to be similar The sequences
homology to six other amidotransferases (Fig 38) which are
(named after the purF-encoded l phosphoribosylpyrophosphate
amidotransferase) and Weng (1987)
The glutamine amide transfer domain of approximately 194 amino acid residues is at the
of protein chain Zalkin and Mei (1989) using site-directed to
the 9 invariant amino acids in the glutamine amide transfer domain of
phosphoribosylpyrophosphated indicated in their a
catalytic triad is involved glutamine amide transfer function of
73
Comparison of the ammo acid sequence alignment of ghtcosamine-6-phosphate
amidotranferases (GFAT) of Rhizobium meliloti (R_m) Rhizobium leguminnosarum (RJ)
Ecoli (E_c) Hinjluenzae Mycobacterium leprae (M_I) Bsubtilis (B_s) and
Scerevisiae (S_c) to that of Tferrooxidans Amino acids are identified by their single
codes The asterisks () represent homologous amino acids of Tferrooxidans glmS to
at least two of the other Consensus ammo acids to all eight organisms are
highlighted (bold and underlined)
1 50 R m i bullbullbullbullbullbull hkps riag s tf bullbullbullbull R~l i bullbullbullbullbullbull hqps rvaep trod bullbullbullbull a
a bullbullbullbullbullbull aa 1r 1 d bullbullbull a a bullbullbullbullbullbull aa inh g bullbullbullbullbullbull sktIv pmilq 1 1g bullbullbull a MCGIVi V D Gli RI IYRGYPSAi AI D 1 gvmar s 1gsaks Ikek a bullbullbullbull qfcny Iversrgi tvq t dgd bullbull ead
51 100 R m hrae 19ktrIk bull bullbull bullbull s t ateR-l tqrae 19rkIk bullbullbull s t atrE-c htIrI qmaqae bull bull h bullbull h gt sv aae in qicI kads stbull bullbull k bullbull i gt T f- Ivsvr aetav bullbullbull r bullbull gq qg c
DL R R G V L AV E L G VGI lTRW E H ntvrrar Isesv1a mv bull sa nIgi rtr gihvfkekr adrvd bullbullbull bullbull nve aka g sy1stfiykqi saketk qnnrdvtv she reqv
101 150 R m ft g skka aevtkqt R 1 ft g ak agaqt E-c v hv hpr krtv aae in sg tf hrI ksrvlq T f- m i h q harah eatt
S E Eamp L A GY l SE TDTEVIAHL ratg eiavv av
qsaIg lqdvk vqv cqrs inkke cky
151 200 bullbull ltk bullbull dgcm hmcve fe a v n bull Iak bullbull dgrrm hmrvk fe st bull bull nweI bullbull kqgtr Iripqr gtvrh 1 s bull bull ewem bullbullbull tds kkvqt gvrh hlvs bull bull hh bullbull at rrvgdr iisg evm
V YR T DLF A A L AV S D PETV AR G M 1 eagvgs lvrrqh tvfana gvrs B s tef rkttIks iInn rvknk 5 c Ihlyntnlqn ~lt klvlees glckschy neitk
74
201 R m ph middot middot middot R 1 ah- middot middot middot middot middot middot E c 1 middot middot middot middot middot middot middot Hae in 1 middot middot middot middot middot T f - c11v middot middotmiddot middot middot middot middot middot M 1 tli middot middot middot middot middot middot middot middot middot B s 11 middot middot middot middot middot S c lvkse kk1kvdfvdv
251 R m middot middot middot middot middot middot middot middot middot middot middot middot middot middot
middot middot middot middot R-1 middot middot middot middot middot middot E c middot middot middot middot middot middot middot middot Hae in middot middot middot middot middot middot middot middot middot middot middot middot T f shy middot middot middot middot middot middot middot middot middot middot middot middot 1M middot middot middot middot middot middot middot middot middot middot middot middot middot middot B s middot middot middot middot middot middot middot middot middot middot middot middot middot middot middot S c feagsqnan1 1piaanefn1
301 R m fndtn wgkt R-1 fneti wgkt E c rf er Hae in srf er T f- rl mlqq
PVTR V YE DGDVA R M 1 ehqaeg qdqavad B s qneyem kmvdd S c khkkl dlhydg
351 R m nhpe v R-1 nhe v E c i nk Hae in k tli T f- lp hr
~ YRHlrM~ ~I EQP AVA M 1 geyl av B-s tpl tdvvrnr S-c pd stf
401 Rm slasa lk R-1 slasca lk E c hql nssr Hae in hq nar-T f f1s hytrq
RVL A SiIS A VG W M 1 ka slakt B s g kqS c lim scatrai
T ferrooxidans (Tf) Scerevisiae (Saee) Mleprae (Myele) and Bsublilis (Baesu)
tr1 ()-ltashyc 8 ~ er (11
-l l
CIgt
~ r (11-CIgt (11
-
$ -0 0shy
a c
omiddot s
S 0 0shyC iii omiddot $
ttl ~ () tI)
I-ltashyt ()
0
~ smiddot (11
81
CHAPTER 4
8341 Summary
8442 Introduction
8643 Materials and methods
431 Bacterial strains and plasmids 86
432 DNA constructs subclones and shortenings 86
864321 Contruct p81852
874322 Construct p81809
874323 Plasmid p8I809 and its shortenings
874324 p8I8II
8844 Results and discussion
441 Analysis of the sequences downstream the glmS gene 88
442 TnsBC homology 96
99443 Homology to the TnsD protein
118444 Analysis of DNA downstream of the region with TnsD homology
LIST OF FIGURES
87Fig 41 Alignment of Tn7-like sequence of T jerrooxidans to Ecoli Tn7
Fig 42 DNA sequences at the proximal end of Tn5468 and Ecoli glmS gene 88
Fig 43 Comparison of the inverted repeats of Tn7 and Tn5468 89
Fig 44 BLAST results of the sequence downtream of T jerrooxidans glmS 90
Fig 45 Alignment of the amino acid sequence of Tn5468 TnsA-like protein 92 to TnsA of Tn7
82
Fig 46 Restriction map of the region of cosmid p8181 with amino acid 95 sequence homology to TnsABC and part of TnsD of Tn7
Fig 47 Restriction map of cosmid p8181Llli with its subclonesmiddot and 96 shortenings
98 Fig 48 BLAST results and DNA sequence of p81852r
100 Fig 49 BLAST results and DNA sequence of p81852f
102 Fig 410 BLAST results and DNA sequence of p81809
Fig 411 Diagrammatic representation of the rearrangement of Tn5468 TnsDshy 106 like protein
107Fig 412 Restriction digests on p818lLlli
108 Fig 413 BLAST results and DNA sequence of p818lOEM
109 Fig 414 BLAST results on joined sequences of p818104 and p818103
111 Fig 415 BLAST results and DNA sequence of p818102
112 Fig 416 BLAST results and nucleotide sequence of p818101
Fig 417 BLAST results and DNA sequence of the combined sequence of 113p818l1f and p81810r
Fig 418 Results of the BLAST search on p81811r and the DNA sequence 117obtained from p81811r
Fig 419 Comparison of the spa operons of Ecali Hinjluenzae and 121 probable structure of T ferraaxidans spa operon
83
CHAPTER FOUR
TN7-LlKE TRANSPOSON OF TFERROOXIDANS
41 Summary
Various constructs and subclones including exonuclease ill shortenings were produced to gain
access to DNA fragments downstream of the T ferrooxidans glmS gene (Chapter 3) and
sequenced Homology searches were performed against sequences in GenBank and EMBL
databases in an attempt to find out how much of a Tn7-like transposon and its antibiotic
markers were present in the T ferrooxidans chromosome (downstream the glmS gene)
Sequences with high homology to the TnsA TnsB TnsC and TnsD proteins of Tn7 were
found covering a region of about 7 kb from the T ferrooxidans glmS gene
Further sequencing (plusmn 45 kb) beyond where TnsD protein homology had been found
revealed no sequences homologous to TnsE protein of Tn7 nor to any of the antibiotic
resistance markers associated with Tn7 However DNA sequences very homologous to Ecoli
ATP-dependentDNA helicase (RecG) protein (EC 361) and guanosine-35-bis (diphosphate)
3-pyrophosphohydrolase (EC 3172) stringent response protein of Ecoli HinJluenzae and
Vibrio sp were found about 15 kb and 4 kb respectively downstream of where homology to
the TnsD protein had been detected
84
42 Transposable insertion sequences in Thiobacillus ferrooxidizns
The presence of two families (family 1 and 2) of repetitive DNA sequences in the genome
of T ferrooxidans has been described previously (Yates and Holmes 1987) One member of
the family was shown to be a 15 kb insertion sequence (IST2) containing open reading
frames (ORFs) (Yates et al 1988) Sequence comparisons have shown that the putative
transposase encoded by IST2 has homology with the proteins encoded by IS256 and ISRm3
present in Staphylococcus aureus and Rhizobium meliloti respectively (Wheatcroft and
Laberge 1991) Restriction enzyme analysis and Southern hybridization of the genome of
T ferrooxidans is consistent with the concept that IST2 can transpose within Tferrooxidans
(Holmes and Haq 1990) Additionally it has been suggested that transposition of family 1
insertion sequences (lST1) might be involved in the phenotypic switching between iron and
sulphur oxidizing modes of growth including the reversible loss of the capacity of
T ferrooxidans to oxidise iron (Schrader and Holmes 1988)
The DNA sequence of IST2 has been determined and exhibits structural features of a typical
insertion sequence such as target-site duplications ORFs and imperfectly matched inverted
repeats (Yates et al 1988) A transposon-like element Tn5467 has been detected in
T ferrooxidans plasmid pTF-FC2 (Rawlings et al 1995) This transposon-like element is
bordered by 38 bp inverted repeat sequences which has sequence identity in 37 of 38 and in
38 of 38 to the tnpA distal and tnpA proximal inverted repeats of Tn21 respectively
Additionally Kusano et al (1991) showed that of the five potential ORFs containing merR
genes in T ferrooxidans strain E-15 ORFs 1 to 3 had significant homology to TnsA from
transposon Tn7
85
Analysis of the sequence at the tenninus of the 35 kb BamHI-BamHI fragment of p81820
revealed an ORF with very high homology to TnsA protein of the transposon Tn7 (Fig 44)
Further studies were carried out to detennine how much of the Tn7 transposition genes were
present in the region further downstream of the T ferroxidans glmS gene Tn7 possesses
trimethoprim streptomycin spectinomycin and streptothricin antibiotic resistance markers
Since T ferrooxidans is not exposed to a hospital environment it was of particular interest to
find out whether similar genes were present in the Tn7-like transposon In the Ecoli
chromosome the Tn7 insertion occurs between the glmS and the pho genes (pstS pstC pstA
pstB and phoU) It was also of interest to detennine whether atp-glm-pst operon order holds
for the T ferrooxidans chromosome It was these questions that motivated the study of this
region of the chromosome
43 OJII
strains and plasm ids used were the same as in Chapter Media and solutions (as
in Chapter 3) can in Appendix Plasmid DNA preparations agarose gel
electrophoresis competent cell preparations transformations and
were all carned out as Chapter 3 Probe preparation Southern blotting hybridization and
detection were as in Chapter 2 The same procedures of nucleotide
DNA sequence described 2 and 3 were
4321 ~lllu~~~
Plasmid p8I852 is a KpnI-SalI subclone p8I820 in the IngtUMnt vector kb) and
was described Chapter 2 where it was used to prepare the probe for Southern hybridization
DNA sequencing was from both (p81852r) and from the Sail sites
(p8I852f)
4322 ==---==
Construct p81809 was made by p8I820 The resulting fragment
(about 42 kb) was then ligated to a Bluescript vector KS+ with the same
enzymes) DNA sequencing was out from end of the construct fA
87
4323 ~mlJtLru1lbJmalpoundsectM~lllgsect
The 40 kb EeoRI-ClaI fragment p8181Llli into Bluescript was called p8181O
Exonuclease III shortening of the fragment was based on the method of Heinikoff (1984) The
vector was protected with and ClaI was as the susceptible for exonuclease III
Another construct p818IOEM was by digesting p81810 and pUCBM20 with MluI and
EeoRI and ligating the approximately 1 kb fragment to the vector
4324 p81811
A 25 ApaI-ClaI digest of p8181Llli was cloned into Bluescript vector KS+ and
sequenced both ends
88
44 Results and discussion
of glmS gene termini (approximately 170
downstream) between
comparison of the nucleotide
coli is shown in Fig 41 This region
site of Tn7 insertion within chromosome of E coli and the imperfect gtpound1
repeat sequences of was marked homology at both the andVI
gene but this homology decreased substantially
beyond the stop codon
amino acid sequence
been associated with duplication at
the insertion at attTn7 by (Lichtenstein and as
CCGGG in and underlined in Figs41
CCGGG and are almost equidistant from their respelt~tle
codons The and the Tn7-like transposon BU- there is
some homology transposons in the regions which includes
repeats
11 inverted
appears to be random thereafter 1) The inverted
repeats of to the inverted repeats of element of
(Fig 43) eight repeats
(four traJrlsposcm was registered with Stanford University
California as
A search on the (GenBank and EMBL) with
of 41 showed high homology to the Tn sA protein 44) Comparison
acid sequence of the TnsA-like protein Tn5468 to the predicted of the
89
Fig 41
Alignment of t DNA sequence of the Tn7-li e
Tferrooxi to E i Tn7 sequence at e of
insertion transcriptional t ion s e of
glmS The ent homology shown low occurs within
the repeats (underl of both the
Tn7-li transposon Tn7 (Fig 43) Homology
becomes before the end of t corresponding
impe s
T V E 10 20 30 40 50 60
Ec Tn 7
Tf7shy(like)
70 80 90 100 110 120
Tf7shy(1
130 140 150 160 170 180 Ec Tn 7 ~~~=-
Tf7shy(like)
90
Fig 42 DNA sequences at the 3 end of the Ecoll glmS and the tnsA proximal of Tn7 to the DNA the 3end of the Tferrooxidans gene and end of Tn7-like (a) DNA sequence determined in the Ecoll strain GD92Tn7 in which Tn been inserted into the glmS transcriptional terminator Walker at ai 1986 Nucleotide 38 onwards are the of the left end of Tn DNA
determined in T strain ATCC 33020 with the of 7-like at the termination site of the glmS
gene Also shown are the 22 bp of the Tn7-like transposon as well as the region where homology the protein begins A good Shine
sequence is shown immediately upstream of what appears to be the TTG initiation codon for the transposon
(a)
_ -35 PLE -10 I tr T~ruR~1 truvmnWCIGACUur ~~GCfuA
100 no 110 110 110
4 M S S bull S I Q I amp I I I I bull I I Q I I 1 I Y I W L ~Tnrcmuamaurmcrmrarr~GGQ1lIGTuaDACf~1lIGcrA
DO 200 amp10 220 DG Zlaquot UO ZIG riO
T Q IT I I 1 I I I I I Y sir r I I T I ILL I D L I ilGIIilJAUlAmrC~GlWllCCCAcarr~GGAWtCmCamp1tlnmcrArClGACTAGNJ
Fig 45 (a) Alignment of the amino acid sequence of Tn5468 (which had homology to TnsA of Tn7 Fig 44) to the amino acid sequence of ORF2 (between the merR genes of Tferrooxidans strain E-1S) ORF2 had been found to have significant homology to Tn sA of Tn7 (Kusano et ai 1991) (b) Alignment of the amino acids translation of Tn5468 (above) to Tn sA of Tn7 (c) Alignment of the amino acid sequence of TnsA of Tn7 to the hypothetical ORF2 protein of Tferrooxidans strain E-1S
(al
Percent Similarity 60000 Percent Identity 44444
Tf Tn5468 1 LARQRYGVDEDRVARFQKEGRGQGRGADYHPWLTIQDVPSQGRSHRLKGI SO 11 1111111 I 1111 1111 I I 111
Fig 49 (a) Results of the BLAST search on the inverted and complemented nucleotide sequence of 1852f (from the Sall site) Results indicate amino acid sequence to the TnsC of Tn7 (protein E is the same as TnsC protein) (b) The nucleotide sequence and open reading frame of p81852f
High Prob producing Segment Pairs Frame Score P(N)
IB255431QQECE7 protein E - Escher +1 176 15e-20 gplX044921lSTN7EUR 1 Tn7 E with +1 176 15e-20 splP058461 ECOLI TRANSPOSON TN7 TRANSPOSITION PR +1 176 64e-20 gplU410111 4 D20248 gene product [Caenorhab +3 59 17e-06
IB255431QQECE7 ical protein E - Escherichia coli transposon Tn7 (fragment)
417 (al BLAST results obtained from combined sequence of (inverted and complemented) and 1810r good sequence to Ecoli and Hinfluenzae DNA RecG (EC 361) was found (b) The combined sequence and open frame which was homologous to RecG
444 Analysis of DNA downstream of region with TnsD homology
The location of the of p81811 ApaI-CLaI construct is shown in Fig 47 The single strand
sequence from the C LaI site was joined to p8181Or and searched using BLAST against the
GenBank and EMBL databases Good homology to Ecoli and Hinjluezae A TP-dependent
DNA helicase recombinase proteins (EC 361) was obtained (Fig 417) The BLAST search
with the sequence from the ApaI end showed high sequence homology to the stringent
response protein guanosine-35-bis (diphosphate) 3-pyrophosphohydrolase (EC 3172) of
Ecoli Hinjluenzae and Scoelicolor (Fig 418) In both Ecoli and Hinjluenzae the two
proteins RecG helicase recombinase and ppGpp stringent response protein constitute part of
the spo operon
445 Spo operon
A brief description will be given on the spo operons of Ecoli and Hinjluenzae which appear
to differ in their arrangements In Ecoli spoT gene encodes guanosine-35-bis
pyrophosphohydrolase (ppGpp) which is synthesized during stringent response to amino acid
starvation It is also known to be responsible for cellular ppGpp degradation (Gentry and
Cashel 1995) The RecG protein is required for normal levels of recombination and DNA
repair RecG protein is a junction specific DNA helicase that acts post-synaptically to drive
branch migration of Holliday junction intermediate made by RecA during the strand
exchange stage of recombination (Whitby and Lloyd 1995)
The spoS (also called rpoZ) encodes the omega subunit of RNA polymerase which is found
associated with core and holoenzyme of RNA polymerase The physiological function of the
omega subunit is unknown Nevertheless it binds stoichiometrically to RNA polymerase
119
Fig 418 BLAST search nucleotide sequence of 1811r I restrict ) inverted and complement high sequence homology to st in s 3 5 1 -bis ( e) 3 I ase (EC 31 7 2) [(ppGpp)shyase] -3-pyropho ase) of Ecoli Hinfluenzae (b) The nucleot and the open frame with n
Add 4 ml 52 mix by inversion at room temp for 5 mins
Add 4 ml 53 Mix by to homogenous suspension
Spin at 15 K 40 mins at 4
remove to fresh tube
Equilibrate column with 2 ml N2
Load supernatant 2 to 4 ml amounts
Wash column 2 X 4 of N3 Elute the DNA the first bed
each) add 07
volumes of isopropanol
Spin at 4 Wash with 70 Ethanol Resuspended pellet in n rv 100 AU of TE and scan
volume of about 8 to 10 drops) To the eluent (two
to
140
SEQUITHERM CYCLE SEQUENCING
Alf-express Cy5 end labelled promer method
only DNA transformed into end- Ecoli
The label is sensitive to light do all with fluorescent lights off
3-5 kb
3-7 kb
7-10 kb
Thaw all reagents from kit at well before use and keep on
1) Label 200 PCR tubes on or little cap flap
(The heated lid removes from the top of the tubes)
2) Add 3 Jtl of termination mixes to labelled tubes
3) On ice with off using 12 ml Eppendorf DNA up to
125 lll with MilliQ water
Add 1 ltl
Add lll of lOX
polymeraseAdd 1 ltl
Mix well Aliquot 38 lll from the eppendorf to Spin down
Push caps on nrnnp1
141
Hybaid thermal Cycler
Program
93 DC for 5 mins 1 cycle
93degC for 30 sees
55 DC for 30 secs
70 DC for 60 secs 30 cycle 93 DC_ 30s 55 DC-30s
70degC for 5 mins 1 cycle
Primer must be min 20 bp long and min 50 GC content if the annealing step is to be
omitted Incubate at 95 DC for 5 mins to denature before running Spin down Load 3 U)
SEQUITHERM CYCLE SEQUENCING
Ordinary method
Use only DNA transformed into end- Ecoli strain
3-5 kb 3J
3-7 kb 44
7-10 kb 6J
Thaw all reagents from kit at RT mix well before use and keep on ice
1) Label 200 PCR tubes on side or little cap flap
(The heated lid removes markings from the top of the tubes)
2) Add 3 AU of termination mixes to labelled tubes
3) On ice using l2 ml Eppendorf tubes make DNA up to 125 41 with MilliQ water
142
Add I Jtl of Primer
Add 25 ttl of lOX sequencing buffer
Add 1 Jtl Sequitherm DNA polymerase
Mix well spin Aliquot 38 ttl from the eppendorf to each termination tube Spin down
Push caps on properly
Hybaid thermal Cycler
Program
93 DC for 5 mins 1 cycle
93 DC for 30 secs
55 DC for 30 secs
70 DC for 60 secs 30 cycles
93 DC_ 30s 55 DC-30s 70 DC for 5 mins 1 cycle
Primer must be minimum of 20 bp long and min 50 GC content if the annealing step is
to be omitted Incubate at 95 DC for 5 mins to denature before running
Spin down Load 3 ttl for short and medium gels 21t1 for long gel runs
143
REFERENCES
144
REFERENCES
Abrahams J P Lutter R Todd R J van Raaij M J Leslie A G W and Walker J E 1993 Inherent asymmetry of the structure of F1-ATPase from bovine heart mitochondria at 65 Aresolution EMBO J 121775-1780
Abrahams J P Leslie A G W Lutter R and Walker 1 E 1994 Structure at 28 A resolution of FeATPase from bovine heart mitochondria Nature 370621-628
Adzumah K and Mizuuchi K 1988 Target immunity of Mu transposition reflects a differential distribution of Mu B protein Cell 53257-266
Akey C W Crepeau R H Dunn S D McCarty R E and Edelstein S J 1983 Electron microscopy of single molecules and crystals of F1-ATPases EMBO J 21409-1415
Allet B 1979 Mu insertion duplicates a 5 bp sequence at the host inserted site Cell 16 123shy129
Amzel L M and Pedersen P L 1983 Proton ATP-ases structure and mechanism Ann Rev Biochem 52801-824
Andersson L Mac Neela J and Wolfenden R 1985 Use of secondary isotope effects and varying pH to investigate mode of binding of inhibitory amino aldehydes by leucine aminopeptidase Biochem 24330-333
Andrews G F Dugan R P and Stevens C J 1992 Combining physical and bacterial treatment for removing pyritic sulfur from coal In Processing and Utilization of High-sulfur coals IV Dugan P R D Quigley and Y Attia (eds) p 515 Elsevier New York
Andrulis I L Chen J and Ray P N 1987 Isolation of human cDNAs for asparagine synthetase and expression in Jansen rat sarcoama cells Mol Cell BioI 72435-2443
Andruszkiewicz R Milewsky S Zieniawa T and Borowski E 1990 Anticandidal properties of N3 -(4-methoxy-fumaroyl)-L-2 3-diamino propanoic acid oligopeptides 1 Med Chern 33132-135
Arciszewska L K Drake D and Craig NL 1989 Transposon Tn7 cis-acting sequences in transposition and transposition immunity J Mol BioI 20735-42
Bachmann B J 1983 Linkage map of Escherichia coli K-12 edition 7 Microbiol Rev 47180-230
145
B Plumbridge 1 A Cochet D Souza J M M M and Calcagno M 1988 Cordinated regulation of amino J
Bacteriol 1754951-4956
0 B Vermoote P V and Le Goffic F 1993 synthetase from Ecoli lUllL- mechanism and inhibition by N3-fumaroyl-2 3-diaminopropionic derivatives Biochem 27 2282-2287
Vermoote P Haumont PY syrlthl~ta~e from Escherichia coli purification site location Biochemistry 26 1940-1948
Badet B Inagaki K Soda K Walsh dependent inhibition of Bacillus stearothermophilus alanine racemase by phosphonate isomers by isomerization to non covalent enzyme-(1-aminoethyl) complexes Biochem 253275-3282
Badet-Denisot M A and Badet of glucoseamine-6shyphosphate synthetase by diethylpyrocarbonates histidine requirement for enzymatic activity Arch Biochem Biophys
Bainton R Gamas P and transposition in vitro proceeds through an excised transposon intermediate (JpnIPtlltpi1 111 breaks in DNA Cell 65805-816
Barry G 1986 Permanent insertion of v~u into the chromosomes of soil bacteria BioTechnology 4446-449
Barth P T Datta N Grinter N S 1976 Transposition a deoxyribonucleic acid sequence trimethoprim and streptomycin resistances from R483 to other replicons 1 Bacteriol 125800-810
Bates C J Adams W and R R 1968 Control of the formation of uri dine diphospho-N-acetyl and glycoprotein synthesis in rat liver J Chern 2411705-17
Benjamin N 1989 Intramolecular transposition of TnlD Cell 59373shy383
Bennet R L and Malamy M resistant mutants of Escgerichia coli and phosphate Commun 40496-503
Benneth1 1978 Bacterial leaching patterns on pyrite crystal J Bacteriol
146
Berg D E Davies 1 Allet B and Rochaix 1 D 1975 Transposition of R factor genes to bacteriophage lambda Proc Natl Acad Sci USA 723268-3632
Berg D E and Drummond M1978 Absence of DNA sequences homologous to transposable element Tn5 (Kan) in the chromosome of Ecoli K-12 J Bcateriol 136419-422
Birkenhager R Hoppert M Deckers-Hebestriet G Mayer F and Altendorf K 1995 The Fo complex of the Ecoli ATP synthase investigation by electron imaging and immunoelectron microscopy Eur J Biochem 23058-67
Bjqgtrbaek C Foersom V and Michelsen O 1990 The transmembrane topology of the a subunit from ATPase in Escherichia coli analysed by PhoA protein fusions FEBS lett 26031-34
Boekemia E J Berden JA and van Heel MG 1986 sructure of mitochondrial F1-ATPase studied by electron microscopy and image processing Biochim Biophys Acta 851353-360
Bolton E Glynn P and OGara F 1984 Site specific transposition of Tn7 into a Rhizobiwn meliloti megaplasmid Mol Gen Genet 193153-157
Boos W 1974 Bacterial transport Annu Rev Biochem 43123-146
Boursaux-Eude C Girons I S and Zuerner R 1995 IS1500 an IS3-like element from Leptospira interrogans Microbiol 1412165-2173
Boyer P D 1993 The binding change mechanism for ATP synthase-some probabilities and possibilities Biochim Biophys Acta 1140215-250
Brachet P Eisen H and Rambach A 1970 Mutations in coliphage lambda affecting the expression of replicative functions 0 and P Mol Gen Genet 108266-276
Brink J Boekemia E 1 and van Bruggen E F 1987 The structure of NADH ubiquitone oxidoreductase fron beef heart mitochondria Crystals containing an octameric arrangement of iron-sulphur protein fragments Eur J Biochem 166287-294
Brock T D and Gustafson J 1976 Ferric iron reduction by sulphur- and iron-oxidizing bacteria Appl Environ Microbiol 32 567-571
Brown L D Dennehy M E and Rawlings D E 1994 The FI genes of the FIFo ATP synthase from the acidophilic bacterium Thiobacillus jerrooxidans complement Escherichia coli FI unc mutants FEMS Microbiol Lett 12219-25
Buchanan 1 1 Adv The Amidotransferases Enzymol 3991-183
Buckley Science
Caruso M Pseudomonas nOVHrn
oxidation of pyrite Applied
1 1982 Interactions Mol Gen Genet
phage 1
Chmara H by Biophysica
synthetase from bacteria antibiotic tetaine Biochimica et
Microbiol Lett 11197-206 controls on the oxidation of refractory Barberton
genetic elements and evolution Nature 263731shyCohen S N 1976 738
Corbet C M and 1 Ingledew 1987 Is oxidation by Thiobacillus ferrooxidans
Couillard D and ~~b 118(5)808-81
Metallurgical residue V UlllLltHIH of metals from sewage sludge J
Cox G B a reassessment of the
F and Hatch L 1986 The mechanism of ATP synthase of the b and a subunits Biochim Acta 84962-69
Craig N L 1995 Unity in Science 270253-254
Craig N and Gamas P 1 Purification and characterization of a transposition protein that binds ATP DNA Nuc Acids Res
1 2873-97 C A 1990 in response to environmental stress Advances in
alUIUH~ on the activity subunit b-specific polyc1onal
G Simoni and Altendorf K 1992 Influence vlJnu~ of the ATP synthase of t-S(nel~lCn
1 BioI Chern 26712364shy
M A Le Goffic F and 1991 Glucosamine- 6P two proteins on limited proteolysis ltVU Biophys 288225-230
148
Dobrogosz W J 1968 _l in Escherichia coli and its to catabolite repression J
Doublet P 1 van Heijenoort and 1993 The murI gene of is an essential gene that encodes klUltUllU~v racemase activity J Bacteriol 1752970-2979
P J van Heijenoort 1992 Identification of the Ecoli which is required for the D-glutamic acid a specific component peptidoglycan 1 Bacteriol
Garren A Garen and Torri ani 1961 Genetic control of repression UCUlllV phosphatase in Ecoli 1 Mol BioI
D J and Boeke 1 D 1990 A 1-1-0- structure is required for Ty1 transposition Genes Develop 4324-330
1982 Transposition of Tn7 occurs at a on Caulobacter crescentus 10SIClmle 1 Bacteriol 151 1056-1 058
H 1990 Molecular IHovUUU)11 -transporting 345-391 In T 12 Bacterial
Press Ltd London
transport and coupling by the synthase insights mechanism of function J Bioenerg 24485-491
1992b Subunit c of FIFo ATP role m transduction Biochim Biophys Acta 1101 v--rJ
Eliopoulos E E Jackson P 1 Keen IN HUIUVI L Thompson P and 1 Structure of a 16 kDa integral AU-UUl that identity to
of the vacuolar H+ -ATPase Protein 15
Fling M 1 C 1985 Nucleotide sequence of encoding the amino glycoside-modifying enzyme 3(9)-O-nucleotidyltransferase Res 137095-7106
Fling M and C l-1UUJIU nucleotide sequence of dihydrofolate rhnrpri by Tn7 Nucleic Acids Res 115
Flores Llchtenstem C P 1990 DNA sequence anlysis of tnsA for the Tn7 transposition Nucleic Acids 18901 911
149
149
Foster D L and Fillingame R H 1982 Stoichiometry of subunits in the H+-ATPase complex of Escherichia coli J BioI Chern 2572009-2015
Fraga D Hermolin J Oldenburg M Miller MJ and Fillingame R H 1994 Arginine 41 of subunit c of Escherichia coli H+-A TP synthase is essential in binding and coupling of F to Fo J BioI Chern 2697532-7537
Friedl P Hoppe J Gunsalus R P Michelsen 0 von Meyenburg K and Schairer H U 1983 Membrane integration and function of the three Fo subunits of the A TP-synthase of Escherichia coli K12 EMBO 1 299-103
Frisa P S and Sonneborn D R 1982 Developmentally regulated interconversion between end product inhibitable and non-inhibitable forms of a first pathway-specific enzyme activity can be mimicked in vitro by dephosphorylation reactions Proc Natl Acad Sci USA 796289-6293
Fujiwara T and Mizuuchi K 1988 Retroviral DNA integration Structure of an integration intermediate Cell 54497-504
Galas D J and Chandler M 1989 Bacterial insertion sequences In Mobile DNA Berg D E and Howe M M (eds) Washington D C American Society for Microbiology pp 109shy162
Gay N J Tybulewicz V L1 Walker JE 1986 Insertion of transposon Tn7 into the Ecoli gmS transcriptional terminator Biochem J 234 111-117
Gentry D R and Cashel M 1995 Cellular localization of the Escherichia coli SpoT protein J Bacteriol 177 3890-3893
Gentry D R Xiao H Burgess R R and Cashel M 1991 The omega subunit of Ecoli K-12 RNA polymerase is not required for stringent RNA control in vivo J Bacteriol 173 3901-3903
Girvin M E and Fillingame R H 1993 Helical structure and folding of subunit c of FIFo ATP synthase IH NMR resonace assignments and NOE analysis Biochemistry 3212167shy12177
Gogol E P Lucken U Bork T and Capaldi R A 1989 Molecular architecture of Escherichia coli F Adenosinetriphosphatase Biochemistry 284709-4716
Gogol E P Aggeler R Sagermann M and Capaldi R A 1989 Cryoelectronic Microscopy of Escherichia coli FI Adenosinetriphosphatase Decorated with Monoclonal Antibodies to Individual Subunits of the complex Biochemistry 284717-4724
150
Heinikoff S 1984
Golinelli-Pimpaneaux B and Badet involvement of Lys603 from Ecoli glucosamoine-6-phosphate synthase substrate fructose-6-phosphate 1 Biochem 201175-182
Gottesman M M and Rosner 1 L of a detenninant of uvu_bullu
resistance by coliphage lambda Sci USA 725041-5045
Grunstein M and Hogness D
Colony hybridization a method for isolation cloned DNAs that contain a 1Jbullu Proc NatL Acad Sci USA 723961-3965
Hauer B and Shapiro 1 A 1984 Control of Tn7 transposition Mol Gen Genet 194
Hedges R W and Jacob Transposition of ampicillin resistance from to other replicons MoL 1-40
Hennolin 1 Gallant 1 psi subunit in the Fo sector of the H+ -ATPase of Ecoli J Bioi
R H 1983 Topology organization and function
Hennolin J and R 1989 Assembly of Fo sector of synthase Interdepenndence of subunit insertion into the membrane 1 2817
van Montagu M Holsters Zambryski P Beuckeleer M Willmitzer and Schell 1 M 1980 interaction of
DNA plant cells Proc Soc Lond 210351-365
P 1972 Insertion mutations control region of Physical characterization of the mutants Mol Gen Genet
115266-276
Holmes applications pI
UI Haq 1990 Adaptation of Thiobacillus vu)nuu for industrial In J Salley R G McCready Wichlacz (ed)
1989 CANMET Ottawa Canada
Holtje J V and U Schwarz 1985 Biosynthesis and growth murein sacculus p 77shy119 InN (ed) Molecular cytology of Escherichia Academic Press Inc New
Acad Sci USA
Heffron E Reubens C and which mediates ampicillin
Translocation of a plasmid DNA sequence nature and specificity of Natl
digestion with exonuclease III creates fl tr breakpoints 1-369
Hernalsteens J M Agrobacterium
Hirsch H the galactose nnnn fJu
151
Holzenburg A Jones P c Franklin T Padi J B and Finbow M E 1993 Evidence for a common structure 1 Biochem 21321-30
Hoppe 1 and Sebald W 1986 Topological pathway of the protons through Fo is provided by amino acid the lipid phase Biochimie (Paris) 68427-434
S Otsubo H Davidson N and 1975 Electron microscope heteroduplex of sequence relations among plasmids identification and mapping of the
insertion sequences IS] and IS2 in F and R plasmids J Bacteriol 22762-775
Inoue c Sugawara K and 1991 regulatory gene in Thiobacillus ferrooxidans is spaced apart from Mol Microbiol 52707-2718
Ish-Horowicz D and Burke and cosmid cloning Nucleic Acids Res 92989-2998
Johnston B Clennell M and D 1995 Structure and function of Tn5467 a Tn2l-like transposon located on T jerrooxidans broad host range plasmid Appl Envir Micro
Kahmann R and Kamp Nucleotide sequences of the attachment bacteriophage Mu DNA Nature 280247-250
Kahn K and Schaefer M R Characterization of trans po son 5469 from cyanobacterium Fremyella diplosiphon J 1777026-7032
Karavaiko G Golovacheva S Pivovarova T A Tzaplina I A and Vartanjan 1988 Thermophilic ltPM Sulfobacillus page 29-41 In Biohydrometallurgyshy87 Norris P and Science and Technology Letters Kew 1
Kennedy J and Humpphreys J D 1976 Microbial cells living immobilised on Nature 261242-244
Koonin 1993 SpoU protein of Escherichia coli belongs to a new family of Nucleic Acids Res 19
Kopecko J and site specific recA mdependtm recombination between bacterial Pt of palindromes at the N ad Acad Sci USA
on L-glutamine D-fructose ud()transtiera~e 1 BioI
152
Krumholz L R Esser U and Simoni RD 1989 Nucleotide sequence of the unc operon of Vibrio alginolyticus Nucleic Acids Res 177993-7994
Kubo K and Craig N 1990 Bacterial transposon Tn7 utilizes two classes of target sites 1 Bacteriol 1722774-2778
Kucharczyk N Denisot M A Le Goffic F and Badet B 1990 Glucosarnine-6-phosphate synthase from Ecoli detennination of the mechanism of inactivation by N3 fumaroyl-L-2-3 diarninoproprionic derivatives Biochemistry 293668-3676
Kusano M Takeshima T Inoue C and Sugawara K 1991 Evidence for two sets of structural genes coding for Ribulose biphosphate carboxylase in Thiobacillus ferrooxidans 1 Bacteriol 1737313-7323
Lane Dl A P Harrison lnr D Stahl B Pace SJ Giovannoni GJ Olsen and N R Pace 1992 Evolutionary relationship among sulphur- and iron-oxidizing eubacteria 1 Bacteriol 174267-278
Lee C H Bhagwhat A and Heffron F 1983 Identification of a transposon TnJ sequence required for transposition immunity Natl Acad Sci USA 806765-6769
Lewis M 1 Chang 1 A and Somoni R D 1990 A topological analysis of subunit a from Escherichia coli F1Fo-ATP synthase predicts eight transmembrane segments 1 BioI Chern 265 10541-10550
Lichtenstein C P and Brenner S 1982 Unique insertion site of Tn7 in the Ecoli chromosome Nature (London) 297601-603
Lichtenstein C P and Brenner S 1981 Site-specific properties of transposition to the Ecoli chromosome Mol Gen Genet 183380-387
Liu X Petersson S and Sandstrom A Mesophilic versus moderate thennophilic bioleaching Biohydrometallurgy Technologies Vol 1 A E Tonna 1 E Wey and Lakshmanan V 1 (ed) pp 29-38
Lizarna H M and Sankey B M 1993 Oxidation of H2S by Thiobacillus thiooxidans is inhibited by substrate and methane BiohydrometaHurgy Technologies Vol II A E Tonna 1 E Wey and C L Brierley (ed) pp 339-364
Lundgren D G and Silver M 1980 Ore leaching by bacteria Ann Rev Microbiol 34263shy268
Makino K Amemura M Shinagawa H Kobayashi A and Nakata A 1985 Sequence of the genes involved in phosphate transport and regulation of the phosphate regulon in Escherichia coli 1 Mol BioI 184231-240
Makino K Shinagawa Amemura M Kimura S Nakata A and Ishihama 1988 Euuv1J of the phosphate regulon of Ecoli Activation of pstS by the PhoB
protein in vitro 1 Mol BioI 20385-95
Makino K Shinagawa H Amemura M Yamada M and 1990 Signal transduction in the phosphate regulon of the Escherichia coli involves phosphotransfer nprlrJp~middotn PhoR and PhoB proteins J Mol BioI 21055
Malamy 1966 Frameshift mutations in the nnlnn of Escherichia coli Cold Harb Symp Quant BioI 31189-201
Malamy M H 1972 Electron microscopy insertions in the lac operon of Escherichia coli MoL Gen
Malamy M H and Bennett R L 1970 mutants of Ecoli and phosphate transport Biochem Biophys Res Commun
Maniatis T Fritsch EF Sambrook J 1982 nn A laboratory manual Cold Spring Harbor Laboratory Press Cold
McKnight G L S L Mudri SH Mathewest R S Marshall P O Sheppard and P J OHara 1990 Molecular cloning synthesis and bacterial expression of human glutaminefructose-6-phosphate UUAUU u J Chem 26725208-25212
Medveczky N and H Rosenburg 1970 phosphate-binding protein of Escherichia Biochim Biophys Acta 211 158-168
Mei B and Zalkin H 1989 A Cysteine-Histidine-Aspartate catalytic triad is involved in Glutamide Amide Transfer Function purF-type Glutamine amodotransferases 1 BioI Chem 264 16613-16619
Mengin-Lecreulx D and J van 1993 Identification of glmU gene encoding Nshyacetylglucosamine in Escherichia coli J Bacteriol 1756150shy6157
Michaelis M J and Criddle M J 1970 Mitochondrial DNA and mutants in Saccharomyces cerevisiae Biochem Genet 5487-495
Michaelis Starlinger P 1969 Two insertions in the jO v
operon having different homologous DNA sequences MoL Gen Genet 10437 377
Miller J H Calos Transposable elements Cell 20579-595
154
Miller J Oldenburg M and Fillingame R 1990 The essential carboxyl group of in subunit c the FIFo ATP synthase can be and H( +)-translocating function retained Proc NatL Acad 874900-4904
Mitcell P 1966 Chemiosmotic coupling in - and phosphorylation BioL Rev 41455-502 (1966)
Mizuuchi K 1984 Mechanism of transposition of bacteriophage Mu Polarity of the strand reaction at the initiation of the transposon Cell 39395-404
C Ph de Donato Berthelin J Surface oxidized species a key factor in the study of bioleaching processes Biohydrometallurgical In A J
Weyand V I Lakshmanan ed 1993 Vol 1 175-184
A Ishino Shinagawa H Makino K and M 1987 Nucleotide of the iap gene responsible for alkaline phosphatase isoenzyme conversion in Ecoli
identification of product 1 1695429-5433
K 1989 The tsr gene-coding prevents thiopeptin from inhibiting ppGpp synthesis in Streptomyces lividans FEMS Microbiol Lett
Ogasawara N and Yoshikawa H 1992 and their organIzatIon in the repnc()n of the bacterial chromosome Mol Microbiol 6(5)629-634
Ohtsubo Davidson N and 1975 microscope heteroduplex studies of sequence relations among plasmids identification and mapping of the insertion sequences 1 and IS2 in F and R plasmids 1 122746-775
Olson G J 1994 Microbial oxidation gold ores and gold bioleaching FEMS un Lett 119 1-6
Orle K A N L 1991 Identification of transposition prroteins by the bacterial transposon [corrected republished originally printed in Gene 1990 Nov 30 96(1) Gene 10425-31
Ouellette M Roy P Homology of ORFs from and from to site specific recombinases 1987 Nucl Acids 10055-10059
Murein synthesis p663-671ln Neidhardt J L Ingraham B Louw M~ M Schaechter H E Umbarger (ed) Escherichia coli and Salmonella
ryphimurium cellular and bioI voL 1 American Society Microbiology Washington
Perlin D S and Senior A Functional and cross-reactivity of antibody to purified subunit b (uncF protein) of Escherichia coli proton-ATPase Arch Biochem Biophys 236603-611
155
Plumbridge J A O Cochet Souza J M Altramirano M M Calcagno M L and Badet B 1993 Coordinated regulation of amino sugar-synthesizing and -degrading enzymes in Escherichia coli K-12 J Bacteriol 175(16)4951-4956
Pretorius I M Rawlings D E and Woods D R 1986 Identification and cloning of Thiobacillus jerrooxidans structural Nif genes in Escherichia coli Gene 4559-65
Qadri M I Flores C c Davis J and Lichtenstein C 1989 Genetic analysis of attTn7 the transposon Tn7 attachment site in Escherichia coli using a novel M13-based transduction assay 1 Mol BioI 20785-98
Radstrom P Skold 0 Swedberg J Roy P H and Sundstrom L 1994 Tn5090 of plasmid R751 which carries integron is related to Tn7 Mu and retroelements J Bacteriol 1763257-3268
Raetz C R H 1987 Structure and biosynthesis of lipid A in Escherichia coli pp 498-503 In F C Niedhardt J L Ingraham K B Low B Magasanik M Schaechter and H E Umbarger (ed) Escherichia coli and Salmonella typhimurium cellular and molecular biology vol 1 American Society for Microbiology Washington DC
Rao N N and Torriani A 1990 Molecular aspects of phosphate transport in Escherichia coli Mol Microbiol 4(7) 1083-1090
Rawlings DE D R Woods and NP Mjoli 1991 The cloning and structre of genes from the autotrophic biomining bacterium Thiobacillusjerrooxidans p 215-237 In PJ Greenaway (ed) Advances in gene technology vol 2 JAI Press London
Richet E and O Raibaud 1989 MalT the regulatory protein of the Escherichia coli maltose system is an A TP-dependent transcriptional activator EMBO 1 8981-987
Rogers M Ekaterrinaki N Nimmo E and Sherratt D 1986 Analysis of Tn7 transposition Mol Gen Genet 205550-556
Ronson C W Nixon B T Albright L M anf Ausubel F M 1987 Rhizobium meliloti ntrA (rpoN) gene is required for diverse metabolic functions J Bacteriol 1692424-2431
Rosenburg H Gerdes R G and Chegwidden K 1977 Two systems for the uptake of phosphate in Ecoli JBacterioL 131505-511
Rosenburg H 1987 In ion transport in Prokaryotes Rosen B P and Silver S (eds) New York Academic Press pp 205-248
Ross D G Swan 1 and N Klecker 1979 Physical structures of TnlO-promoted deletions and inversions role of 1400 base pairs inverted repetitions Cell 16721-731
156
Saedler H and P 19670 mutation in the galactose operon in Ecoli II Physiological characterization MoL Gen Genet 100 190-202
Saedler P 1968 00 and strong polar mutations in the Genet 102353-363
Sambrook J Fritsch Maniatis 1989 Molecular cloning a laboratory manual Cold Harbor Laboratory Cold Spring Harbor New York
Sand W Gehrke T Hallmann Rohde K Sobotke B and Wintzien S 1993 Bioleaching metal sulphides The importance of Leptospirillum ferrooxidans Biohydromemetallurgical Technologies Voll pp 15-25
Sanger Nicklen and Coulson A DNA sequencing with chain-tennination inhibitors Natl Acad USA 745463-5467
Schneider E and Altendorf K All the three subunits are required for an active proton channel (Fo) of Escherichia coli synthase (FIFo) ~-
518
Schneider E and Allendorf K 1987 Bacterial adenosine 5-triphosphate synthase purification and reconstitution complexes and biochemical and functional characterization of subunits Microbio Rev 51477-497
Schrader 1 A and D S Holmes 1988 Phenotypic switching Thiobacillus ferrooxidans J BacterioL 1703915-3023
Scordilis G E H and Lassie 1987 Identification of transposable elements which activates gene expression in Pseudomonas cepacia J Bacteriol 1698-13
Sekine Eisaki N and Ohtsubo E 1996 Identification and characterization of the lS3 molecules generated by breaks 1 BioL Chern 271197-202
Senior A E 1990 The proton-trans locating of Escherichia coli Annu Rev Biophys Chern 194-41
Shapiro 1 and Adhya S 1969 The jLU operon of K-12 n A deletion analysis of the structure polarity 62249-264
J A 1969 Mutations caused by insertion genenc material the galactose operon Escherichia 1 MoL BioI 4093-105
Silvennan M P 1967 Mechanism of bacterial pyrite oxidation 1 Bacteriol 941046-1051
157
C C ElIson and Levinson 1983 Identification of the typel trimethoprim resistance reductase specified by the R-plasmid R43 comparison with procaryotic and eucaryotic dihydrofolate reductase 1 Bacteriol 155 100 1-1008
Smith and Jones P transposition a multigene process Identification of a regulatory product 147915-7927
Sprague Jr Bell R M Cronan 1 A mutant of Ecoli auxotrophic for organic phosphates evidence two defects in phosphate Mol Gen Genet 14371-77
Starlinger and Michaelis 1968 Suppression the sequential aO[)ealame of the galactose enzymes in a transferase amber mutant coli Mol Genet 102367-369
Starlinger 1977 IS elements in microorganisms MicrobioL Immunol
Steffens K Schneider E Herkernhoff B Schmid Altendorf K portion of Escherichia coli A TP synthase resolution of from subunit b J BioI Chern 2625866-5869
Steffens A Deckers-hebestreit G and Altendorf K 1987b The and functional relationship of A TP synthases (FoFI) from Ecoii and the thermophilic bacterium PS3 J Biol 2626334-6338
Strominger J and M S Smith 1959 Uridine diphosphoacetylglucosamine pyrophosphorylase 1 BioI Chern 2341822-1827
Sundstrom Skold 1990 dhfrI trimethoprim resistance gene of can be found at in other genetic surroundings Antimicrob Agents Chemother 34642shy650
Sundstrom L Roy P H and Skold Site-specific of three cassettes in Tn7 J Bacteriol 1733025-3028
Surin B P and Downie JA 1988 of the Rhizobium leguminosarum nodLMN involved efficient host-specific nodulation Mol Microbiol 2 173-183
B P Dixon N and Rosenburg 1986 Purification PhoU protein a OClItnl
regulator of the pho regulon of Ecoli K-1 J Bacteriol 168631
B P D A Jans A L Fimmel Shaw GB Cox Rosenburg Structural gene for the phosphate-repressible phosphate binding of Escherichia coli
own promoter nucleotide of the phoS 1 Bacteriol
158
Suzuki I Chang W and Takeuchi 1994 Oxidation of organic compounds by Thiobacilli Symp Series 55060-67
Takeyama M S Y Noumi T Maeda Ishibashi S and Futai M 1988 Beta subunit of Ecoli amino acid replacement within a conserved sequence G-X-X-X-X-GshyK-TS) v~u binding proteins lett 218222-226
Thomson JA M Hendson and R M 1981 Mutagenesis by insertion of drug resistance Tn7 into a vibrio 11 J Bacteriol
Tichy R Grotenhuis J T c Janssen van Houten R Rulkens and Lettinga 1993 Application of the sulphur cycle bioremediation of soils polluted with heavy metals In Int Conf Contaminated 93 ed F Arendt G J Annokee R Bosman and W J van der Brink Kluwer Academic Publishers Dordrecht pp 1461-1462
G and Mayer An electron approach to the quaternary structure of mitochondrial Eur J Biochem 13237-45
J and Tschape streptothricin resistance transposons Tn1825 and Tn1826 and transposon Tn 7 Plasmidnuu
18246-249
Tso J Y Zalkin H van Cleemput M Yanofsky C and JM 1982 Nucleotide of Escherichia coli and deduced amino glutamine
phosphribosylpyrophosphate am idotransferase J BioI Chern
V Mesyanzhinova I V Koslov I A and Orlova 1984 Structure of studied by electron and image processing Lett 167285-289
P Barber C and transposons Tn5 and Tn7 in Xanthomonas campestris pv campestris MoL Gen 157
Ullrich J and van Putten P Identification of the Gonococcal glmU gene UVUUIJ
the enzyme N-acetylglucosamine 1-Phosphate Uridyltransferase involved in the synthesis UDP-GlcNAc J of Bact 1776902-6909
M 1992 Eight bacterial proteins includin]g UDP-N -acetylglucosamine acyltransferase three vother of Escherichia coli of a six-residue n tVI
theme FEMS MicrobioL 97249-254
van der Steen J J D Doddema J and de Uidoging van zware metalen afvalstromen met behulp van thiobacilli (Removal metals from waste streams
using thiobacilli) Ministry of Housing Physical and Enviromental (VROM) Directoraat-Generaal Milieubeheer Rapport 199210 Hague
Vermoote P 1988 Universite Paris VI Paris Hrllnro
159
Vignais P V Lunard Issartel J P and Dupuis A 1985 Interaction n l1f middotn
oligomycin sensitivity protein (OSCP) beef heart mitochondrial FeATPase 2 Identification of interacting Fl subunits cross-linking Biochem J
Vik S and Dao NN Prediction of transmembrane topology of Fo proteins from Ecoli ATP synthase variational hydrophobic moment analyses Biochim Biophys Acta 1140 199-207
von Meyenburg B B Jcentrgensen J Nielsen and F Hansen 1982 Promoters of the atp operon for the membrane-bound ATP synthase of Ecoli mapped by TnlO insertion mutations MoL Gen Genet 188240-248
von Meyenberg F G Hansen 1980 The origin of replication oriC the Ecoli chromosome near oriC and construction of oriC mutants ICN-UCLA Symp MoLCellBiol 191 159
Waddell S and Craig N 1989 Tn7 transposition recognition of the attTn7 Proc Natl Sci USA 863958-3962
Waddell C S and Craig N L 1988 transposition two transposition pathways directed by five Tn7-encoded genes Develop 21
J E Gay N J Saraste M and A N 1984 DNA sequence the Escherichia coli unc operon Completion of the sequence of a kilobase segment containing
oriC unc and phoS J224799-815
and Collinson 1 1994 the role the stalk in the coupling mechanism of FEBS 34639-43
Walker 1 E Gay N J and Tybulewicz V L 1986 of transposon Tn7 into the Escherichia glmS transcriptional terminator Biochem 1 243 111-1
Wanner Land McSharry 1982 Phosphate-controlled gene in Escherichia coli using Mudl-directed lacZ fusions J Mol BioI 158347-363
Weng M and Zalkin H 1987 Structural role for a region the CTP synthetase glutamide amide transfer domain J Bacteriol 1693023-3028
Wheatcroft R and S and nucleotide sequence of Rhizobiwn meliloti insertion between the transposase encoded by ISRm3 and those encoded by Staphylococcus aureus and Thiobacillus ferrooxidans IST2 J 1732530-2538
160
Whitby M C and Lloyd R 1995 Branch of three-strand recombination intennediates by RecG a possible pathway for securing middotIULl initiated by duplex DNA 1 143303-3310
Wilkens and Capaldi R A Assymmetry and changes in examined by cryoelectronmicroscopy BioI Chern Hoppe-Seyler 37543-51
and Malamy M 1974 The loss of the PhoS periplasmic protein leads to a the specificity a constitutive phosphate transport system in Escherichia coli Biochem Res Commun 60226-233
WiUsky G Rand Malamy M 1980 of two separable inorganic phosphate transport systems in Escherichia coli J BacterioL 144356-365
P J and and C F 1973 enzyme of metabolism and Stadtman R eds) pp 343-363 Academic Press New York
Winterbum P J and Phelps F 197L control of hexosamine biosynthesis by glucosamine synthetase Biochem 1 121711
Wolf-Watz H and Norquist A 1979 Deoxyribonucleic acid and outer membrane protein to outer membrane protein involves a protein J 14043-49
Wolf-Watz H and M 1979 Deoxyribonucleic acid and outer membrane strains for oriC elevated levels deoxyribonucleic acid-binding protein and
11 for specific UU6 of the oriC region to outer membrane J Bacteriol 14050-58
Wolf-Watz H 1984 Affinity of two different chromosome to the outer membrane of Ecoli J Bacteriol 157968-970
Wu C and Te Wu 197 L Isolation and characterization of a glucosamine-requiring mutant of Escherichia 12 defective in glucoamine-6-phosphate synthetase 1 Bacteriol 105455-466
Yamada M Makino Shinagawa Nakata A 1990 Regulation of the phosphate regulon of Escherichia coli properties the pooR mutants and subcellular localization of protein Mol Genet 220366-372
Yates J R and Holmes D S 1987 Two families of repeatcXl DNA sequences Thiobacillus 1 Bacteriol 169 1861-1870
Yates J R R P and D S 1988 an insertion sequence from Thiobacillus errooxidans Proc Natl 857284-7287
161
Youvan D c Elder 1 T Sandlin D E Zsebo K Alder D P Panopoulos N 1 Marrs B L and Hearst 1 E 1982 R-prime site-directed transposon Tn7 mutagenesis of the photosynthetic apparatus in Rhodopseudomonas capsulata 1 Mol BioI 162 17-41
Zalkin H and Mei B 1990 Amino terminal deletions define a glutamide transfer domain in glutamine phosphoribosylpyrophosphate ami do transferase and other purF-type amidotransferases 1 Bacteriol 1723512-3514
Zalkin H and Weng M L 1987 Structural role for a conserved region III the CTP synthetase glutamide amide transfer domain 1 Bacteriol 169 (7)3023-3028
Zhang Y and Fillingame R H 1994 Essential aspartate in subunit c of FIF0 ATP synthase Effect of position 61 substitutions in helix-2 on function of Asp24 in helix-I 1 BioI Chern 2695473-5479
iv
LIST OF TABLES
Table 21 Source and media of iron-sulphur oxidizing bacteria used 44
Table 22 Contructs and subclones of pS1S1 45
Table 31 Contructs and subclones used to study
the complementation of Ecoli CGSC 5392 79
v
ABSTRACT
A Tn7-like element was found in a region downstream of a cosmid (p8181) isolated from a
genomic library of Thiobacillus ferrooxidans ATCC 33020 A probe made from the Tn7-like
element hybridized to restriction fragments of identical size from both cosmid p8181 and
T ferrooxidans chromosomal DNA The same probe hybridized to restricted chromosomal
DNA from two other T ferrooxidans strains (ATCC 23270 and 19859) There were no positive
signals when an attempt was made to hybridize the probe to chromosomal DNA from two
Thiobacillus thiooxidans strains (ATCC 19733 and DSM504) and a Leptospirillum
ferrooxidans strain DSM 2705
A 35 kb BamHI-BamHI fragment was subcloned from p8181 downstream the T ferrooxidans
unc operon and sequenced in both directions One partial open reading frame (ORFl) and two
complete open reading frames (ORF2 and ORF3) were found On the basis of high homology
to previously published sequences ORFI was found to be the C-terminus of the
T ferrooxidans glmU gene encoding the enzyme GlcNAc I-P uridyltransferase (EC 17723)
The ORF2 was identified as the T ferrooxidans glmS gene encoding the amidotransferase
glucosamine synthetase (EC 26116) The third open reading frame (ORF3) was found to
have very good amino acid sequence homology to TnsA of transposon Tn7 Inverted repeats
very similar to the imperfect inverted repeat sequences of Tn7 were found upstream of ORF3
The cloned T ferrooxidans glmS gene was successfully used to complement an Ecoli glmS
mutant CGSC 5392 when placed behind a vector promoter but was otherwise not expressed
in Ecoli
Subc10ning and single strand sequencing of DNA fragments covering a region of about 7 kb
beyond the 35 kb BamHI-BamHI fragment were carried out and the sequences searched
against the GenBank and EMBL databases Sequences homologous to the TnsBCD proteins
of Tn7 were found The TnsD-like protein of the Tn7-like element (registered as Tn5468) was
found to be shuffled truncated and rearranged Homologous sequence to the TnsE and the
antibiotic resistance markers of Tn7 were not found Instead single strand sequencing of a
further 35 kb revealed sequences which suggested the T ferrooxidans spo operon had been
encountered High amino acid sequence homology to two of the four genes of the spo operon
from Ecoli and Hinjluenzae namely spoT encoding guanosine-35-bis (diphosphate) 3shy
pyrophosphohydrolase (EC 3172) and recG encoding ATP-dependent DNA helicase RecG
(EC 361) was found This suggests that Tn5468 is incomplete and appears to terminate with
the reshuffled TnsD-like protein The orientation of the spoT and recG genes with respect to
each other was found to be different in T ferrooxidans compared to those of Ecoli and
Hinjluenzae
11 Bioleaching 1
112 Organism-substrate raction 1
113 Leaching reactions 2
114 Industrial applicat 3
12 Thiobacillus ferrooxidans 4
13 Region around the Ecoli unc operon 5
131 Region immediately Ie of the unc operon 5
132 The unc operon 8
1321 Fe subun 8
1322 subun 10
133 Region proximate right of the unc operon (EcoURF 1) 13
1331 Metabolic link between glmU and glmS 14
134 Glucosamine synthetase (GlmS) 15
135 The pho 18
1351 Phosphate uptake in Ecoli 18
1352 The Pst system 19
1353 Pst operon 20
1354 Modulation of Pho uptake 21
136 Transposable elements 25
1361 Transposons and Insertion sequences (IS) 27
1362 Tn7 27
13621 Insertion of Tn7 into Ecoli chromosome 29
13622 The Tn7 transposition mechanism 32
137 Region downstream unc operons
of Ecoli and Tferrooxidans 35
LIST OF FIGURES
Fig
Fig
Fig
Fig
Fig
Fig
Fig
Fig
la
Ib
Ie
Id
Ie
If
Ig
Ih
6
11
14
22
23
28
30
33
1
CHAPTER ONE
INTRODUCTION
11 BIOLEACHING
The elements iron and sulphur circulate in the biosphere through specific paths from the
environment to organism and back to the environment Certain paths involve only
microorganisms and it is here that biological reactions of relevance in leaching of metals from
mineral ores occur (Sand et al 1993 Liu et aI 1993) These organisms have evolved an
unusual mode of existence and it is known that their oxidative reactions have assisted
mankind over the centuries Of major importance are the biological oxidation of iron
elemental sulphur and mineral sulphides Metals can be dissolved from insoluble minerals
directly by the metabolism of these microorganisms or indrectly by the products of their
metabolism Many metals may be leached from the corresponding sulphides and it is this
process that has been utilized in the commercial leaching operations using microorganisms
112 Or2anismSubstrate interaction
Some microorganisms are capable of direct oxidative attack on mineral sulphides Scanning
electron micrographs have revealed that numerous bacteria attach themselves to the surface
of sulphide minerals in solution when supplemented with nutrients (Benneth and Tributsch
1978) Direct observation has indicated that bacteria dissolve a sulphide surface of the crystal
by means of cell contact (Buckley and Woods 1987) Microbial cells have also been shown
to attach to metal hydroxides (Kennedy et ai 1976) Silverman (1967) concluded that at
least two roles were performed by the bacteria in the solubilization of minerals One role
involved the ferric-ferrous cycle (indirect mechanism) whereas the other involved the physical
2
contact of the microrganism with the insoluble sulfide crystals and was independent of the
the ferric-ferrous cycle Insoluble sulphide minerals can be degraded by microrganisms in the
absence of ferric iron under conditions that preclude any likely involvement of a ferrous-ferric
cycle (Lizama and Sackey 1993) Although many aspects of the direct attack by bacteria on
mineral sulphides remain unknown it is apparent that specific iron and sulphide oxidizers
must play a part (Mustin et aI 1993 Suzuki et al 1994) Microbial involvement is
influenced by the chemical nature of both the aqueous and solid crystal phases (Mustin et al
1993) The extent of surface corrosion varies from crystal to crystal and is related to the
orientation of the mineral (Benneth and Tributsch 1978 Claassen 1993)
113 Leachinamp reactions
Attachment of the leaching bacteria to surfaces of pyrite (FeS2) and chalcopyrite (CuFeS2) is
pSIS1 construct was further subcloned to plasm ids IS30 IS40 pSlS50
pS183S p81841 and p81 (Fig Some of subclones were extensively mapped
although not all sites are shown in 22 exact positions of the ends of
T jerrooxidans plasmids pTfatpl and pTfatp2 on the cosmid p8I81 were identified
21)
Tfeooxidans
that the cosmid was
unrearranged DNA digests of cosmid pSISI and T jerrooxidans chromosomal DNA were
with pSI The of the bands gave a positive hybridization signal were
identical each of the three different restriction enzyme digests of p8I8l and chromosomal
In order to confirm of pSI81 and to
49
kb kb ~
(A)
11g 50Sts5
middot 2S4
17 bull tU (probe)
- tosect 0S1
(e)
kb
+- 10 kb
- 46 kb
28---+ 26-+
17---
Fig 23 Hybrdization of cosmid pSISI and T jerrooxitians (A TCC 33020) chromosomal
DNA by the KpnI-SalI fragment of pSIS52 (probe) (a) Autoradiographic image of the
restriction digests Lane x contains the probe lane 13 and 5 contain pSISI restricted with BamHl HindIII and Bgffi respectively Lanes 2 4 and 6 contain T errooxitians chromosomal DNA also restricted with same enzymes in similar order (b) The sizes of restricted pSISI
and T jerrooxitians chromosome hybridized by the probe The lanes correspond to those in Fig 23a A DNA digested with Pst served as the molecular weight nlUkermiddot
50
DNA (Fig BamHI digests SHklC at 26 and 2S kb 1 2) HindIII
at 10 kb 3 and 4) and BgnI at 46 (lanes 5 and 6) The signals in
the 181 was because the purified KpnI-San probe from p81 had a small quantity
ofcontaminating vector DNA which has regions ofhomology to the cosmid
vector The observation that the band sizes of hybridizing
for both pS181 and the T ferrooxidans ATCC 33020 same
digest that the region from BgnI site at to HindIII site at 16
kb on -VJjUIU 1 represents unrearranged chromosomal DNA from T ferrooxidans
ATCC there were no hybridization signals in to those predicted
the map with the 2 4 and 6 one can that are no
multiple copies of the probe (a Tn7-like segment) in chromosome of
and Lferrooxidans
of plasmid pSI was found to fall entirely within a Tn7-like trallSDOS()n nrpn
on chromosome 33020 4) A Southern hybridizationUUAcpoundUU
was out to determine whether Tn7-like element is on other
ofT ferrooxidans of T and Lferrooxidans results of this
experiment are shown 24 Lanes D E and F 24 represent strains
19377 DSM and Lferrooxidans DSM 2705 digested to
with BgnI enzyme A hybridization was obtained for
the three strains (lane A) ATCC 19859 (lane B) and UUHUltn
51
(A) AAB CD E F kb
140
115 shy
284 -244 = 199 -
_ I
116 -109 -
08
os -
(8)) A B c o E F
46 kb -----
Fig 24 (a) Autoradiographic image of chromosomal DNA of T ferrooxidans strains ATCC 33020 19859 23270 (lanes A B C) Tthiooxidans strains ATCC 19377 and DSM 504 (D and E) and Lferrooxidans strain DSM 2705 (lane F) all restricted with Bgffi A Pst molecular weight marker was used for sizing (b) Hybridization of the 17 kb KpnI-San piece of p81852 (probe) to the chromosomal DNA of the organisms mentioned 1 Fig 24a The lanes in Fig 24a correspond to those in Fig 24b
(lane C) uuvu (Fig 24) The same size BgllI fragments (46 kb) were
lanes A Band C This result
different countries and grown on two different
they Tn7-like transposon in apparently the same location
no hybridization signal was obtained for T thiooxidans
1 504 or Lferrooxidans DSM 2705 (lanes D E and F of
T thiooxidans have been found to be very closely related based on 1
rRNA et 1992) Since all Tferrooxidans and no Tthiooxidans
~~AA~~ have it implies either T ferrooxidans and
diverged before Tn7-like transposon or Tthiooxidans does not have
an attTn7 attachment the Tn7-like element Though strains
T ferrooxidans were 10catH)uS as apart as the USA and Japan
it is difficult to estimate acquired the Tn7-like transposon as
bacteria get around the world are horizontally transmitted It
remains to be is a general property
of all Lferrooxidans T ferrooxidans strains
harbour this Tn7-like element their
CHAPTER 3
5431 Summary
54Introduction
56Materials and methods
56L Bacterial and plasmid vectors
Media and solutions
56333 DNA
57334 gel electrophoresis
Competent preparation
57336 Transformation of DNA into
58337 Recombinant DNA techniques
58ExonucleaseIII shortening
339 DNA sequencing
633310 Complementation studies
64Results discussion
1 DNA analysis
64Analysis of ORF-l
72343 Glucosamine gene
77344 Complementation of by T ferrooxidans glmS
57cosmid pSIS1 pSlS20
58Plasmidmap of p81816r
Fig Plasmidmap of lS16f
60Fig 34 Restriction digest p81S1Sr and pSIS16f with
63DNA kb BamHI-BamHI
Fig Codon and bias plot of the DNA sequence of pSlS16
Fig 37 Alignment of C-terminal sequences of and Bsubtilis
38 Alignment of amino sequences organisms with high
homology to that of T ferrooxidans 71
Fig Phylogenetic relationship organisms high glmS
sequence homology to Tferrooxidans
31 Summary
A 35 kb BamHI-BamHI p81820 was cloned into pUCBM20 and pUCBM21 to
produce constructs p818 and p81816r respectively This was completely
sequenced both rrelct1()ns and shown to cover the entire gmS (184 kb) Fig 31
and 34 The derived sequence of the T jerrooxidans synthetase was
compared to similar nrvnC ~ other organisms and found to have high sequence
homology was to the glucosamine the best studied
eubacterium EcoU Both constructs p81816f p81 the entire
glmS gene of T jerrooxidans also complemented an Ecoli glmS mutant for growth on medium
lacking N-acetyl glucosamine
32 =-====
Cell wall is vital to every microorganism In most procaryotes this process starts
with amino which are made from rructClsemiddotmiddotn-[)ll()S by the transfer of amide group
to a hexose sugar to form an This reaction is by
glucosamine synthetase the product of the gmS part of the catalysis
to the 40-residue N-terminal glutamine-binding domain (Denisot et al 1991)
participation of Cys 1 to generate a glutamyl thiol ester and nascent ammonia (Buchanan
368-residue C-terminal domain is responsible for the second part of the ClUUU
glucosamine 6-phosphate This been shown to require the ltgtttrltgtt1iltn
of Clpro-R hydrogen of a putative rrulctoseam 6-phosphate to fonn a cis-enolamine
intennediate which upon reprotonation to the gives rise to the product (Golinelli-
Pimpaneau et al 1989)
bacterial enzyme mn two can be separated by limited chymotryptic
proteolysis (Denisot et 199 binding domain encompassing residues 1 to
240 has the same capacity to hydrolyse (and the corresponding p-nitroanilide
derivative) into glutamate as amino acid porI11
binding domain is highly cOllserveu among members of the F-type
ases Enzymes in this include amidophosphophoribosyl et al
1982) asparagine synthetase (Andrulis et al 1987) glucosamine-6-P synmellase (Walker et
aI 1984) and the NodM protein of Rhizobium leguminosarum (Surin and 1988) The
368-residue carboxyl-tenninal domain retains the ability to bind fructose-6-phosphate
The complete DNA sequence of T ferrooxidans glmS a third of the
glmU gene (preceding glmS) sequences downstream of glmS are reported in this
chapter This contains a comparison of the VVA r glmS gene
and Mycobacterium (422 ) An interesting observation was that the T ferrooxidans
glmS gene had comparatively high homology sequences to the Rhizobium leguminosarum and
Rhizobium meliloti M the amino to was 44 and 436
respectively A consensus Dalgarno upstream of the start codon
(ATG) is in 35 No Ecoli a70-type n r~ consensus sequence was
detected in the bp preceding the start codon on intergenic distance
between the two and the absence of any promoter consensus sequence Plumbridge et
al (1993) that the Ecoli glmU and glmS were co-transcribed In the case
the glmU-glmS --n the T errooxidans the to be similar The sequences
homology to six other amidotransferases (Fig 38) which are
(named after the purF-encoded l phosphoribosylpyrophosphate
amidotransferase) and Weng (1987)
The glutamine amide transfer domain of approximately 194 amino acid residues is at the
of protein chain Zalkin and Mei (1989) using site-directed to
the 9 invariant amino acids in the glutamine amide transfer domain of
phosphoribosylpyrophosphated indicated in their a
catalytic triad is involved glutamine amide transfer function of
73
Comparison of the ammo acid sequence alignment of ghtcosamine-6-phosphate
amidotranferases (GFAT) of Rhizobium meliloti (R_m) Rhizobium leguminnosarum (RJ)
Ecoli (E_c) Hinjluenzae Mycobacterium leprae (M_I) Bsubtilis (B_s) and
Scerevisiae (S_c) to that of Tferrooxidans Amino acids are identified by their single
codes The asterisks () represent homologous amino acids of Tferrooxidans glmS to
at least two of the other Consensus ammo acids to all eight organisms are
highlighted (bold and underlined)
1 50 R m i bullbullbullbullbullbull hkps riag s tf bullbullbullbull R~l i bullbullbullbullbullbull hqps rvaep trod bullbullbullbull a
a bullbullbullbullbullbull aa 1r 1 d bullbullbull a a bullbullbullbullbullbull aa inh g bullbullbullbullbullbull sktIv pmilq 1 1g bullbullbull a MCGIVi V D Gli RI IYRGYPSAi AI D 1 gvmar s 1gsaks Ikek a bullbullbullbull qfcny Iversrgi tvq t dgd bullbull ead
51 100 R m hrae 19ktrIk bull bullbull bullbull s t ateR-l tqrae 19rkIk bullbullbull s t atrE-c htIrI qmaqae bull bull h bullbull h gt sv aae in qicI kads stbull bullbull k bullbull i gt T f- Ivsvr aetav bullbullbull r bullbull gq qg c
DL R R G V L AV E L G VGI lTRW E H ntvrrar Isesv1a mv bull sa nIgi rtr gihvfkekr adrvd bullbullbull bullbull nve aka g sy1stfiykqi saketk qnnrdvtv she reqv
101 150 R m ft g skka aevtkqt R 1 ft g ak agaqt E-c v hv hpr krtv aae in sg tf hrI ksrvlq T f- m i h q harah eatt
S E Eamp L A GY l SE TDTEVIAHL ratg eiavv av
qsaIg lqdvk vqv cqrs inkke cky
151 200 bullbull ltk bullbull dgcm hmcve fe a v n bull Iak bullbull dgrrm hmrvk fe st bull bull nweI bullbull kqgtr Iripqr gtvrh 1 s bull bull ewem bullbullbull tds kkvqt gvrh hlvs bull bull hh bullbull at rrvgdr iisg evm
V YR T DLF A A L AV S D PETV AR G M 1 eagvgs lvrrqh tvfana gvrs B s tef rkttIks iInn rvknk 5 c Ihlyntnlqn ~lt klvlees glckschy neitk
74
201 R m ph middot middot middot R 1 ah- middot middot middot middot middot middot E c 1 middot middot middot middot middot middot middot Hae in 1 middot middot middot middot middot T f - c11v middot middotmiddot middot middot middot middot middot M 1 tli middot middot middot middot middot middot middot middot middot B s 11 middot middot middot middot middot S c lvkse kk1kvdfvdv
251 R m middot middot middot middot middot middot middot middot middot middot middot middot middot middot
middot middot middot middot R-1 middot middot middot middot middot middot E c middot middot middot middot middot middot middot middot Hae in middot middot middot middot middot middot middot middot middot middot middot middot T f shy middot middot middot middot middot middot middot middot middot middot middot middot 1M middot middot middot middot middot middot middot middot middot middot middot middot middot middot B s middot middot middot middot middot middot middot middot middot middot middot middot middot middot middot S c feagsqnan1 1piaanefn1
301 R m fndtn wgkt R-1 fneti wgkt E c rf er Hae in srf er T f- rl mlqq
PVTR V YE DGDVA R M 1 ehqaeg qdqavad B s qneyem kmvdd S c khkkl dlhydg
351 R m nhpe v R-1 nhe v E c i nk Hae in k tli T f- lp hr
~ YRHlrM~ ~I EQP AVA M 1 geyl av B-s tpl tdvvrnr S-c pd stf
401 Rm slasa lk R-1 slasca lk E c hql nssr Hae in hq nar-T f f1s hytrq
RVL A SiIS A VG W M 1 ka slakt B s g kqS c lim scatrai
T ferrooxidans (Tf) Scerevisiae (Saee) Mleprae (Myele) and Bsublilis (Baesu)
tr1 ()-ltashyc 8 ~ er (11
-l l
CIgt
~ r (11-CIgt (11
-
$ -0 0shy
a c
omiddot s
S 0 0shyC iii omiddot $
ttl ~ () tI)
I-ltashyt ()
0
~ smiddot (11
81
CHAPTER 4
8341 Summary
8442 Introduction
8643 Materials and methods
431 Bacterial strains and plasmids 86
432 DNA constructs subclones and shortenings 86
864321 Contruct p81852
874322 Construct p81809
874323 Plasmid p8I809 and its shortenings
874324 p8I8II
8844 Results and discussion
441 Analysis of the sequences downstream the glmS gene 88
442 TnsBC homology 96
99443 Homology to the TnsD protein
118444 Analysis of DNA downstream of the region with TnsD homology
LIST OF FIGURES
87Fig 41 Alignment of Tn7-like sequence of T jerrooxidans to Ecoli Tn7
Fig 42 DNA sequences at the proximal end of Tn5468 and Ecoli glmS gene 88
Fig 43 Comparison of the inverted repeats of Tn7 and Tn5468 89
Fig 44 BLAST results of the sequence downtream of T jerrooxidans glmS 90
Fig 45 Alignment of the amino acid sequence of Tn5468 TnsA-like protein 92 to TnsA of Tn7
82
Fig 46 Restriction map of the region of cosmid p8181 with amino acid 95 sequence homology to TnsABC and part of TnsD of Tn7
Fig 47 Restriction map of cosmid p8181Llli with its subclonesmiddot and 96 shortenings
98 Fig 48 BLAST results and DNA sequence of p81852r
100 Fig 49 BLAST results and DNA sequence of p81852f
102 Fig 410 BLAST results and DNA sequence of p81809
Fig 411 Diagrammatic representation of the rearrangement of Tn5468 TnsDshy 106 like protein
107Fig 412 Restriction digests on p818lLlli
108 Fig 413 BLAST results and DNA sequence of p818lOEM
109 Fig 414 BLAST results on joined sequences of p818104 and p818103
111 Fig 415 BLAST results and DNA sequence of p818102
112 Fig 416 BLAST results and nucleotide sequence of p818101
Fig 417 BLAST results and DNA sequence of the combined sequence of 113p818l1f and p81810r
Fig 418 Results of the BLAST search on p81811r and the DNA sequence 117obtained from p81811r
Fig 419 Comparison of the spa operons of Ecali Hinjluenzae and 121 probable structure of T ferraaxidans spa operon
83
CHAPTER FOUR
TN7-LlKE TRANSPOSON OF TFERROOXIDANS
41 Summary
Various constructs and subclones including exonuclease ill shortenings were produced to gain
access to DNA fragments downstream of the T ferrooxidans glmS gene (Chapter 3) and
sequenced Homology searches were performed against sequences in GenBank and EMBL
databases in an attempt to find out how much of a Tn7-like transposon and its antibiotic
markers were present in the T ferrooxidans chromosome (downstream the glmS gene)
Sequences with high homology to the TnsA TnsB TnsC and TnsD proteins of Tn7 were
found covering a region of about 7 kb from the T ferrooxidans glmS gene
Further sequencing (plusmn 45 kb) beyond where TnsD protein homology had been found
revealed no sequences homologous to TnsE protein of Tn7 nor to any of the antibiotic
resistance markers associated with Tn7 However DNA sequences very homologous to Ecoli
ATP-dependentDNA helicase (RecG) protein (EC 361) and guanosine-35-bis (diphosphate)
3-pyrophosphohydrolase (EC 3172) stringent response protein of Ecoli HinJluenzae and
Vibrio sp were found about 15 kb and 4 kb respectively downstream of where homology to
the TnsD protein had been detected
84
42 Transposable insertion sequences in Thiobacillus ferrooxidizns
The presence of two families (family 1 and 2) of repetitive DNA sequences in the genome
of T ferrooxidans has been described previously (Yates and Holmes 1987) One member of
the family was shown to be a 15 kb insertion sequence (IST2) containing open reading
frames (ORFs) (Yates et al 1988) Sequence comparisons have shown that the putative
transposase encoded by IST2 has homology with the proteins encoded by IS256 and ISRm3
present in Staphylococcus aureus and Rhizobium meliloti respectively (Wheatcroft and
Laberge 1991) Restriction enzyme analysis and Southern hybridization of the genome of
T ferrooxidans is consistent with the concept that IST2 can transpose within Tferrooxidans
(Holmes and Haq 1990) Additionally it has been suggested that transposition of family 1
insertion sequences (lST1) might be involved in the phenotypic switching between iron and
sulphur oxidizing modes of growth including the reversible loss of the capacity of
T ferrooxidans to oxidise iron (Schrader and Holmes 1988)
The DNA sequence of IST2 has been determined and exhibits structural features of a typical
insertion sequence such as target-site duplications ORFs and imperfectly matched inverted
repeats (Yates et al 1988) A transposon-like element Tn5467 has been detected in
T ferrooxidans plasmid pTF-FC2 (Rawlings et al 1995) This transposon-like element is
bordered by 38 bp inverted repeat sequences which has sequence identity in 37 of 38 and in
38 of 38 to the tnpA distal and tnpA proximal inverted repeats of Tn21 respectively
Additionally Kusano et al (1991) showed that of the five potential ORFs containing merR
genes in T ferrooxidans strain E-15 ORFs 1 to 3 had significant homology to TnsA from
transposon Tn7
85
Analysis of the sequence at the tenninus of the 35 kb BamHI-BamHI fragment of p81820
revealed an ORF with very high homology to TnsA protein of the transposon Tn7 (Fig 44)
Further studies were carried out to detennine how much of the Tn7 transposition genes were
present in the region further downstream of the T ferroxidans glmS gene Tn7 possesses
trimethoprim streptomycin spectinomycin and streptothricin antibiotic resistance markers
Since T ferrooxidans is not exposed to a hospital environment it was of particular interest to
find out whether similar genes were present in the Tn7-like transposon In the Ecoli
chromosome the Tn7 insertion occurs between the glmS and the pho genes (pstS pstC pstA
pstB and phoU) It was also of interest to detennine whether atp-glm-pst operon order holds
for the T ferrooxidans chromosome It was these questions that motivated the study of this
region of the chromosome
43 OJII
strains and plasm ids used were the same as in Chapter Media and solutions (as
in Chapter 3) can in Appendix Plasmid DNA preparations agarose gel
electrophoresis competent cell preparations transformations and
were all carned out as Chapter 3 Probe preparation Southern blotting hybridization and
detection were as in Chapter 2 The same procedures of nucleotide
DNA sequence described 2 and 3 were
4321 ~lllu~~~
Plasmid p8I852 is a KpnI-SalI subclone p8I820 in the IngtUMnt vector kb) and
was described Chapter 2 where it was used to prepare the probe for Southern hybridization
DNA sequencing was from both (p81852r) and from the Sail sites
(p8I852f)
4322 ==---==
Construct p81809 was made by p8I820 The resulting fragment
(about 42 kb) was then ligated to a Bluescript vector KS+ with the same
enzymes) DNA sequencing was out from end of the construct fA
87
4323 ~mlJtLru1lbJmalpoundsectM~lllgsect
The 40 kb EeoRI-ClaI fragment p8181Llli into Bluescript was called p8181O
Exonuclease III shortening of the fragment was based on the method of Heinikoff (1984) The
vector was protected with and ClaI was as the susceptible for exonuclease III
Another construct p818IOEM was by digesting p81810 and pUCBM20 with MluI and
EeoRI and ligating the approximately 1 kb fragment to the vector
4324 p81811
A 25 ApaI-ClaI digest of p8181Llli was cloned into Bluescript vector KS+ and
sequenced both ends
88
44 Results and discussion
of glmS gene termini (approximately 170
downstream) between
comparison of the nucleotide
coli is shown in Fig 41 This region
site of Tn7 insertion within chromosome of E coli and the imperfect gtpound1
repeat sequences of was marked homology at both the andVI
gene but this homology decreased substantially
beyond the stop codon
amino acid sequence
been associated with duplication at
the insertion at attTn7 by (Lichtenstein and as
CCGGG in and underlined in Figs41
CCGGG and are almost equidistant from their respelt~tle
codons The and the Tn7-like transposon BU- there is
some homology transposons in the regions which includes
repeats
11 inverted
appears to be random thereafter 1) The inverted
repeats of to the inverted repeats of element of
(Fig 43) eight repeats
(four traJrlsposcm was registered with Stanford University
California as
A search on the (GenBank and EMBL) with
of 41 showed high homology to the Tn sA protein 44) Comparison
acid sequence of the TnsA-like protein Tn5468 to the predicted of the
89
Fig 41
Alignment of t DNA sequence of the Tn7-li e
Tferrooxi to E i Tn7 sequence at e of
insertion transcriptional t ion s e of
glmS The ent homology shown low occurs within
the repeats (underl of both the
Tn7-li transposon Tn7 (Fig 43) Homology
becomes before the end of t corresponding
impe s
T V E 10 20 30 40 50 60
Ec Tn 7
Tf7shy(like)
70 80 90 100 110 120
Tf7shy(1
130 140 150 160 170 180 Ec Tn 7 ~~~=-
Tf7shy(like)
90
Fig 42 DNA sequences at the 3 end of the Ecoll glmS and the tnsA proximal of Tn7 to the DNA the 3end of the Tferrooxidans gene and end of Tn7-like (a) DNA sequence determined in the Ecoll strain GD92Tn7 in which Tn been inserted into the glmS transcriptional terminator Walker at ai 1986 Nucleotide 38 onwards are the of the left end of Tn DNA
determined in T strain ATCC 33020 with the of 7-like at the termination site of the glmS
gene Also shown are the 22 bp of the Tn7-like transposon as well as the region where homology the protein begins A good Shine
sequence is shown immediately upstream of what appears to be the TTG initiation codon for the transposon
(a)
_ -35 PLE -10 I tr T~ruR~1 truvmnWCIGACUur ~~GCfuA
100 no 110 110 110
4 M S S bull S I Q I amp I I I I bull I I Q I I 1 I Y I W L ~Tnrcmuamaurmcrmrarr~GGQ1lIGTuaDACf~1lIGcrA
DO 200 amp10 220 DG Zlaquot UO ZIG riO
T Q IT I I 1 I I I I I Y sir r I I T I ILL I D L I ilGIIilJAUlAmrC~GlWllCCCAcarr~GGAWtCmCamp1tlnmcrArClGACTAGNJ
Fig 45 (a) Alignment of the amino acid sequence of Tn5468 (which had homology to TnsA of Tn7 Fig 44) to the amino acid sequence of ORF2 (between the merR genes of Tferrooxidans strain E-1S) ORF2 had been found to have significant homology to Tn sA of Tn7 (Kusano et ai 1991) (b) Alignment of the amino acids translation of Tn5468 (above) to Tn sA of Tn7 (c) Alignment of the amino acid sequence of TnsA of Tn7 to the hypothetical ORF2 protein of Tferrooxidans strain E-1S
(al
Percent Similarity 60000 Percent Identity 44444
Tf Tn5468 1 LARQRYGVDEDRVARFQKEGRGQGRGADYHPWLTIQDVPSQGRSHRLKGI SO 11 1111111 I 1111 1111 I I 111
Fig 49 (a) Results of the BLAST search on the inverted and complemented nucleotide sequence of 1852f (from the Sall site) Results indicate amino acid sequence to the TnsC of Tn7 (protein E is the same as TnsC protein) (b) The nucleotide sequence and open reading frame of p81852f
High Prob producing Segment Pairs Frame Score P(N)
IB255431QQECE7 protein E - Escher +1 176 15e-20 gplX044921lSTN7EUR 1 Tn7 E with +1 176 15e-20 splP058461 ECOLI TRANSPOSON TN7 TRANSPOSITION PR +1 176 64e-20 gplU410111 4 D20248 gene product [Caenorhab +3 59 17e-06
IB255431QQECE7 ical protein E - Escherichia coli transposon Tn7 (fragment)
417 (al BLAST results obtained from combined sequence of (inverted and complemented) and 1810r good sequence to Ecoli and Hinfluenzae DNA RecG (EC 361) was found (b) The combined sequence and open frame which was homologous to RecG
444 Analysis of DNA downstream of region with TnsD homology
The location of the of p81811 ApaI-CLaI construct is shown in Fig 47 The single strand
sequence from the C LaI site was joined to p8181Or and searched using BLAST against the
GenBank and EMBL databases Good homology to Ecoli and Hinjluezae A TP-dependent
DNA helicase recombinase proteins (EC 361) was obtained (Fig 417) The BLAST search
with the sequence from the ApaI end showed high sequence homology to the stringent
response protein guanosine-35-bis (diphosphate) 3-pyrophosphohydrolase (EC 3172) of
Ecoli Hinjluenzae and Scoelicolor (Fig 418) In both Ecoli and Hinjluenzae the two
proteins RecG helicase recombinase and ppGpp stringent response protein constitute part of
the spo operon
445 Spo operon
A brief description will be given on the spo operons of Ecoli and Hinjluenzae which appear
to differ in their arrangements In Ecoli spoT gene encodes guanosine-35-bis
pyrophosphohydrolase (ppGpp) which is synthesized during stringent response to amino acid
starvation It is also known to be responsible for cellular ppGpp degradation (Gentry and
Cashel 1995) The RecG protein is required for normal levels of recombination and DNA
repair RecG protein is a junction specific DNA helicase that acts post-synaptically to drive
branch migration of Holliday junction intermediate made by RecA during the strand
exchange stage of recombination (Whitby and Lloyd 1995)
The spoS (also called rpoZ) encodes the omega subunit of RNA polymerase which is found
associated with core and holoenzyme of RNA polymerase The physiological function of the
omega subunit is unknown Nevertheless it binds stoichiometrically to RNA polymerase
119
Fig 418 BLAST search nucleotide sequence of 1811r I restrict ) inverted and complement high sequence homology to st in s 3 5 1 -bis ( e) 3 I ase (EC 31 7 2) [(ppGpp)shyase] -3-pyropho ase) of Ecoli Hinfluenzae (b) The nucleot and the open frame with n
Add 4 ml 52 mix by inversion at room temp for 5 mins
Add 4 ml 53 Mix by to homogenous suspension
Spin at 15 K 40 mins at 4
remove to fresh tube
Equilibrate column with 2 ml N2
Load supernatant 2 to 4 ml amounts
Wash column 2 X 4 of N3 Elute the DNA the first bed
each) add 07
volumes of isopropanol
Spin at 4 Wash with 70 Ethanol Resuspended pellet in n rv 100 AU of TE and scan
volume of about 8 to 10 drops) To the eluent (two
to
140
SEQUITHERM CYCLE SEQUENCING
Alf-express Cy5 end labelled promer method
only DNA transformed into end- Ecoli
The label is sensitive to light do all with fluorescent lights off
3-5 kb
3-7 kb
7-10 kb
Thaw all reagents from kit at well before use and keep on
1) Label 200 PCR tubes on or little cap flap
(The heated lid removes from the top of the tubes)
2) Add 3 Jtl of termination mixes to labelled tubes
3) On ice with off using 12 ml Eppendorf DNA up to
125 lll with MilliQ water
Add 1 ltl
Add lll of lOX
polymeraseAdd 1 ltl
Mix well Aliquot 38 lll from the eppendorf to Spin down
Push caps on nrnnp1
141
Hybaid thermal Cycler
Program
93 DC for 5 mins 1 cycle
93degC for 30 sees
55 DC for 30 secs
70 DC for 60 secs 30 cycle 93 DC_ 30s 55 DC-30s
70degC for 5 mins 1 cycle
Primer must be min 20 bp long and min 50 GC content if the annealing step is to be
omitted Incubate at 95 DC for 5 mins to denature before running Spin down Load 3 U)
SEQUITHERM CYCLE SEQUENCING
Ordinary method
Use only DNA transformed into end- Ecoli strain
3-5 kb 3J
3-7 kb 44
7-10 kb 6J
Thaw all reagents from kit at RT mix well before use and keep on ice
1) Label 200 PCR tubes on side or little cap flap
(The heated lid removes markings from the top of the tubes)
2) Add 3 AU of termination mixes to labelled tubes
3) On ice using l2 ml Eppendorf tubes make DNA up to 125 41 with MilliQ water
142
Add I Jtl of Primer
Add 25 ttl of lOX sequencing buffer
Add 1 Jtl Sequitherm DNA polymerase
Mix well spin Aliquot 38 ttl from the eppendorf to each termination tube Spin down
Push caps on properly
Hybaid thermal Cycler
Program
93 DC for 5 mins 1 cycle
93 DC for 30 secs
55 DC for 30 secs
70 DC for 60 secs 30 cycles
93 DC_ 30s 55 DC-30s 70 DC for 5 mins 1 cycle
Primer must be minimum of 20 bp long and min 50 GC content if the annealing step is
to be omitted Incubate at 95 DC for 5 mins to denature before running
Spin down Load 3 ttl for short and medium gels 21t1 for long gel runs
143
REFERENCES
144
REFERENCES
Abrahams J P Lutter R Todd R J van Raaij M J Leslie A G W and Walker J E 1993 Inherent asymmetry of the structure of F1-ATPase from bovine heart mitochondria at 65 Aresolution EMBO J 121775-1780
Abrahams J P Leslie A G W Lutter R and Walker 1 E 1994 Structure at 28 A resolution of FeATPase from bovine heart mitochondria Nature 370621-628
Adzumah K and Mizuuchi K 1988 Target immunity of Mu transposition reflects a differential distribution of Mu B protein Cell 53257-266
Akey C W Crepeau R H Dunn S D McCarty R E and Edelstein S J 1983 Electron microscopy of single molecules and crystals of F1-ATPases EMBO J 21409-1415
Allet B 1979 Mu insertion duplicates a 5 bp sequence at the host inserted site Cell 16 123shy129
Amzel L M and Pedersen P L 1983 Proton ATP-ases structure and mechanism Ann Rev Biochem 52801-824
Andersson L Mac Neela J and Wolfenden R 1985 Use of secondary isotope effects and varying pH to investigate mode of binding of inhibitory amino aldehydes by leucine aminopeptidase Biochem 24330-333
Andrews G F Dugan R P and Stevens C J 1992 Combining physical and bacterial treatment for removing pyritic sulfur from coal In Processing and Utilization of High-sulfur coals IV Dugan P R D Quigley and Y Attia (eds) p 515 Elsevier New York
Andrulis I L Chen J and Ray P N 1987 Isolation of human cDNAs for asparagine synthetase and expression in Jansen rat sarcoama cells Mol Cell BioI 72435-2443
Andruszkiewicz R Milewsky S Zieniawa T and Borowski E 1990 Anticandidal properties of N3 -(4-methoxy-fumaroyl)-L-2 3-diamino propanoic acid oligopeptides 1 Med Chern 33132-135
Arciszewska L K Drake D and Craig NL 1989 Transposon Tn7 cis-acting sequences in transposition and transposition immunity J Mol BioI 20735-42
Bachmann B J 1983 Linkage map of Escherichia coli K-12 edition 7 Microbiol Rev 47180-230
145
B Plumbridge 1 A Cochet D Souza J M M M and Calcagno M 1988 Cordinated regulation of amino J
Bacteriol 1754951-4956
0 B Vermoote P V and Le Goffic F 1993 synthetase from Ecoli lUllL- mechanism and inhibition by N3-fumaroyl-2 3-diaminopropionic derivatives Biochem 27 2282-2287
Vermoote P Haumont PY syrlthl~ta~e from Escherichia coli purification site location Biochemistry 26 1940-1948
Badet B Inagaki K Soda K Walsh dependent inhibition of Bacillus stearothermophilus alanine racemase by phosphonate isomers by isomerization to non covalent enzyme-(1-aminoethyl) complexes Biochem 253275-3282
Badet-Denisot M A and Badet of glucoseamine-6shyphosphate synthetase by diethylpyrocarbonates histidine requirement for enzymatic activity Arch Biochem Biophys
Bainton R Gamas P and transposition in vitro proceeds through an excised transposon intermediate (JpnIPtlltpi1 111 breaks in DNA Cell 65805-816
Barry G 1986 Permanent insertion of v~u into the chromosomes of soil bacteria BioTechnology 4446-449
Barth P T Datta N Grinter N S 1976 Transposition a deoxyribonucleic acid sequence trimethoprim and streptomycin resistances from R483 to other replicons 1 Bacteriol 125800-810
Bates C J Adams W and R R 1968 Control of the formation of uri dine diphospho-N-acetyl and glycoprotein synthesis in rat liver J Chern 2411705-17
Benjamin N 1989 Intramolecular transposition of TnlD Cell 59373shy383
Bennet R L and Malamy M resistant mutants of Escgerichia coli and phosphate Commun 40496-503
Benneth1 1978 Bacterial leaching patterns on pyrite crystal J Bacteriol
146
Berg D E Davies 1 Allet B and Rochaix 1 D 1975 Transposition of R factor genes to bacteriophage lambda Proc Natl Acad Sci USA 723268-3632
Berg D E and Drummond M1978 Absence of DNA sequences homologous to transposable element Tn5 (Kan) in the chromosome of Ecoli K-12 J Bcateriol 136419-422
Birkenhager R Hoppert M Deckers-Hebestriet G Mayer F and Altendorf K 1995 The Fo complex of the Ecoli ATP synthase investigation by electron imaging and immunoelectron microscopy Eur J Biochem 23058-67
Bjqgtrbaek C Foersom V and Michelsen O 1990 The transmembrane topology of the a subunit from ATPase in Escherichia coli analysed by PhoA protein fusions FEBS lett 26031-34
Boekemia E J Berden JA and van Heel MG 1986 sructure of mitochondrial F1-ATPase studied by electron microscopy and image processing Biochim Biophys Acta 851353-360
Bolton E Glynn P and OGara F 1984 Site specific transposition of Tn7 into a Rhizobiwn meliloti megaplasmid Mol Gen Genet 193153-157
Boos W 1974 Bacterial transport Annu Rev Biochem 43123-146
Boursaux-Eude C Girons I S and Zuerner R 1995 IS1500 an IS3-like element from Leptospira interrogans Microbiol 1412165-2173
Boyer P D 1993 The binding change mechanism for ATP synthase-some probabilities and possibilities Biochim Biophys Acta 1140215-250
Brachet P Eisen H and Rambach A 1970 Mutations in coliphage lambda affecting the expression of replicative functions 0 and P Mol Gen Genet 108266-276
Brink J Boekemia E 1 and van Bruggen E F 1987 The structure of NADH ubiquitone oxidoreductase fron beef heart mitochondria Crystals containing an octameric arrangement of iron-sulphur protein fragments Eur J Biochem 166287-294
Brock T D and Gustafson J 1976 Ferric iron reduction by sulphur- and iron-oxidizing bacteria Appl Environ Microbiol 32 567-571
Brown L D Dennehy M E and Rawlings D E 1994 The FI genes of the FIFo ATP synthase from the acidophilic bacterium Thiobacillus jerrooxidans complement Escherichia coli FI unc mutants FEMS Microbiol Lett 12219-25
Buchanan 1 1 Adv The Amidotransferases Enzymol 3991-183
Buckley Science
Caruso M Pseudomonas nOVHrn
oxidation of pyrite Applied
1 1982 Interactions Mol Gen Genet
phage 1
Chmara H by Biophysica
synthetase from bacteria antibiotic tetaine Biochimica et
Microbiol Lett 11197-206 controls on the oxidation of refractory Barberton
genetic elements and evolution Nature 263731shyCohen S N 1976 738
Corbet C M and 1 Ingledew 1987 Is oxidation by Thiobacillus ferrooxidans
Couillard D and ~~b 118(5)808-81
Metallurgical residue V UlllLltHIH of metals from sewage sludge J
Cox G B a reassessment of the
F and Hatch L 1986 The mechanism of ATP synthase of the b and a subunits Biochim Acta 84962-69
Craig N L 1995 Unity in Science 270253-254
Craig N and Gamas P 1 Purification and characterization of a transposition protein that binds ATP DNA Nuc Acids Res
1 2873-97 C A 1990 in response to environmental stress Advances in
alUIUH~ on the activity subunit b-specific polyc1onal
G Simoni and Altendorf K 1992 Influence vlJnu~ of the ATP synthase of t-S(nel~lCn
1 BioI Chern 26712364shy
M A Le Goffic F and 1991 Glucosamine- 6P two proteins on limited proteolysis ltVU Biophys 288225-230
148
Dobrogosz W J 1968 _l in Escherichia coli and its to catabolite repression J
Doublet P 1 van Heijenoort and 1993 The murI gene of is an essential gene that encodes klUltUllU~v racemase activity J Bacteriol 1752970-2979
P J van Heijenoort 1992 Identification of the Ecoli which is required for the D-glutamic acid a specific component peptidoglycan 1 Bacteriol
Garren A Garen and Torri ani 1961 Genetic control of repression UCUlllV phosphatase in Ecoli 1 Mol BioI
D J and Boeke 1 D 1990 A 1-1-0- structure is required for Ty1 transposition Genes Develop 4324-330
1982 Transposition of Tn7 occurs at a on Caulobacter crescentus 10SIClmle 1 Bacteriol 151 1056-1 058
H 1990 Molecular IHovUUU)11 -transporting 345-391 In T 12 Bacterial
Press Ltd London
transport and coupling by the synthase insights mechanism of function J Bioenerg 24485-491
1992b Subunit c of FIFo ATP role m transduction Biochim Biophys Acta 1101 v--rJ
Eliopoulos E E Jackson P 1 Keen IN HUIUVI L Thompson P and 1 Structure of a 16 kDa integral AU-UUl that identity to
of the vacuolar H+ -ATPase Protein 15
Fling M 1 C 1985 Nucleotide sequence of encoding the amino glycoside-modifying enzyme 3(9)-O-nucleotidyltransferase Res 137095-7106
Fling M and C l-1UUJIU nucleotide sequence of dihydrofolate rhnrpri by Tn7 Nucleic Acids Res 115
Flores Llchtenstem C P 1990 DNA sequence anlysis of tnsA for the Tn7 transposition Nucleic Acids 18901 911
149
149
Foster D L and Fillingame R H 1982 Stoichiometry of subunits in the H+-ATPase complex of Escherichia coli J BioI Chern 2572009-2015
Fraga D Hermolin J Oldenburg M Miller MJ and Fillingame R H 1994 Arginine 41 of subunit c of Escherichia coli H+-A TP synthase is essential in binding and coupling of F to Fo J BioI Chern 2697532-7537
Friedl P Hoppe J Gunsalus R P Michelsen 0 von Meyenburg K and Schairer H U 1983 Membrane integration and function of the three Fo subunits of the A TP-synthase of Escherichia coli K12 EMBO 1 299-103
Frisa P S and Sonneborn D R 1982 Developmentally regulated interconversion between end product inhibitable and non-inhibitable forms of a first pathway-specific enzyme activity can be mimicked in vitro by dephosphorylation reactions Proc Natl Acad Sci USA 796289-6293
Fujiwara T and Mizuuchi K 1988 Retroviral DNA integration Structure of an integration intermediate Cell 54497-504
Galas D J and Chandler M 1989 Bacterial insertion sequences In Mobile DNA Berg D E and Howe M M (eds) Washington D C American Society for Microbiology pp 109shy162
Gay N J Tybulewicz V L1 Walker JE 1986 Insertion of transposon Tn7 into the Ecoli gmS transcriptional terminator Biochem J 234 111-117
Gentry D R and Cashel M 1995 Cellular localization of the Escherichia coli SpoT protein J Bacteriol 177 3890-3893
Gentry D R Xiao H Burgess R R and Cashel M 1991 The omega subunit of Ecoli K-12 RNA polymerase is not required for stringent RNA control in vivo J Bacteriol 173 3901-3903
Girvin M E and Fillingame R H 1993 Helical structure and folding of subunit c of FIFo ATP synthase IH NMR resonace assignments and NOE analysis Biochemistry 3212167shy12177
Gogol E P Lucken U Bork T and Capaldi R A 1989 Molecular architecture of Escherichia coli F Adenosinetriphosphatase Biochemistry 284709-4716
Gogol E P Aggeler R Sagermann M and Capaldi R A 1989 Cryoelectronic Microscopy of Escherichia coli FI Adenosinetriphosphatase Decorated with Monoclonal Antibodies to Individual Subunits of the complex Biochemistry 284717-4724
150
Heinikoff S 1984
Golinelli-Pimpaneaux B and Badet involvement of Lys603 from Ecoli glucosamoine-6-phosphate synthase substrate fructose-6-phosphate 1 Biochem 201175-182
Gottesman M M and Rosner 1 L of a detenninant of uvu_bullu
resistance by coliphage lambda Sci USA 725041-5045
Grunstein M and Hogness D
Colony hybridization a method for isolation cloned DNAs that contain a 1Jbullu Proc NatL Acad Sci USA 723961-3965
Hauer B and Shapiro 1 A 1984 Control of Tn7 transposition Mol Gen Genet 194
Hedges R W and Jacob Transposition of ampicillin resistance from to other replicons MoL 1-40
Hennolin 1 Gallant 1 psi subunit in the Fo sector of the H+ -ATPase of Ecoli J Bioi
R H 1983 Topology organization and function
Hennolin J and R 1989 Assembly of Fo sector of synthase Interdepenndence of subunit insertion into the membrane 1 2817
van Montagu M Holsters Zambryski P Beuckeleer M Willmitzer and Schell 1 M 1980 interaction of
DNA plant cells Proc Soc Lond 210351-365
P 1972 Insertion mutations control region of Physical characterization of the mutants Mol Gen Genet
115266-276
Holmes applications pI
UI Haq 1990 Adaptation of Thiobacillus vu)nuu for industrial In J Salley R G McCready Wichlacz (ed)
1989 CANMET Ottawa Canada
Holtje J V and U Schwarz 1985 Biosynthesis and growth murein sacculus p 77shy119 InN (ed) Molecular cytology of Escherichia Academic Press Inc New
Acad Sci USA
Heffron E Reubens C and which mediates ampicillin
Translocation of a plasmid DNA sequence nature and specificity of Natl
digestion with exonuclease III creates fl tr breakpoints 1-369
Hernalsteens J M Agrobacterium
Hirsch H the galactose nnnn fJu
151
Holzenburg A Jones P c Franklin T Padi J B and Finbow M E 1993 Evidence for a common structure 1 Biochem 21321-30
Hoppe 1 and Sebald W 1986 Topological pathway of the protons through Fo is provided by amino acid the lipid phase Biochimie (Paris) 68427-434
S Otsubo H Davidson N and 1975 Electron microscope heteroduplex of sequence relations among plasmids identification and mapping of the
insertion sequences IS] and IS2 in F and R plasmids J Bacteriol 22762-775
Inoue c Sugawara K and 1991 regulatory gene in Thiobacillus ferrooxidans is spaced apart from Mol Microbiol 52707-2718
Ish-Horowicz D and Burke and cosmid cloning Nucleic Acids Res 92989-2998
Johnston B Clennell M and D 1995 Structure and function of Tn5467 a Tn2l-like transposon located on T jerrooxidans broad host range plasmid Appl Envir Micro
Kahmann R and Kamp Nucleotide sequences of the attachment bacteriophage Mu DNA Nature 280247-250
Kahn K and Schaefer M R Characterization of trans po son 5469 from cyanobacterium Fremyella diplosiphon J 1777026-7032
Karavaiko G Golovacheva S Pivovarova T A Tzaplina I A and Vartanjan 1988 Thermophilic ltPM Sulfobacillus page 29-41 In Biohydrometallurgyshy87 Norris P and Science and Technology Letters Kew 1
Kennedy J and Humpphreys J D 1976 Microbial cells living immobilised on Nature 261242-244
Koonin 1993 SpoU protein of Escherichia coli belongs to a new family of Nucleic Acids Res 19
Kopecko J and site specific recA mdependtm recombination between bacterial Pt of palindromes at the N ad Acad Sci USA
on L-glutamine D-fructose ud()transtiera~e 1 BioI
152
Krumholz L R Esser U and Simoni RD 1989 Nucleotide sequence of the unc operon of Vibrio alginolyticus Nucleic Acids Res 177993-7994
Kubo K and Craig N 1990 Bacterial transposon Tn7 utilizes two classes of target sites 1 Bacteriol 1722774-2778
Kucharczyk N Denisot M A Le Goffic F and Badet B 1990 Glucosarnine-6-phosphate synthase from Ecoli detennination of the mechanism of inactivation by N3 fumaroyl-L-2-3 diarninoproprionic derivatives Biochemistry 293668-3676
Kusano M Takeshima T Inoue C and Sugawara K 1991 Evidence for two sets of structural genes coding for Ribulose biphosphate carboxylase in Thiobacillus ferrooxidans 1 Bacteriol 1737313-7323
Lane Dl A P Harrison lnr D Stahl B Pace SJ Giovannoni GJ Olsen and N R Pace 1992 Evolutionary relationship among sulphur- and iron-oxidizing eubacteria 1 Bacteriol 174267-278
Lee C H Bhagwhat A and Heffron F 1983 Identification of a transposon TnJ sequence required for transposition immunity Natl Acad Sci USA 806765-6769
Lewis M 1 Chang 1 A and Somoni R D 1990 A topological analysis of subunit a from Escherichia coli F1Fo-ATP synthase predicts eight transmembrane segments 1 BioI Chern 265 10541-10550
Lichtenstein C P and Brenner S 1982 Unique insertion site of Tn7 in the Ecoli chromosome Nature (London) 297601-603
Lichtenstein C P and Brenner S 1981 Site-specific properties of transposition to the Ecoli chromosome Mol Gen Genet 183380-387
Liu X Petersson S and Sandstrom A Mesophilic versus moderate thennophilic bioleaching Biohydrometallurgy Technologies Vol 1 A E Tonna 1 E Wey and Lakshmanan V 1 (ed) pp 29-38
Lizarna H M and Sankey B M 1993 Oxidation of H2S by Thiobacillus thiooxidans is inhibited by substrate and methane BiohydrometaHurgy Technologies Vol II A E Tonna 1 E Wey and C L Brierley (ed) pp 339-364
Lundgren D G and Silver M 1980 Ore leaching by bacteria Ann Rev Microbiol 34263shy268
Makino K Amemura M Shinagawa H Kobayashi A and Nakata A 1985 Sequence of the genes involved in phosphate transport and regulation of the phosphate regulon in Escherichia coli 1 Mol BioI 184231-240
Makino K Shinagawa Amemura M Kimura S Nakata A and Ishihama 1988 Euuv1J of the phosphate regulon of Ecoli Activation of pstS by the PhoB
protein in vitro 1 Mol BioI 20385-95
Makino K Shinagawa H Amemura M Yamada M and 1990 Signal transduction in the phosphate regulon of the Escherichia coli involves phosphotransfer nprlrJp~middotn PhoR and PhoB proteins J Mol BioI 21055
Malamy 1966 Frameshift mutations in the nnlnn of Escherichia coli Cold Harb Symp Quant BioI 31189-201
Malamy M H 1972 Electron microscopy insertions in the lac operon of Escherichia coli MoL Gen
Malamy M H and Bennett R L 1970 mutants of Ecoli and phosphate transport Biochem Biophys Res Commun
Maniatis T Fritsch EF Sambrook J 1982 nn A laboratory manual Cold Spring Harbor Laboratory Press Cold
McKnight G L S L Mudri SH Mathewest R S Marshall P O Sheppard and P J OHara 1990 Molecular cloning synthesis and bacterial expression of human glutaminefructose-6-phosphate UUAUU u J Chem 26725208-25212
Medveczky N and H Rosenburg 1970 phosphate-binding protein of Escherichia Biochim Biophys Acta 211 158-168
Mei B and Zalkin H 1989 A Cysteine-Histidine-Aspartate catalytic triad is involved in Glutamide Amide Transfer Function purF-type Glutamine amodotransferases 1 BioI Chem 264 16613-16619
Mengin-Lecreulx D and J van 1993 Identification of glmU gene encoding Nshyacetylglucosamine in Escherichia coli J Bacteriol 1756150shy6157
Michaelis M J and Criddle M J 1970 Mitochondrial DNA and mutants in Saccharomyces cerevisiae Biochem Genet 5487-495
Michaelis Starlinger P 1969 Two insertions in the jO v
operon having different homologous DNA sequences MoL Gen Genet 10437 377
Miller J H Calos Transposable elements Cell 20579-595
154
Miller J Oldenburg M and Fillingame R 1990 The essential carboxyl group of in subunit c the FIFo ATP synthase can be and H( +)-translocating function retained Proc NatL Acad 874900-4904
Mitcell P 1966 Chemiosmotic coupling in - and phosphorylation BioL Rev 41455-502 (1966)
Mizuuchi K 1984 Mechanism of transposition of bacteriophage Mu Polarity of the strand reaction at the initiation of the transposon Cell 39395-404
C Ph de Donato Berthelin J Surface oxidized species a key factor in the study of bioleaching processes Biohydrometallurgical In A J
Weyand V I Lakshmanan ed 1993 Vol 1 175-184
A Ishino Shinagawa H Makino K and M 1987 Nucleotide of the iap gene responsible for alkaline phosphatase isoenzyme conversion in Ecoli
identification of product 1 1695429-5433
K 1989 The tsr gene-coding prevents thiopeptin from inhibiting ppGpp synthesis in Streptomyces lividans FEMS Microbiol Lett
Ogasawara N and Yoshikawa H 1992 and their organIzatIon in the repnc()n of the bacterial chromosome Mol Microbiol 6(5)629-634
Ohtsubo Davidson N and 1975 microscope heteroduplex studies of sequence relations among plasmids identification and mapping of the insertion sequences 1 and IS2 in F and R plasmids 1 122746-775
Olson G J 1994 Microbial oxidation gold ores and gold bioleaching FEMS un Lett 119 1-6
Orle K A N L 1991 Identification of transposition prroteins by the bacterial transposon [corrected republished originally printed in Gene 1990 Nov 30 96(1) Gene 10425-31
Ouellette M Roy P Homology of ORFs from and from to site specific recombinases 1987 Nucl Acids 10055-10059
Murein synthesis p663-671ln Neidhardt J L Ingraham B Louw M~ M Schaechter H E Umbarger (ed) Escherichia coli and Salmonella
ryphimurium cellular and bioI voL 1 American Society Microbiology Washington
Perlin D S and Senior A Functional and cross-reactivity of antibody to purified subunit b (uncF protein) of Escherichia coli proton-ATPase Arch Biochem Biophys 236603-611
155
Plumbridge J A O Cochet Souza J M Altramirano M M Calcagno M L and Badet B 1993 Coordinated regulation of amino sugar-synthesizing and -degrading enzymes in Escherichia coli K-12 J Bacteriol 175(16)4951-4956
Pretorius I M Rawlings D E and Woods D R 1986 Identification and cloning of Thiobacillus jerrooxidans structural Nif genes in Escherichia coli Gene 4559-65
Qadri M I Flores C c Davis J and Lichtenstein C 1989 Genetic analysis of attTn7 the transposon Tn7 attachment site in Escherichia coli using a novel M13-based transduction assay 1 Mol BioI 20785-98
Radstrom P Skold 0 Swedberg J Roy P H and Sundstrom L 1994 Tn5090 of plasmid R751 which carries integron is related to Tn7 Mu and retroelements J Bacteriol 1763257-3268
Raetz C R H 1987 Structure and biosynthesis of lipid A in Escherichia coli pp 498-503 In F C Niedhardt J L Ingraham K B Low B Magasanik M Schaechter and H E Umbarger (ed) Escherichia coli and Salmonella typhimurium cellular and molecular biology vol 1 American Society for Microbiology Washington DC
Rao N N and Torriani A 1990 Molecular aspects of phosphate transport in Escherichia coli Mol Microbiol 4(7) 1083-1090
Rawlings DE D R Woods and NP Mjoli 1991 The cloning and structre of genes from the autotrophic biomining bacterium Thiobacillusjerrooxidans p 215-237 In PJ Greenaway (ed) Advances in gene technology vol 2 JAI Press London
Richet E and O Raibaud 1989 MalT the regulatory protein of the Escherichia coli maltose system is an A TP-dependent transcriptional activator EMBO 1 8981-987
Rogers M Ekaterrinaki N Nimmo E and Sherratt D 1986 Analysis of Tn7 transposition Mol Gen Genet 205550-556
Ronson C W Nixon B T Albright L M anf Ausubel F M 1987 Rhizobium meliloti ntrA (rpoN) gene is required for diverse metabolic functions J Bacteriol 1692424-2431
Rosenburg H Gerdes R G and Chegwidden K 1977 Two systems for the uptake of phosphate in Ecoli JBacterioL 131505-511
Rosenburg H 1987 In ion transport in Prokaryotes Rosen B P and Silver S (eds) New York Academic Press pp 205-248
Ross D G Swan 1 and N Klecker 1979 Physical structures of TnlO-promoted deletions and inversions role of 1400 base pairs inverted repetitions Cell 16721-731
156
Saedler H and P 19670 mutation in the galactose operon in Ecoli II Physiological characterization MoL Gen Genet 100 190-202
Saedler P 1968 00 and strong polar mutations in the Genet 102353-363
Sambrook J Fritsch Maniatis 1989 Molecular cloning a laboratory manual Cold Harbor Laboratory Cold Spring Harbor New York
Sand W Gehrke T Hallmann Rohde K Sobotke B and Wintzien S 1993 Bioleaching metal sulphides The importance of Leptospirillum ferrooxidans Biohydromemetallurgical Technologies Voll pp 15-25
Sanger Nicklen and Coulson A DNA sequencing with chain-tennination inhibitors Natl Acad USA 745463-5467
Schneider E and Altendorf K All the three subunits are required for an active proton channel (Fo) of Escherichia coli synthase (FIFo) ~-
518
Schneider E and Allendorf K 1987 Bacterial adenosine 5-triphosphate synthase purification and reconstitution complexes and biochemical and functional characterization of subunits Microbio Rev 51477-497
Schrader 1 A and D S Holmes 1988 Phenotypic switching Thiobacillus ferrooxidans J BacterioL 1703915-3023
Scordilis G E H and Lassie 1987 Identification of transposable elements which activates gene expression in Pseudomonas cepacia J Bacteriol 1698-13
Sekine Eisaki N and Ohtsubo E 1996 Identification and characterization of the lS3 molecules generated by breaks 1 BioL Chern 271197-202
Senior A E 1990 The proton-trans locating of Escherichia coli Annu Rev Biophys Chern 194-41
Shapiro 1 and Adhya S 1969 The jLU operon of K-12 n A deletion analysis of the structure polarity 62249-264
J A 1969 Mutations caused by insertion genenc material the galactose operon Escherichia 1 MoL BioI 4093-105
Silvennan M P 1967 Mechanism of bacterial pyrite oxidation 1 Bacteriol 941046-1051
157
C C ElIson and Levinson 1983 Identification of the typel trimethoprim resistance reductase specified by the R-plasmid R43 comparison with procaryotic and eucaryotic dihydrofolate reductase 1 Bacteriol 155 100 1-1008
Smith and Jones P transposition a multigene process Identification of a regulatory product 147915-7927
Sprague Jr Bell R M Cronan 1 A mutant of Ecoli auxotrophic for organic phosphates evidence two defects in phosphate Mol Gen Genet 14371-77
Starlinger and Michaelis 1968 Suppression the sequential aO[)ealame of the galactose enzymes in a transferase amber mutant coli Mol Genet 102367-369
Starlinger 1977 IS elements in microorganisms MicrobioL Immunol
Steffens K Schneider E Herkernhoff B Schmid Altendorf K portion of Escherichia coli A TP synthase resolution of from subunit b J BioI Chern 2625866-5869
Steffens A Deckers-hebestreit G and Altendorf K 1987b The and functional relationship of A TP synthases (FoFI) from Ecoii and the thermophilic bacterium PS3 J Biol 2626334-6338
Strominger J and M S Smith 1959 Uridine diphosphoacetylglucosamine pyrophosphorylase 1 BioI Chern 2341822-1827
Sundstrom Skold 1990 dhfrI trimethoprim resistance gene of can be found at in other genetic surroundings Antimicrob Agents Chemother 34642shy650
Sundstrom L Roy P H and Skold Site-specific of three cassettes in Tn7 J Bacteriol 1733025-3028
Surin B P and Downie JA 1988 of the Rhizobium leguminosarum nodLMN involved efficient host-specific nodulation Mol Microbiol 2 173-183
B P Dixon N and Rosenburg 1986 Purification PhoU protein a OClItnl
regulator of the pho regulon of Ecoli K-1 J Bacteriol 168631
B P D A Jans A L Fimmel Shaw GB Cox Rosenburg Structural gene for the phosphate-repressible phosphate binding of Escherichia coli
own promoter nucleotide of the phoS 1 Bacteriol
158
Suzuki I Chang W and Takeuchi 1994 Oxidation of organic compounds by Thiobacilli Symp Series 55060-67
Takeyama M S Y Noumi T Maeda Ishibashi S and Futai M 1988 Beta subunit of Ecoli amino acid replacement within a conserved sequence G-X-X-X-X-GshyK-TS) v~u binding proteins lett 218222-226
Thomson JA M Hendson and R M 1981 Mutagenesis by insertion of drug resistance Tn7 into a vibrio 11 J Bacteriol
Tichy R Grotenhuis J T c Janssen van Houten R Rulkens and Lettinga 1993 Application of the sulphur cycle bioremediation of soils polluted with heavy metals In Int Conf Contaminated 93 ed F Arendt G J Annokee R Bosman and W J van der Brink Kluwer Academic Publishers Dordrecht pp 1461-1462
G and Mayer An electron approach to the quaternary structure of mitochondrial Eur J Biochem 13237-45
J and Tschape streptothricin resistance transposons Tn1825 and Tn1826 and transposon Tn 7 Plasmidnuu
18246-249
Tso J Y Zalkin H van Cleemput M Yanofsky C and JM 1982 Nucleotide of Escherichia coli and deduced amino glutamine
phosphribosylpyrophosphate am idotransferase J BioI Chern
V Mesyanzhinova I V Koslov I A and Orlova 1984 Structure of studied by electron and image processing Lett 167285-289
P Barber C and transposons Tn5 and Tn7 in Xanthomonas campestris pv campestris MoL Gen 157
Ullrich J and van Putten P Identification of the Gonococcal glmU gene UVUUIJ
the enzyme N-acetylglucosamine 1-Phosphate Uridyltransferase involved in the synthesis UDP-GlcNAc J of Bact 1776902-6909
M 1992 Eight bacterial proteins includin]g UDP-N -acetylglucosamine acyltransferase three vother of Escherichia coli of a six-residue n tVI
theme FEMS MicrobioL 97249-254
van der Steen J J D Doddema J and de Uidoging van zware metalen afvalstromen met behulp van thiobacilli (Removal metals from waste streams
using thiobacilli) Ministry of Housing Physical and Enviromental (VROM) Directoraat-Generaal Milieubeheer Rapport 199210 Hague
Vermoote P 1988 Universite Paris VI Paris Hrllnro
159
Vignais P V Lunard Issartel J P and Dupuis A 1985 Interaction n l1f middotn
oligomycin sensitivity protein (OSCP) beef heart mitochondrial FeATPase 2 Identification of interacting Fl subunits cross-linking Biochem J
Vik S and Dao NN Prediction of transmembrane topology of Fo proteins from Ecoli ATP synthase variational hydrophobic moment analyses Biochim Biophys Acta 1140 199-207
von Meyenburg B B Jcentrgensen J Nielsen and F Hansen 1982 Promoters of the atp operon for the membrane-bound ATP synthase of Ecoli mapped by TnlO insertion mutations MoL Gen Genet 188240-248
von Meyenberg F G Hansen 1980 The origin of replication oriC the Ecoli chromosome near oriC and construction of oriC mutants ICN-UCLA Symp MoLCellBiol 191 159
Waddell S and Craig N 1989 Tn7 transposition recognition of the attTn7 Proc Natl Sci USA 863958-3962
Waddell C S and Craig N L 1988 transposition two transposition pathways directed by five Tn7-encoded genes Develop 21
J E Gay N J Saraste M and A N 1984 DNA sequence the Escherichia coli unc operon Completion of the sequence of a kilobase segment containing
oriC unc and phoS J224799-815
and Collinson 1 1994 the role the stalk in the coupling mechanism of FEBS 34639-43
Walker 1 E Gay N J and Tybulewicz V L 1986 of transposon Tn7 into the Escherichia glmS transcriptional terminator Biochem 1 243 111-1
Wanner Land McSharry 1982 Phosphate-controlled gene in Escherichia coli using Mudl-directed lacZ fusions J Mol BioI 158347-363
Weng M and Zalkin H 1987 Structural role for a region the CTP synthetase glutamide amide transfer domain J Bacteriol 1693023-3028
Wheatcroft R and S and nucleotide sequence of Rhizobiwn meliloti insertion between the transposase encoded by ISRm3 and those encoded by Staphylococcus aureus and Thiobacillus ferrooxidans IST2 J 1732530-2538
160
Whitby M C and Lloyd R 1995 Branch of three-strand recombination intennediates by RecG a possible pathway for securing middotIULl initiated by duplex DNA 1 143303-3310
Wilkens and Capaldi R A Assymmetry and changes in examined by cryoelectronmicroscopy BioI Chern Hoppe-Seyler 37543-51
and Malamy M 1974 The loss of the PhoS periplasmic protein leads to a the specificity a constitutive phosphate transport system in Escherichia coli Biochem Res Commun 60226-233
WiUsky G Rand Malamy M 1980 of two separable inorganic phosphate transport systems in Escherichia coli J BacterioL 144356-365
P J and and C F 1973 enzyme of metabolism and Stadtman R eds) pp 343-363 Academic Press New York
Winterbum P J and Phelps F 197L control of hexosamine biosynthesis by glucosamine synthetase Biochem 1 121711
Wolf-Watz H and Norquist A 1979 Deoxyribonucleic acid and outer membrane protein to outer membrane protein involves a protein J 14043-49
Wolf-Watz H and M 1979 Deoxyribonucleic acid and outer membrane strains for oriC elevated levels deoxyribonucleic acid-binding protein and
11 for specific UU6 of the oriC region to outer membrane J Bacteriol 14050-58
Wolf-Watz H 1984 Affinity of two different chromosome to the outer membrane of Ecoli J Bacteriol 157968-970
Wu C and Te Wu 197 L Isolation and characterization of a glucosamine-requiring mutant of Escherichia 12 defective in glucoamine-6-phosphate synthetase 1 Bacteriol 105455-466
Yamada M Makino Shinagawa Nakata A 1990 Regulation of the phosphate regulon of Escherichia coli properties the pooR mutants and subcellular localization of protein Mol Genet 220366-372
Yates J R and Holmes D S 1987 Two families of repeatcXl DNA sequences Thiobacillus 1 Bacteriol 169 1861-1870
Yates J R R P and D S 1988 an insertion sequence from Thiobacillus errooxidans Proc Natl 857284-7287
161
Youvan D c Elder 1 T Sandlin D E Zsebo K Alder D P Panopoulos N 1 Marrs B L and Hearst 1 E 1982 R-prime site-directed transposon Tn7 mutagenesis of the photosynthetic apparatus in Rhodopseudomonas capsulata 1 Mol BioI 162 17-41
Zalkin H and Mei B 1990 Amino terminal deletions define a glutamide transfer domain in glutamine phosphoribosylpyrophosphate ami do transferase and other purF-type amidotransferases 1 Bacteriol 1723512-3514
Zalkin H and Weng M L 1987 Structural role for a conserved region III the CTP synthetase glutamide amide transfer domain 1 Bacteriol 169 (7)3023-3028
Zhang Y and Fillingame R H 1994 Essential aspartate in subunit c of FIF0 ATP synthase Effect of position 61 substitutions in helix-2 on function of Asp24 in helix-I 1 BioI Chern 2695473-5479
v
ABSTRACT
A Tn7-like element was found in a region downstream of a cosmid (p8181) isolated from a
genomic library of Thiobacillus ferrooxidans ATCC 33020 A probe made from the Tn7-like
element hybridized to restriction fragments of identical size from both cosmid p8181 and
T ferrooxidans chromosomal DNA The same probe hybridized to restricted chromosomal
DNA from two other T ferrooxidans strains (ATCC 23270 and 19859) There were no positive
signals when an attempt was made to hybridize the probe to chromosomal DNA from two
Thiobacillus thiooxidans strains (ATCC 19733 and DSM504) and a Leptospirillum
ferrooxidans strain DSM 2705
A 35 kb BamHI-BamHI fragment was subcloned from p8181 downstream the T ferrooxidans
unc operon and sequenced in both directions One partial open reading frame (ORFl) and two
complete open reading frames (ORF2 and ORF3) were found On the basis of high homology
to previously published sequences ORFI was found to be the C-terminus of the
T ferrooxidans glmU gene encoding the enzyme GlcNAc I-P uridyltransferase (EC 17723)
The ORF2 was identified as the T ferrooxidans glmS gene encoding the amidotransferase
glucosamine synthetase (EC 26116) The third open reading frame (ORF3) was found to
have very good amino acid sequence homology to TnsA of transposon Tn7 Inverted repeats
very similar to the imperfect inverted repeat sequences of Tn7 were found upstream of ORF3
The cloned T ferrooxidans glmS gene was successfully used to complement an Ecoli glmS
mutant CGSC 5392 when placed behind a vector promoter but was otherwise not expressed
in Ecoli
Subc10ning and single strand sequencing of DNA fragments covering a region of about 7 kb
beyond the 35 kb BamHI-BamHI fragment were carried out and the sequences searched
against the GenBank and EMBL databases Sequences homologous to the TnsBCD proteins
of Tn7 were found The TnsD-like protein of the Tn7-like element (registered as Tn5468) was
found to be shuffled truncated and rearranged Homologous sequence to the TnsE and the
antibiotic resistance markers of Tn7 were not found Instead single strand sequencing of a
further 35 kb revealed sequences which suggested the T ferrooxidans spo operon had been
encountered High amino acid sequence homology to two of the four genes of the spo operon
from Ecoli and Hinjluenzae namely spoT encoding guanosine-35-bis (diphosphate) 3shy
pyrophosphohydrolase (EC 3172) and recG encoding ATP-dependent DNA helicase RecG
(EC 361) was found This suggests that Tn5468 is incomplete and appears to terminate with
the reshuffled TnsD-like protein The orientation of the spoT and recG genes with respect to
each other was found to be different in T ferrooxidans compared to those of Ecoli and
Hinjluenzae
11 Bioleaching 1
112 Organism-substrate raction 1
113 Leaching reactions 2
114 Industrial applicat 3
12 Thiobacillus ferrooxidans 4
13 Region around the Ecoli unc operon 5
131 Region immediately Ie of the unc operon 5
132 The unc operon 8
1321 Fe subun 8
1322 subun 10
133 Region proximate right of the unc operon (EcoURF 1) 13
1331 Metabolic link between glmU and glmS 14
134 Glucosamine synthetase (GlmS) 15
135 The pho 18
1351 Phosphate uptake in Ecoli 18
1352 The Pst system 19
1353 Pst operon 20
1354 Modulation of Pho uptake 21
136 Transposable elements 25
1361 Transposons and Insertion sequences (IS) 27
1362 Tn7 27
13621 Insertion of Tn7 into Ecoli chromosome 29
13622 The Tn7 transposition mechanism 32
137 Region downstream unc operons
of Ecoli and Tferrooxidans 35
LIST OF FIGURES
Fig
Fig
Fig
Fig
Fig
Fig
Fig
Fig
la
Ib
Ie
Id
Ie
If
Ig
Ih
6
11
14
22
23
28
30
33
1
CHAPTER ONE
INTRODUCTION
11 BIOLEACHING
The elements iron and sulphur circulate in the biosphere through specific paths from the
environment to organism and back to the environment Certain paths involve only
microorganisms and it is here that biological reactions of relevance in leaching of metals from
mineral ores occur (Sand et al 1993 Liu et aI 1993) These organisms have evolved an
unusual mode of existence and it is known that their oxidative reactions have assisted
mankind over the centuries Of major importance are the biological oxidation of iron
elemental sulphur and mineral sulphides Metals can be dissolved from insoluble minerals
directly by the metabolism of these microorganisms or indrectly by the products of their
metabolism Many metals may be leached from the corresponding sulphides and it is this
process that has been utilized in the commercial leaching operations using microorganisms
112 Or2anismSubstrate interaction
Some microorganisms are capable of direct oxidative attack on mineral sulphides Scanning
electron micrographs have revealed that numerous bacteria attach themselves to the surface
of sulphide minerals in solution when supplemented with nutrients (Benneth and Tributsch
1978) Direct observation has indicated that bacteria dissolve a sulphide surface of the crystal
by means of cell contact (Buckley and Woods 1987) Microbial cells have also been shown
to attach to metal hydroxides (Kennedy et ai 1976) Silverman (1967) concluded that at
least two roles were performed by the bacteria in the solubilization of minerals One role
involved the ferric-ferrous cycle (indirect mechanism) whereas the other involved the physical
2
contact of the microrganism with the insoluble sulfide crystals and was independent of the
the ferric-ferrous cycle Insoluble sulphide minerals can be degraded by microrganisms in the
absence of ferric iron under conditions that preclude any likely involvement of a ferrous-ferric
cycle (Lizama and Sackey 1993) Although many aspects of the direct attack by bacteria on
mineral sulphides remain unknown it is apparent that specific iron and sulphide oxidizers
must play a part (Mustin et aI 1993 Suzuki et al 1994) Microbial involvement is
influenced by the chemical nature of both the aqueous and solid crystal phases (Mustin et al
1993) The extent of surface corrosion varies from crystal to crystal and is related to the
orientation of the mineral (Benneth and Tributsch 1978 Claassen 1993)
113 Leachinamp reactions
Attachment of the leaching bacteria to surfaces of pyrite (FeS2) and chalcopyrite (CuFeS2) is
pSIS1 construct was further subcloned to plasm ids IS30 IS40 pSlS50
pS183S p81841 and p81 (Fig Some of subclones were extensively mapped
although not all sites are shown in 22 exact positions of the ends of
T jerrooxidans plasmids pTfatpl and pTfatp2 on the cosmid p8I81 were identified
21)
Tfeooxidans
that the cosmid was
unrearranged DNA digests of cosmid pSISI and T jerrooxidans chromosomal DNA were
with pSI The of the bands gave a positive hybridization signal were
identical each of the three different restriction enzyme digests of p8I8l and chromosomal
In order to confirm of pSI81 and to
49
kb kb ~
(A)
11g 50Sts5
middot 2S4
17 bull tU (probe)
- tosect 0S1
(e)
kb
+- 10 kb
- 46 kb
28---+ 26-+
17---
Fig 23 Hybrdization of cosmid pSISI and T jerrooxitians (A TCC 33020) chromosomal
DNA by the KpnI-SalI fragment of pSIS52 (probe) (a) Autoradiographic image of the
restriction digests Lane x contains the probe lane 13 and 5 contain pSISI restricted with BamHl HindIII and Bgffi respectively Lanes 2 4 and 6 contain T errooxitians chromosomal DNA also restricted with same enzymes in similar order (b) The sizes of restricted pSISI
and T jerrooxitians chromosome hybridized by the probe The lanes correspond to those in Fig 23a A DNA digested with Pst served as the molecular weight nlUkermiddot
50
DNA (Fig BamHI digests SHklC at 26 and 2S kb 1 2) HindIII
at 10 kb 3 and 4) and BgnI at 46 (lanes 5 and 6) The signals in
the 181 was because the purified KpnI-San probe from p81 had a small quantity
ofcontaminating vector DNA which has regions ofhomology to the cosmid
vector The observation that the band sizes of hybridizing
for both pS181 and the T ferrooxidans ATCC 33020 same
digest that the region from BgnI site at to HindIII site at 16
kb on -VJjUIU 1 represents unrearranged chromosomal DNA from T ferrooxidans
ATCC there were no hybridization signals in to those predicted
the map with the 2 4 and 6 one can that are no
multiple copies of the probe (a Tn7-like segment) in chromosome of
and Lferrooxidans
of plasmid pSI was found to fall entirely within a Tn7-like trallSDOS()n nrpn
on chromosome 33020 4) A Southern hybridizationUUAcpoundUU
was out to determine whether Tn7-like element is on other
ofT ferrooxidans of T and Lferrooxidans results of this
experiment are shown 24 Lanes D E and F 24 represent strains
19377 DSM and Lferrooxidans DSM 2705 digested to
with BgnI enzyme A hybridization was obtained for
the three strains (lane A) ATCC 19859 (lane B) and UUHUltn
51
(A) AAB CD E F kb
140
115 shy
284 -244 = 199 -
_ I
116 -109 -
08
os -
(8)) A B c o E F
46 kb -----
Fig 24 (a) Autoradiographic image of chromosomal DNA of T ferrooxidans strains ATCC 33020 19859 23270 (lanes A B C) Tthiooxidans strains ATCC 19377 and DSM 504 (D and E) and Lferrooxidans strain DSM 2705 (lane F) all restricted with Bgffi A Pst molecular weight marker was used for sizing (b) Hybridization of the 17 kb KpnI-San piece of p81852 (probe) to the chromosomal DNA of the organisms mentioned 1 Fig 24a The lanes in Fig 24a correspond to those in Fig 24b
(lane C) uuvu (Fig 24) The same size BgllI fragments (46 kb) were
lanes A Band C This result
different countries and grown on two different
they Tn7-like transposon in apparently the same location
no hybridization signal was obtained for T thiooxidans
1 504 or Lferrooxidans DSM 2705 (lanes D E and F of
T thiooxidans have been found to be very closely related based on 1
rRNA et 1992) Since all Tferrooxidans and no Tthiooxidans
~~AA~~ have it implies either T ferrooxidans and
diverged before Tn7-like transposon or Tthiooxidans does not have
an attTn7 attachment the Tn7-like element Though strains
T ferrooxidans were 10catH)uS as apart as the USA and Japan
it is difficult to estimate acquired the Tn7-like transposon as
bacteria get around the world are horizontally transmitted It
remains to be is a general property
of all Lferrooxidans T ferrooxidans strains
harbour this Tn7-like element their
CHAPTER 3
5431 Summary
54Introduction
56Materials and methods
56L Bacterial and plasmid vectors
Media and solutions
56333 DNA
57334 gel electrophoresis
Competent preparation
57336 Transformation of DNA into
58337 Recombinant DNA techniques
58ExonucleaseIII shortening
339 DNA sequencing
633310 Complementation studies
64Results discussion
1 DNA analysis
64Analysis of ORF-l
72343 Glucosamine gene
77344 Complementation of by T ferrooxidans glmS
57cosmid pSIS1 pSlS20
58Plasmidmap of p81816r
Fig Plasmidmap of lS16f
60Fig 34 Restriction digest p81S1Sr and pSIS16f with
63DNA kb BamHI-BamHI
Fig Codon and bias plot of the DNA sequence of pSlS16
Fig 37 Alignment of C-terminal sequences of and Bsubtilis
38 Alignment of amino sequences organisms with high
homology to that of T ferrooxidans 71
Fig Phylogenetic relationship organisms high glmS
sequence homology to Tferrooxidans
31 Summary
A 35 kb BamHI-BamHI p81820 was cloned into pUCBM20 and pUCBM21 to
produce constructs p818 and p81816r respectively This was completely
sequenced both rrelct1()ns and shown to cover the entire gmS (184 kb) Fig 31
and 34 The derived sequence of the T jerrooxidans synthetase was
compared to similar nrvnC ~ other organisms and found to have high sequence
homology was to the glucosamine the best studied
eubacterium EcoU Both constructs p81816f p81 the entire
glmS gene of T jerrooxidans also complemented an Ecoli glmS mutant for growth on medium
lacking N-acetyl glucosamine
32 =-====
Cell wall is vital to every microorganism In most procaryotes this process starts
with amino which are made from rructClsemiddotmiddotn-[)ll()S by the transfer of amide group
to a hexose sugar to form an This reaction is by
glucosamine synthetase the product of the gmS part of the catalysis
to the 40-residue N-terminal glutamine-binding domain (Denisot et al 1991)
participation of Cys 1 to generate a glutamyl thiol ester and nascent ammonia (Buchanan
368-residue C-terminal domain is responsible for the second part of the ClUUU
glucosamine 6-phosphate This been shown to require the ltgtttrltgtt1iltn
of Clpro-R hydrogen of a putative rrulctoseam 6-phosphate to fonn a cis-enolamine
intennediate which upon reprotonation to the gives rise to the product (Golinelli-
Pimpaneau et al 1989)
bacterial enzyme mn two can be separated by limited chymotryptic
proteolysis (Denisot et 199 binding domain encompassing residues 1 to
240 has the same capacity to hydrolyse (and the corresponding p-nitroanilide
derivative) into glutamate as amino acid porI11
binding domain is highly cOllserveu among members of the F-type
ases Enzymes in this include amidophosphophoribosyl et al
1982) asparagine synthetase (Andrulis et al 1987) glucosamine-6-P synmellase (Walker et
aI 1984) and the NodM protein of Rhizobium leguminosarum (Surin and 1988) The
368-residue carboxyl-tenninal domain retains the ability to bind fructose-6-phosphate
The complete DNA sequence of T ferrooxidans glmS a third of the
glmU gene (preceding glmS) sequences downstream of glmS are reported in this
chapter This contains a comparison of the VVA r glmS gene
and Mycobacterium (422 ) An interesting observation was that the T ferrooxidans
glmS gene had comparatively high homology sequences to the Rhizobium leguminosarum and
Rhizobium meliloti M the amino to was 44 and 436
respectively A consensus Dalgarno upstream of the start codon
(ATG) is in 35 No Ecoli a70-type n r~ consensus sequence was
detected in the bp preceding the start codon on intergenic distance
between the two and the absence of any promoter consensus sequence Plumbridge et
al (1993) that the Ecoli glmU and glmS were co-transcribed In the case
the glmU-glmS --n the T errooxidans the to be similar The sequences
homology to six other amidotransferases (Fig 38) which are
(named after the purF-encoded l phosphoribosylpyrophosphate
amidotransferase) and Weng (1987)
The glutamine amide transfer domain of approximately 194 amino acid residues is at the
of protein chain Zalkin and Mei (1989) using site-directed to
the 9 invariant amino acids in the glutamine amide transfer domain of
phosphoribosylpyrophosphated indicated in their a
catalytic triad is involved glutamine amide transfer function of
73
Comparison of the ammo acid sequence alignment of ghtcosamine-6-phosphate
amidotranferases (GFAT) of Rhizobium meliloti (R_m) Rhizobium leguminnosarum (RJ)
Ecoli (E_c) Hinjluenzae Mycobacterium leprae (M_I) Bsubtilis (B_s) and
Scerevisiae (S_c) to that of Tferrooxidans Amino acids are identified by their single
codes The asterisks () represent homologous amino acids of Tferrooxidans glmS to
at least two of the other Consensus ammo acids to all eight organisms are
highlighted (bold and underlined)
1 50 R m i bullbullbullbullbullbull hkps riag s tf bullbullbullbull R~l i bullbullbullbullbullbull hqps rvaep trod bullbullbullbull a
a bullbullbullbullbullbull aa 1r 1 d bullbullbull a a bullbullbullbullbullbull aa inh g bullbullbullbullbullbull sktIv pmilq 1 1g bullbullbull a MCGIVi V D Gli RI IYRGYPSAi AI D 1 gvmar s 1gsaks Ikek a bullbullbullbull qfcny Iversrgi tvq t dgd bullbull ead
51 100 R m hrae 19ktrIk bull bullbull bullbull s t ateR-l tqrae 19rkIk bullbullbull s t atrE-c htIrI qmaqae bull bull h bullbull h gt sv aae in qicI kads stbull bullbull k bullbull i gt T f- Ivsvr aetav bullbullbull r bullbull gq qg c
DL R R G V L AV E L G VGI lTRW E H ntvrrar Isesv1a mv bull sa nIgi rtr gihvfkekr adrvd bullbullbull bullbull nve aka g sy1stfiykqi saketk qnnrdvtv she reqv
101 150 R m ft g skka aevtkqt R 1 ft g ak agaqt E-c v hv hpr krtv aae in sg tf hrI ksrvlq T f- m i h q harah eatt
S E Eamp L A GY l SE TDTEVIAHL ratg eiavv av
qsaIg lqdvk vqv cqrs inkke cky
151 200 bullbull ltk bullbull dgcm hmcve fe a v n bull Iak bullbull dgrrm hmrvk fe st bull bull nweI bullbull kqgtr Iripqr gtvrh 1 s bull bull ewem bullbullbull tds kkvqt gvrh hlvs bull bull hh bullbull at rrvgdr iisg evm
V YR T DLF A A L AV S D PETV AR G M 1 eagvgs lvrrqh tvfana gvrs B s tef rkttIks iInn rvknk 5 c Ihlyntnlqn ~lt klvlees glckschy neitk
74
201 R m ph middot middot middot R 1 ah- middot middot middot middot middot middot E c 1 middot middot middot middot middot middot middot Hae in 1 middot middot middot middot middot T f - c11v middot middotmiddot middot middot middot middot middot M 1 tli middot middot middot middot middot middot middot middot middot B s 11 middot middot middot middot middot S c lvkse kk1kvdfvdv
251 R m middot middot middot middot middot middot middot middot middot middot middot middot middot middot
middot middot middot middot R-1 middot middot middot middot middot middot E c middot middot middot middot middot middot middot middot Hae in middot middot middot middot middot middot middot middot middot middot middot middot T f shy middot middot middot middot middot middot middot middot middot middot middot middot 1M middot middot middot middot middot middot middot middot middot middot middot middot middot middot B s middot middot middot middot middot middot middot middot middot middot middot middot middot middot middot S c feagsqnan1 1piaanefn1
301 R m fndtn wgkt R-1 fneti wgkt E c rf er Hae in srf er T f- rl mlqq
PVTR V YE DGDVA R M 1 ehqaeg qdqavad B s qneyem kmvdd S c khkkl dlhydg
351 R m nhpe v R-1 nhe v E c i nk Hae in k tli T f- lp hr
~ YRHlrM~ ~I EQP AVA M 1 geyl av B-s tpl tdvvrnr S-c pd stf
401 Rm slasa lk R-1 slasca lk E c hql nssr Hae in hq nar-T f f1s hytrq
RVL A SiIS A VG W M 1 ka slakt B s g kqS c lim scatrai
T ferrooxidans (Tf) Scerevisiae (Saee) Mleprae (Myele) and Bsublilis (Baesu)
tr1 ()-ltashyc 8 ~ er (11
-l l
CIgt
~ r (11-CIgt (11
-
$ -0 0shy
a c
omiddot s
S 0 0shyC iii omiddot $
ttl ~ () tI)
I-ltashyt ()
0
~ smiddot (11
81
CHAPTER 4
8341 Summary
8442 Introduction
8643 Materials and methods
431 Bacterial strains and plasmids 86
432 DNA constructs subclones and shortenings 86
864321 Contruct p81852
874322 Construct p81809
874323 Plasmid p8I809 and its shortenings
874324 p8I8II
8844 Results and discussion
441 Analysis of the sequences downstream the glmS gene 88
442 TnsBC homology 96
99443 Homology to the TnsD protein
118444 Analysis of DNA downstream of the region with TnsD homology
LIST OF FIGURES
87Fig 41 Alignment of Tn7-like sequence of T jerrooxidans to Ecoli Tn7
Fig 42 DNA sequences at the proximal end of Tn5468 and Ecoli glmS gene 88
Fig 43 Comparison of the inverted repeats of Tn7 and Tn5468 89
Fig 44 BLAST results of the sequence downtream of T jerrooxidans glmS 90
Fig 45 Alignment of the amino acid sequence of Tn5468 TnsA-like protein 92 to TnsA of Tn7
82
Fig 46 Restriction map of the region of cosmid p8181 with amino acid 95 sequence homology to TnsABC and part of TnsD of Tn7
Fig 47 Restriction map of cosmid p8181Llli with its subclonesmiddot and 96 shortenings
98 Fig 48 BLAST results and DNA sequence of p81852r
100 Fig 49 BLAST results and DNA sequence of p81852f
102 Fig 410 BLAST results and DNA sequence of p81809
Fig 411 Diagrammatic representation of the rearrangement of Tn5468 TnsDshy 106 like protein
107Fig 412 Restriction digests on p818lLlli
108 Fig 413 BLAST results and DNA sequence of p818lOEM
109 Fig 414 BLAST results on joined sequences of p818104 and p818103
111 Fig 415 BLAST results and DNA sequence of p818102
112 Fig 416 BLAST results and nucleotide sequence of p818101
Fig 417 BLAST results and DNA sequence of the combined sequence of 113p818l1f and p81810r
Fig 418 Results of the BLAST search on p81811r and the DNA sequence 117obtained from p81811r
Fig 419 Comparison of the spa operons of Ecali Hinjluenzae and 121 probable structure of T ferraaxidans spa operon
83
CHAPTER FOUR
TN7-LlKE TRANSPOSON OF TFERROOXIDANS
41 Summary
Various constructs and subclones including exonuclease ill shortenings were produced to gain
access to DNA fragments downstream of the T ferrooxidans glmS gene (Chapter 3) and
sequenced Homology searches were performed against sequences in GenBank and EMBL
databases in an attempt to find out how much of a Tn7-like transposon and its antibiotic
markers were present in the T ferrooxidans chromosome (downstream the glmS gene)
Sequences with high homology to the TnsA TnsB TnsC and TnsD proteins of Tn7 were
found covering a region of about 7 kb from the T ferrooxidans glmS gene
Further sequencing (plusmn 45 kb) beyond where TnsD protein homology had been found
revealed no sequences homologous to TnsE protein of Tn7 nor to any of the antibiotic
resistance markers associated with Tn7 However DNA sequences very homologous to Ecoli
ATP-dependentDNA helicase (RecG) protein (EC 361) and guanosine-35-bis (diphosphate)
3-pyrophosphohydrolase (EC 3172) stringent response protein of Ecoli HinJluenzae and
Vibrio sp were found about 15 kb and 4 kb respectively downstream of where homology to
the TnsD protein had been detected
84
42 Transposable insertion sequences in Thiobacillus ferrooxidizns
The presence of two families (family 1 and 2) of repetitive DNA sequences in the genome
of T ferrooxidans has been described previously (Yates and Holmes 1987) One member of
the family was shown to be a 15 kb insertion sequence (IST2) containing open reading
frames (ORFs) (Yates et al 1988) Sequence comparisons have shown that the putative
transposase encoded by IST2 has homology with the proteins encoded by IS256 and ISRm3
present in Staphylococcus aureus and Rhizobium meliloti respectively (Wheatcroft and
Laberge 1991) Restriction enzyme analysis and Southern hybridization of the genome of
T ferrooxidans is consistent with the concept that IST2 can transpose within Tferrooxidans
(Holmes and Haq 1990) Additionally it has been suggested that transposition of family 1
insertion sequences (lST1) might be involved in the phenotypic switching between iron and
sulphur oxidizing modes of growth including the reversible loss of the capacity of
T ferrooxidans to oxidise iron (Schrader and Holmes 1988)
The DNA sequence of IST2 has been determined and exhibits structural features of a typical
insertion sequence such as target-site duplications ORFs and imperfectly matched inverted
repeats (Yates et al 1988) A transposon-like element Tn5467 has been detected in
T ferrooxidans plasmid pTF-FC2 (Rawlings et al 1995) This transposon-like element is
bordered by 38 bp inverted repeat sequences which has sequence identity in 37 of 38 and in
38 of 38 to the tnpA distal and tnpA proximal inverted repeats of Tn21 respectively
Additionally Kusano et al (1991) showed that of the five potential ORFs containing merR
genes in T ferrooxidans strain E-15 ORFs 1 to 3 had significant homology to TnsA from
transposon Tn7
85
Analysis of the sequence at the tenninus of the 35 kb BamHI-BamHI fragment of p81820
revealed an ORF with very high homology to TnsA protein of the transposon Tn7 (Fig 44)
Further studies were carried out to detennine how much of the Tn7 transposition genes were
present in the region further downstream of the T ferroxidans glmS gene Tn7 possesses
trimethoprim streptomycin spectinomycin and streptothricin antibiotic resistance markers
Since T ferrooxidans is not exposed to a hospital environment it was of particular interest to
find out whether similar genes were present in the Tn7-like transposon In the Ecoli
chromosome the Tn7 insertion occurs between the glmS and the pho genes (pstS pstC pstA
pstB and phoU) It was also of interest to detennine whether atp-glm-pst operon order holds
for the T ferrooxidans chromosome It was these questions that motivated the study of this
region of the chromosome
43 OJII
strains and plasm ids used were the same as in Chapter Media and solutions (as
in Chapter 3) can in Appendix Plasmid DNA preparations agarose gel
electrophoresis competent cell preparations transformations and
were all carned out as Chapter 3 Probe preparation Southern blotting hybridization and
detection were as in Chapter 2 The same procedures of nucleotide
DNA sequence described 2 and 3 were
4321 ~lllu~~~
Plasmid p8I852 is a KpnI-SalI subclone p8I820 in the IngtUMnt vector kb) and
was described Chapter 2 where it was used to prepare the probe for Southern hybridization
DNA sequencing was from both (p81852r) and from the Sail sites
(p8I852f)
4322 ==---==
Construct p81809 was made by p8I820 The resulting fragment
(about 42 kb) was then ligated to a Bluescript vector KS+ with the same
enzymes) DNA sequencing was out from end of the construct fA
87
4323 ~mlJtLru1lbJmalpoundsectM~lllgsect
The 40 kb EeoRI-ClaI fragment p8181Llli into Bluescript was called p8181O
Exonuclease III shortening of the fragment was based on the method of Heinikoff (1984) The
vector was protected with and ClaI was as the susceptible for exonuclease III
Another construct p818IOEM was by digesting p81810 and pUCBM20 with MluI and
EeoRI and ligating the approximately 1 kb fragment to the vector
4324 p81811
A 25 ApaI-ClaI digest of p8181Llli was cloned into Bluescript vector KS+ and
sequenced both ends
88
44 Results and discussion
of glmS gene termini (approximately 170
downstream) between
comparison of the nucleotide
coli is shown in Fig 41 This region
site of Tn7 insertion within chromosome of E coli and the imperfect gtpound1
repeat sequences of was marked homology at both the andVI
gene but this homology decreased substantially
beyond the stop codon
amino acid sequence
been associated with duplication at
the insertion at attTn7 by (Lichtenstein and as
CCGGG in and underlined in Figs41
CCGGG and are almost equidistant from their respelt~tle
codons The and the Tn7-like transposon BU- there is
some homology transposons in the regions which includes
repeats
11 inverted
appears to be random thereafter 1) The inverted
repeats of to the inverted repeats of element of
(Fig 43) eight repeats
(four traJrlsposcm was registered with Stanford University
California as
A search on the (GenBank and EMBL) with
of 41 showed high homology to the Tn sA protein 44) Comparison
acid sequence of the TnsA-like protein Tn5468 to the predicted of the
89
Fig 41
Alignment of t DNA sequence of the Tn7-li e
Tferrooxi to E i Tn7 sequence at e of
insertion transcriptional t ion s e of
glmS The ent homology shown low occurs within
the repeats (underl of both the
Tn7-li transposon Tn7 (Fig 43) Homology
becomes before the end of t corresponding
impe s
T V E 10 20 30 40 50 60
Ec Tn 7
Tf7shy(like)
70 80 90 100 110 120
Tf7shy(1
130 140 150 160 170 180 Ec Tn 7 ~~~=-
Tf7shy(like)
90
Fig 42 DNA sequences at the 3 end of the Ecoll glmS and the tnsA proximal of Tn7 to the DNA the 3end of the Tferrooxidans gene and end of Tn7-like (a) DNA sequence determined in the Ecoll strain GD92Tn7 in which Tn been inserted into the glmS transcriptional terminator Walker at ai 1986 Nucleotide 38 onwards are the of the left end of Tn DNA
determined in T strain ATCC 33020 with the of 7-like at the termination site of the glmS
gene Also shown are the 22 bp of the Tn7-like transposon as well as the region where homology the protein begins A good Shine
sequence is shown immediately upstream of what appears to be the TTG initiation codon for the transposon
(a)
_ -35 PLE -10 I tr T~ruR~1 truvmnWCIGACUur ~~GCfuA
100 no 110 110 110
4 M S S bull S I Q I amp I I I I bull I I Q I I 1 I Y I W L ~Tnrcmuamaurmcrmrarr~GGQ1lIGTuaDACf~1lIGcrA
DO 200 amp10 220 DG Zlaquot UO ZIG riO
T Q IT I I 1 I I I I I Y sir r I I T I ILL I D L I ilGIIilJAUlAmrC~GlWllCCCAcarr~GGAWtCmCamp1tlnmcrArClGACTAGNJ
Fig 45 (a) Alignment of the amino acid sequence of Tn5468 (which had homology to TnsA of Tn7 Fig 44) to the amino acid sequence of ORF2 (between the merR genes of Tferrooxidans strain E-1S) ORF2 had been found to have significant homology to Tn sA of Tn7 (Kusano et ai 1991) (b) Alignment of the amino acids translation of Tn5468 (above) to Tn sA of Tn7 (c) Alignment of the amino acid sequence of TnsA of Tn7 to the hypothetical ORF2 protein of Tferrooxidans strain E-1S
(al
Percent Similarity 60000 Percent Identity 44444
Tf Tn5468 1 LARQRYGVDEDRVARFQKEGRGQGRGADYHPWLTIQDVPSQGRSHRLKGI SO 11 1111111 I 1111 1111 I I 111
Fig 49 (a) Results of the BLAST search on the inverted and complemented nucleotide sequence of 1852f (from the Sall site) Results indicate amino acid sequence to the TnsC of Tn7 (protein E is the same as TnsC protein) (b) The nucleotide sequence and open reading frame of p81852f
High Prob producing Segment Pairs Frame Score P(N)
IB255431QQECE7 protein E - Escher +1 176 15e-20 gplX044921lSTN7EUR 1 Tn7 E with +1 176 15e-20 splP058461 ECOLI TRANSPOSON TN7 TRANSPOSITION PR +1 176 64e-20 gplU410111 4 D20248 gene product [Caenorhab +3 59 17e-06
IB255431QQECE7 ical protein E - Escherichia coli transposon Tn7 (fragment)
417 (al BLAST results obtained from combined sequence of (inverted and complemented) and 1810r good sequence to Ecoli and Hinfluenzae DNA RecG (EC 361) was found (b) The combined sequence and open frame which was homologous to RecG
444 Analysis of DNA downstream of region with TnsD homology
The location of the of p81811 ApaI-CLaI construct is shown in Fig 47 The single strand
sequence from the C LaI site was joined to p8181Or and searched using BLAST against the
GenBank and EMBL databases Good homology to Ecoli and Hinjluezae A TP-dependent
DNA helicase recombinase proteins (EC 361) was obtained (Fig 417) The BLAST search
with the sequence from the ApaI end showed high sequence homology to the stringent
response protein guanosine-35-bis (diphosphate) 3-pyrophosphohydrolase (EC 3172) of
Ecoli Hinjluenzae and Scoelicolor (Fig 418) In both Ecoli and Hinjluenzae the two
proteins RecG helicase recombinase and ppGpp stringent response protein constitute part of
the spo operon
445 Spo operon
A brief description will be given on the spo operons of Ecoli and Hinjluenzae which appear
to differ in their arrangements In Ecoli spoT gene encodes guanosine-35-bis
pyrophosphohydrolase (ppGpp) which is synthesized during stringent response to amino acid
starvation It is also known to be responsible for cellular ppGpp degradation (Gentry and
Cashel 1995) The RecG protein is required for normal levels of recombination and DNA
repair RecG protein is a junction specific DNA helicase that acts post-synaptically to drive
branch migration of Holliday junction intermediate made by RecA during the strand
exchange stage of recombination (Whitby and Lloyd 1995)
The spoS (also called rpoZ) encodes the omega subunit of RNA polymerase which is found
associated with core and holoenzyme of RNA polymerase The physiological function of the
omega subunit is unknown Nevertheless it binds stoichiometrically to RNA polymerase
119
Fig 418 BLAST search nucleotide sequence of 1811r I restrict ) inverted and complement high sequence homology to st in s 3 5 1 -bis ( e) 3 I ase (EC 31 7 2) [(ppGpp)shyase] -3-pyropho ase) of Ecoli Hinfluenzae (b) The nucleot and the open frame with n
Add 4 ml 52 mix by inversion at room temp for 5 mins
Add 4 ml 53 Mix by to homogenous suspension
Spin at 15 K 40 mins at 4
remove to fresh tube
Equilibrate column with 2 ml N2
Load supernatant 2 to 4 ml amounts
Wash column 2 X 4 of N3 Elute the DNA the first bed
each) add 07
volumes of isopropanol
Spin at 4 Wash with 70 Ethanol Resuspended pellet in n rv 100 AU of TE and scan
volume of about 8 to 10 drops) To the eluent (two
to
140
SEQUITHERM CYCLE SEQUENCING
Alf-express Cy5 end labelled promer method
only DNA transformed into end- Ecoli
The label is sensitive to light do all with fluorescent lights off
3-5 kb
3-7 kb
7-10 kb
Thaw all reagents from kit at well before use and keep on
1) Label 200 PCR tubes on or little cap flap
(The heated lid removes from the top of the tubes)
2) Add 3 Jtl of termination mixes to labelled tubes
3) On ice with off using 12 ml Eppendorf DNA up to
125 lll with MilliQ water
Add 1 ltl
Add lll of lOX
polymeraseAdd 1 ltl
Mix well Aliquot 38 lll from the eppendorf to Spin down
Push caps on nrnnp1
141
Hybaid thermal Cycler
Program
93 DC for 5 mins 1 cycle
93degC for 30 sees
55 DC for 30 secs
70 DC for 60 secs 30 cycle 93 DC_ 30s 55 DC-30s
70degC for 5 mins 1 cycle
Primer must be min 20 bp long and min 50 GC content if the annealing step is to be
omitted Incubate at 95 DC for 5 mins to denature before running Spin down Load 3 U)
SEQUITHERM CYCLE SEQUENCING
Ordinary method
Use only DNA transformed into end- Ecoli strain
3-5 kb 3J
3-7 kb 44
7-10 kb 6J
Thaw all reagents from kit at RT mix well before use and keep on ice
1) Label 200 PCR tubes on side or little cap flap
(The heated lid removes markings from the top of the tubes)
2) Add 3 AU of termination mixes to labelled tubes
3) On ice using l2 ml Eppendorf tubes make DNA up to 125 41 with MilliQ water
142
Add I Jtl of Primer
Add 25 ttl of lOX sequencing buffer
Add 1 Jtl Sequitherm DNA polymerase
Mix well spin Aliquot 38 ttl from the eppendorf to each termination tube Spin down
Push caps on properly
Hybaid thermal Cycler
Program
93 DC for 5 mins 1 cycle
93 DC for 30 secs
55 DC for 30 secs
70 DC for 60 secs 30 cycles
93 DC_ 30s 55 DC-30s 70 DC for 5 mins 1 cycle
Primer must be minimum of 20 bp long and min 50 GC content if the annealing step is
to be omitted Incubate at 95 DC for 5 mins to denature before running
Spin down Load 3 ttl for short and medium gels 21t1 for long gel runs
143
REFERENCES
144
REFERENCES
Abrahams J P Lutter R Todd R J van Raaij M J Leslie A G W and Walker J E 1993 Inherent asymmetry of the structure of F1-ATPase from bovine heart mitochondria at 65 Aresolution EMBO J 121775-1780
Abrahams J P Leslie A G W Lutter R and Walker 1 E 1994 Structure at 28 A resolution of FeATPase from bovine heart mitochondria Nature 370621-628
Adzumah K and Mizuuchi K 1988 Target immunity of Mu transposition reflects a differential distribution of Mu B protein Cell 53257-266
Akey C W Crepeau R H Dunn S D McCarty R E and Edelstein S J 1983 Electron microscopy of single molecules and crystals of F1-ATPases EMBO J 21409-1415
Allet B 1979 Mu insertion duplicates a 5 bp sequence at the host inserted site Cell 16 123shy129
Amzel L M and Pedersen P L 1983 Proton ATP-ases structure and mechanism Ann Rev Biochem 52801-824
Andersson L Mac Neela J and Wolfenden R 1985 Use of secondary isotope effects and varying pH to investigate mode of binding of inhibitory amino aldehydes by leucine aminopeptidase Biochem 24330-333
Andrews G F Dugan R P and Stevens C J 1992 Combining physical and bacterial treatment for removing pyritic sulfur from coal In Processing and Utilization of High-sulfur coals IV Dugan P R D Quigley and Y Attia (eds) p 515 Elsevier New York
Andrulis I L Chen J and Ray P N 1987 Isolation of human cDNAs for asparagine synthetase and expression in Jansen rat sarcoama cells Mol Cell BioI 72435-2443
Andruszkiewicz R Milewsky S Zieniawa T and Borowski E 1990 Anticandidal properties of N3 -(4-methoxy-fumaroyl)-L-2 3-diamino propanoic acid oligopeptides 1 Med Chern 33132-135
Arciszewska L K Drake D and Craig NL 1989 Transposon Tn7 cis-acting sequences in transposition and transposition immunity J Mol BioI 20735-42
Bachmann B J 1983 Linkage map of Escherichia coli K-12 edition 7 Microbiol Rev 47180-230
145
B Plumbridge 1 A Cochet D Souza J M M M and Calcagno M 1988 Cordinated regulation of amino J
Bacteriol 1754951-4956
0 B Vermoote P V and Le Goffic F 1993 synthetase from Ecoli lUllL- mechanism and inhibition by N3-fumaroyl-2 3-diaminopropionic derivatives Biochem 27 2282-2287
Vermoote P Haumont PY syrlthl~ta~e from Escherichia coli purification site location Biochemistry 26 1940-1948
Badet B Inagaki K Soda K Walsh dependent inhibition of Bacillus stearothermophilus alanine racemase by phosphonate isomers by isomerization to non covalent enzyme-(1-aminoethyl) complexes Biochem 253275-3282
Badet-Denisot M A and Badet of glucoseamine-6shyphosphate synthetase by diethylpyrocarbonates histidine requirement for enzymatic activity Arch Biochem Biophys
Bainton R Gamas P and transposition in vitro proceeds through an excised transposon intermediate (JpnIPtlltpi1 111 breaks in DNA Cell 65805-816
Barry G 1986 Permanent insertion of v~u into the chromosomes of soil bacteria BioTechnology 4446-449
Barth P T Datta N Grinter N S 1976 Transposition a deoxyribonucleic acid sequence trimethoprim and streptomycin resistances from R483 to other replicons 1 Bacteriol 125800-810
Bates C J Adams W and R R 1968 Control of the formation of uri dine diphospho-N-acetyl and glycoprotein synthesis in rat liver J Chern 2411705-17
Benjamin N 1989 Intramolecular transposition of TnlD Cell 59373shy383
Bennet R L and Malamy M resistant mutants of Escgerichia coli and phosphate Commun 40496-503
Benneth1 1978 Bacterial leaching patterns on pyrite crystal J Bacteriol
146
Berg D E Davies 1 Allet B and Rochaix 1 D 1975 Transposition of R factor genes to bacteriophage lambda Proc Natl Acad Sci USA 723268-3632
Berg D E and Drummond M1978 Absence of DNA sequences homologous to transposable element Tn5 (Kan) in the chromosome of Ecoli K-12 J Bcateriol 136419-422
Birkenhager R Hoppert M Deckers-Hebestriet G Mayer F and Altendorf K 1995 The Fo complex of the Ecoli ATP synthase investigation by electron imaging and immunoelectron microscopy Eur J Biochem 23058-67
Bjqgtrbaek C Foersom V and Michelsen O 1990 The transmembrane topology of the a subunit from ATPase in Escherichia coli analysed by PhoA protein fusions FEBS lett 26031-34
Boekemia E J Berden JA and van Heel MG 1986 sructure of mitochondrial F1-ATPase studied by electron microscopy and image processing Biochim Biophys Acta 851353-360
Bolton E Glynn P and OGara F 1984 Site specific transposition of Tn7 into a Rhizobiwn meliloti megaplasmid Mol Gen Genet 193153-157
Boos W 1974 Bacterial transport Annu Rev Biochem 43123-146
Boursaux-Eude C Girons I S and Zuerner R 1995 IS1500 an IS3-like element from Leptospira interrogans Microbiol 1412165-2173
Boyer P D 1993 The binding change mechanism for ATP synthase-some probabilities and possibilities Biochim Biophys Acta 1140215-250
Brachet P Eisen H and Rambach A 1970 Mutations in coliphage lambda affecting the expression of replicative functions 0 and P Mol Gen Genet 108266-276
Brink J Boekemia E 1 and van Bruggen E F 1987 The structure of NADH ubiquitone oxidoreductase fron beef heart mitochondria Crystals containing an octameric arrangement of iron-sulphur protein fragments Eur J Biochem 166287-294
Brock T D and Gustafson J 1976 Ferric iron reduction by sulphur- and iron-oxidizing bacteria Appl Environ Microbiol 32 567-571
Brown L D Dennehy M E and Rawlings D E 1994 The FI genes of the FIFo ATP synthase from the acidophilic bacterium Thiobacillus jerrooxidans complement Escherichia coli FI unc mutants FEMS Microbiol Lett 12219-25
Buchanan 1 1 Adv The Amidotransferases Enzymol 3991-183
Buckley Science
Caruso M Pseudomonas nOVHrn
oxidation of pyrite Applied
1 1982 Interactions Mol Gen Genet
phage 1
Chmara H by Biophysica
synthetase from bacteria antibiotic tetaine Biochimica et
Microbiol Lett 11197-206 controls on the oxidation of refractory Barberton
genetic elements and evolution Nature 263731shyCohen S N 1976 738
Corbet C M and 1 Ingledew 1987 Is oxidation by Thiobacillus ferrooxidans
Couillard D and ~~b 118(5)808-81
Metallurgical residue V UlllLltHIH of metals from sewage sludge J
Cox G B a reassessment of the
F and Hatch L 1986 The mechanism of ATP synthase of the b and a subunits Biochim Acta 84962-69
Craig N L 1995 Unity in Science 270253-254
Craig N and Gamas P 1 Purification and characterization of a transposition protein that binds ATP DNA Nuc Acids Res
1 2873-97 C A 1990 in response to environmental stress Advances in
alUIUH~ on the activity subunit b-specific polyc1onal
G Simoni and Altendorf K 1992 Influence vlJnu~ of the ATP synthase of t-S(nel~lCn
1 BioI Chern 26712364shy
M A Le Goffic F and 1991 Glucosamine- 6P two proteins on limited proteolysis ltVU Biophys 288225-230
148
Dobrogosz W J 1968 _l in Escherichia coli and its to catabolite repression J
Doublet P 1 van Heijenoort and 1993 The murI gene of is an essential gene that encodes klUltUllU~v racemase activity J Bacteriol 1752970-2979
P J van Heijenoort 1992 Identification of the Ecoli which is required for the D-glutamic acid a specific component peptidoglycan 1 Bacteriol
Garren A Garen and Torri ani 1961 Genetic control of repression UCUlllV phosphatase in Ecoli 1 Mol BioI
D J and Boeke 1 D 1990 A 1-1-0- structure is required for Ty1 transposition Genes Develop 4324-330
1982 Transposition of Tn7 occurs at a on Caulobacter crescentus 10SIClmle 1 Bacteriol 151 1056-1 058
H 1990 Molecular IHovUUU)11 -transporting 345-391 In T 12 Bacterial
Press Ltd London
transport and coupling by the synthase insights mechanism of function J Bioenerg 24485-491
1992b Subunit c of FIFo ATP role m transduction Biochim Biophys Acta 1101 v--rJ
Eliopoulos E E Jackson P 1 Keen IN HUIUVI L Thompson P and 1 Structure of a 16 kDa integral AU-UUl that identity to
of the vacuolar H+ -ATPase Protein 15
Fling M 1 C 1985 Nucleotide sequence of encoding the amino glycoside-modifying enzyme 3(9)-O-nucleotidyltransferase Res 137095-7106
Fling M and C l-1UUJIU nucleotide sequence of dihydrofolate rhnrpri by Tn7 Nucleic Acids Res 115
Flores Llchtenstem C P 1990 DNA sequence anlysis of tnsA for the Tn7 transposition Nucleic Acids 18901 911
149
149
Foster D L and Fillingame R H 1982 Stoichiometry of subunits in the H+-ATPase complex of Escherichia coli J BioI Chern 2572009-2015
Fraga D Hermolin J Oldenburg M Miller MJ and Fillingame R H 1994 Arginine 41 of subunit c of Escherichia coli H+-A TP synthase is essential in binding and coupling of F to Fo J BioI Chern 2697532-7537
Friedl P Hoppe J Gunsalus R P Michelsen 0 von Meyenburg K and Schairer H U 1983 Membrane integration and function of the three Fo subunits of the A TP-synthase of Escherichia coli K12 EMBO 1 299-103
Frisa P S and Sonneborn D R 1982 Developmentally regulated interconversion between end product inhibitable and non-inhibitable forms of a first pathway-specific enzyme activity can be mimicked in vitro by dephosphorylation reactions Proc Natl Acad Sci USA 796289-6293
Fujiwara T and Mizuuchi K 1988 Retroviral DNA integration Structure of an integration intermediate Cell 54497-504
Galas D J and Chandler M 1989 Bacterial insertion sequences In Mobile DNA Berg D E and Howe M M (eds) Washington D C American Society for Microbiology pp 109shy162
Gay N J Tybulewicz V L1 Walker JE 1986 Insertion of transposon Tn7 into the Ecoli gmS transcriptional terminator Biochem J 234 111-117
Gentry D R and Cashel M 1995 Cellular localization of the Escherichia coli SpoT protein J Bacteriol 177 3890-3893
Gentry D R Xiao H Burgess R R and Cashel M 1991 The omega subunit of Ecoli K-12 RNA polymerase is not required for stringent RNA control in vivo J Bacteriol 173 3901-3903
Girvin M E and Fillingame R H 1993 Helical structure and folding of subunit c of FIFo ATP synthase IH NMR resonace assignments and NOE analysis Biochemistry 3212167shy12177
Gogol E P Lucken U Bork T and Capaldi R A 1989 Molecular architecture of Escherichia coli F Adenosinetriphosphatase Biochemistry 284709-4716
Gogol E P Aggeler R Sagermann M and Capaldi R A 1989 Cryoelectronic Microscopy of Escherichia coli FI Adenosinetriphosphatase Decorated with Monoclonal Antibodies to Individual Subunits of the complex Biochemistry 284717-4724
150
Heinikoff S 1984
Golinelli-Pimpaneaux B and Badet involvement of Lys603 from Ecoli glucosamoine-6-phosphate synthase substrate fructose-6-phosphate 1 Biochem 201175-182
Gottesman M M and Rosner 1 L of a detenninant of uvu_bullu
resistance by coliphage lambda Sci USA 725041-5045
Grunstein M and Hogness D
Colony hybridization a method for isolation cloned DNAs that contain a 1Jbullu Proc NatL Acad Sci USA 723961-3965
Hauer B and Shapiro 1 A 1984 Control of Tn7 transposition Mol Gen Genet 194
Hedges R W and Jacob Transposition of ampicillin resistance from to other replicons MoL 1-40
Hennolin 1 Gallant 1 psi subunit in the Fo sector of the H+ -ATPase of Ecoli J Bioi
R H 1983 Topology organization and function
Hennolin J and R 1989 Assembly of Fo sector of synthase Interdepenndence of subunit insertion into the membrane 1 2817
van Montagu M Holsters Zambryski P Beuckeleer M Willmitzer and Schell 1 M 1980 interaction of
DNA plant cells Proc Soc Lond 210351-365
P 1972 Insertion mutations control region of Physical characterization of the mutants Mol Gen Genet
115266-276
Holmes applications pI
UI Haq 1990 Adaptation of Thiobacillus vu)nuu for industrial In J Salley R G McCready Wichlacz (ed)
1989 CANMET Ottawa Canada
Holtje J V and U Schwarz 1985 Biosynthesis and growth murein sacculus p 77shy119 InN (ed) Molecular cytology of Escherichia Academic Press Inc New
Acad Sci USA
Heffron E Reubens C and which mediates ampicillin
Translocation of a plasmid DNA sequence nature and specificity of Natl
digestion with exonuclease III creates fl tr breakpoints 1-369
Hernalsteens J M Agrobacterium
Hirsch H the galactose nnnn fJu
151
Holzenburg A Jones P c Franklin T Padi J B and Finbow M E 1993 Evidence for a common structure 1 Biochem 21321-30
Hoppe 1 and Sebald W 1986 Topological pathway of the protons through Fo is provided by amino acid the lipid phase Biochimie (Paris) 68427-434
S Otsubo H Davidson N and 1975 Electron microscope heteroduplex of sequence relations among plasmids identification and mapping of the
insertion sequences IS] and IS2 in F and R plasmids J Bacteriol 22762-775
Inoue c Sugawara K and 1991 regulatory gene in Thiobacillus ferrooxidans is spaced apart from Mol Microbiol 52707-2718
Ish-Horowicz D and Burke and cosmid cloning Nucleic Acids Res 92989-2998
Johnston B Clennell M and D 1995 Structure and function of Tn5467 a Tn2l-like transposon located on T jerrooxidans broad host range plasmid Appl Envir Micro
Kahmann R and Kamp Nucleotide sequences of the attachment bacteriophage Mu DNA Nature 280247-250
Kahn K and Schaefer M R Characterization of trans po son 5469 from cyanobacterium Fremyella diplosiphon J 1777026-7032
Karavaiko G Golovacheva S Pivovarova T A Tzaplina I A and Vartanjan 1988 Thermophilic ltPM Sulfobacillus page 29-41 In Biohydrometallurgyshy87 Norris P and Science and Technology Letters Kew 1
Kennedy J and Humpphreys J D 1976 Microbial cells living immobilised on Nature 261242-244
Koonin 1993 SpoU protein of Escherichia coli belongs to a new family of Nucleic Acids Res 19
Kopecko J and site specific recA mdependtm recombination between bacterial Pt of palindromes at the N ad Acad Sci USA
on L-glutamine D-fructose ud()transtiera~e 1 BioI
152
Krumholz L R Esser U and Simoni RD 1989 Nucleotide sequence of the unc operon of Vibrio alginolyticus Nucleic Acids Res 177993-7994
Kubo K and Craig N 1990 Bacterial transposon Tn7 utilizes two classes of target sites 1 Bacteriol 1722774-2778
Kucharczyk N Denisot M A Le Goffic F and Badet B 1990 Glucosarnine-6-phosphate synthase from Ecoli detennination of the mechanism of inactivation by N3 fumaroyl-L-2-3 diarninoproprionic derivatives Biochemistry 293668-3676
Kusano M Takeshima T Inoue C and Sugawara K 1991 Evidence for two sets of structural genes coding for Ribulose biphosphate carboxylase in Thiobacillus ferrooxidans 1 Bacteriol 1737313-7323
Lane Dl A P Harrison lnr D Stahl B Pace SJ Giovannoni GJ Olsen and N R Pace 1992 Evolutionary relationship among sulphur- and iron-oxidizing eubacteria 1 Bacteriol 174267-278
Lee C H Bhagwhat A and Heffron F 1983 Identification of a transposon TnJ sequence required for transposition immunity Natl Acad Sci USA 806765-6769
Lewis M 1 Chang 1 A and Somoni R D 1990 A topological analysis of subunit a from Escherichia coli F1Fo-ATP synthase predicts eight transmembrane segments 1 BioI Chern 265 10541-10550
Lichtenstein C P and Brenner S 1982 Unique insertion site of Tn7 in the Ecoli chromosome Nature (London) 297601-603
Lichtenstein C P and Brenner S 1981 Site-specific properties of transposition to the Ecoli chromosome Mol Gen Genet 183380-387
Liu X Petersson S and Sandstrom A Mesophilic versus moderate thennophilic bioleaching Biohydrometallurgy Technologies Vol 1 A E Tonna 1 E Wey and Lakshmanan V 1 (ed) pp 29-38
Lizarna H M and Sankey B M 1993 Oxidation of H2S by Thiobacillus thiooxidans is inhibited by substrate and methane BiohydrometaHurgy Technologies Vol II A E Tonna 1 E Wey and C L Brierley (ed) pp 339-364
Lundgren D G and Silver M 1980 Ore leaching by bacteria Ann Rev Microbiol 34263shy268
Makino K Amemura M Shinagawa H Kobayashi A and Nakata A 1985 Sequence of the genes involved in phosphate transport and regulation of the phosphate regulon in Escherichia coli 1 Mol BioI 184231-240
Makino K Shinagawa Amemura M Kimura S Nakata A and Ishihama 1988 Euuv1J of the phosphate regulon of Ecoli Activation of pstS by the PhoB
protein in vitro 1 Mol BioI 20385-95
Makino K Shinagawa H Amemura M Yamada M and 1990 Signal transduction in the phosphate regulon of the Escherichia coli involves phosphotransfer nprlrJp~middotn PhoR and PhoB proteins J Mol BioI 21055
Malamy 1966 Frameshift mutations in the nnlnn of Escherichia coli Cold Harb Symp Quant BioI 31189-201
Malamy M H 1972 Electron microscopy insertions in the lac operon of Escherichia coli MoL Gen
Malamy M H and Bennett R L 1970 mutants of Ecoli and phosphate transport Biochem Biophys Res Commun
Maniatis T Fritsch EF Sambrook J 1982 nn A laboratory manual Cold Spring Harbor Laboratory Press Cold
McKnight G L S L Mudri SH Mathewest R S Marshall P O Sheppard and P J OHara 1990 Molecular cloning synthesis and bacterial expression of human glutaminefructose-6-phosphate UUAUU u J Chem 26725208-25212
Medveczky N and H Rosenburg 1970 phosphate-binding protein of Escherichia Biochim Biophys Acta 211 158-168
Mei B and Zalkin H 1989 A Cysteine-Histidine-Aspartate catalytic triad is involved in Glutamide Amide Transfer Function purF-type Glutamine amodotransferases 1 BioI Chem 264 16613-16619
Mengin-Lecreulx D and J van 1993 Identification of glmU gene encoding Nshyacetylglucosamine in Escherichia coli J Bacteriol 1756150shy6157
Michaelis M J and Criddle M J 1970 Mitochondrial DNA and mutants in Saccharomyces cerevisiae Biochem Genet 5487-495
Michaelis Starlinger P 1969 Two insertions in the jO v
operon having different homologous DNA sequences MoL Gen Genet 10437 377
Miller J H Calos Transposable elements Cell 20579-595
154
Miller J Oldenburg M and Fillingame R 1990 The essential carboxyl group of in subunit c the FIFo ATP synthase can be and H( +)-translocating function retained Proc NatL Acad 874900-4904
Mitcell P 1966 Chemiosmotic coupling in - and phosphorylation BioL Rev 41455-502 (1966)
Mizuuchi K 1984 Mechanism of transposition of bacteriophage Mu Polarity of the strand reaction at the initiation of the transposon Cell 39395-404
C Ph de Donato Berthelin J Surface oxidized species a key factor in the study of bioleaching processes Biohydrometallurgical In A J
Weyand V I Lakshmanan ed 1993 Vol 1 175-184
A Ishino Shinagawa H Makino K and M 1987 Nucleotide of the iap gene responsible for alkaline phosphatase isoenzyme conversion in Ecoli
identification of product 1 1695429-5433
K 1989 The tsr gene-coding prevents thiopeptin from inhibiting ppGpp synthesis in Streptomyces lividans FEMS Microbiol Lett
Ogasawara N and Yoshikawa H 1992 and their organIzatIon in the repnc()n of the bacterial chromosome Mol Microbiol 6(5)629-634
Ohtsubo Davidson N and 1975 microscope heteroduplex studies of sequence relations among plasmids identification and mapping of the insertion sequences 1 and IS2 in F and R plasmids 1 122746-775
Olson G J 1994 Microbial oxidation gold ores and gold bioleaching FEMS un Lett 119 1-6
Orle K A N L 1991 Identification of transposition prroteins by the bacterial transposon [corrected republished originally printed in Gene 1990 Nov 30 96(1) Gene 10425-31
Ouellette M Roy P Homology of ORFs from and from to site specific recombinases 1987 Nucl Acids 10055-10059
Murein synthesis p663-671ln Neidhardt J L Ingraham B Louw M~ M Schaechter H E Umbarger (ed) Escherichia coli and Salmonella
ryphimurium cellular and bioI voL 1 American Society Microbiology Washington
Perlin D S and Senior A Functional and cross-reactivity of antibody to purified subunit b (uncF protein) of Escherichia coli proton-ATPase Arch Biochem Biophys 236603-611
155
Plumbridge J A O Cochet Souza J M Altramirano M M Calcagno M L and Badet B 1993 Coordinated regulation of amino sugar-synthesizing and -degrading enzymes in Escherichia coli K-12 J Bacteriol 175(16)4951-4956
Pretorius I M Rawlings D E and Woods D R 1986 Identification and cloning of Thiobacillus jerrooxidans structural Nif genes in Escherichia coli Gene 4559-65
Qadri M I Flores C c Davis J and Lichtenstein C 1989 Genetic analysis of attTn7 the transposon Tn7 attachment site in Escherichia coli using a novel M13-based transduction assay 1 Mol BioI 20785-98
Radstrom P Skold 0 Swedberg J Roy P H and Sundstrom L 1994 Tn5090 of plasmid R751 which carries integron is related to Tn7 Mu and retroelements J Bacteriol 1763257-3268
Raetz C R H 1987 Structure and biosynthesis of lipid A in Escherichia coli pp 498-503 In F C Niedhardt J L Ingraham K B Low B Magasanik M Schaechter and H E Umbarger (ed) Escherichia coli and Salmonella typhimurium cellular and molecular biology vol 1 American Society for Microbiology Washington DC
Rao N N and Torriani A 1990 Molecular aspects of phosphate transport in Escherichia coli Mol Microbiol 4(7) 1083-1090
Rawlings DE D R Woods and NP Mjoli 1991 The cloning and structre of genes from the autotrophic biomining bacterium Thiobacillusjerrooxidans p 215-237 In PJ Greenaway (ed) Advances in gene technology vol 2 JAI Press London
Richet E and O Raibaud 1989 MalT the regulatory protein of the Escherichia coli maltose system is an A TP-dependent transcriptional activator EMBO 1 8981-987
Rogers M Ekaterrinaki N Nimmo E and Sherratt D 1986 Analysis of Tn7 transposition Mol Gen Genet 205550-556
Ronson C W Nixon B T Albright L M anf Ausubel F M 1987 Rhizobium meliloti ntrA (rpoN) gene is required for diverse metabolic functions J Bacteriol 1692424-2431
Rosenburg H Gerdes R G and Chegwidden K 1977 Two systems for the uptake of phosphate in Ecoli JBacterioL 131505-511
Rosenburg H 1987 In ion transport in Prokaryotes Rosen B P and Silver S (eds) New York Academic Press pp 205-248
Ross D G Swan 1 and N Klecker 1979 Physical structures of TnlO-promoted deletions and inversions role of 1400 base pairs inverted repetitions Cell 16721-731
156
Saedler H and P 19670 mutation in the galactose operon in Ecoli II Physiological characterization MoL Gen Genet 100 190-202
Saedler P 1968 00 and strong polar mutations in the Genet 102353-363
Sambrook J Fritsch Maniatis 1989 Molecular cloning a laboratory manual Cold Harbor Laboratory Cold Spring Harbor New York
Sand W Gehrke T Hallmann Rohde K Sobotke B and Wintzien S 1993 Bioleaching metal sulphides The importance of Leptospirillum ferrooxidans Biohydromemetallurgical Technologies Voll pp 15-25
Sanger Nicklen and Coulson A DNA sequencing with chain-tennination inhibitors Natl Acad USA 745463-5467
Schneider E and Altendorf K All the three subunits are required for an active proton channel (Fo) of Escherichia coli synthase (FIFo) ~-
518
Schneider E and Allendorf K 1987 Bacterial adenosine 5-triphosphate synthase purification and reconstitution complexes and biochemical and functional characterization of subunits Microbio Rev 51477-497
Schrader 1 A and D S Holmes 1988 Phenotypic switching Thiobacillus ferrooxidans J BacterioL 1703915-3023
Scordilis G E H and Lassie 1987 Identification of transposable elements which activates gene expression in Pseudomonas cepacia J Bacteriol 1698-13
Sekine Eisaki N and Ohtsubo E 1996 Identification and characterization of the lS3 molecules generated by breaks 1 BioL Chern 271197-202
Senior A E 1990 The proton-trans locating of Escherichia coli Annu Rev Biophys Chern 194-41
Shapiro 1 and Adhya S 1969 The jLU operon of K-12 n A deletion analysis of the structure polarity 62249-264
J A 1969 Mutations caused by insertion genenc material the galactose operon Escherichia 1 MoL BioI 4093-105
Silvennan M P 1967 Mechanism of bacterial pyrite oxidation 1 Bacteriol 941046-1051
157
C C ElIson and Levinson 1983 Identification of the typel trimethoprim resistance reductase specified by the R-plasmid R43 comparison with procaryotic and eucaryotic dihydrofolate reductase 1 Bacteriol 155 100 1-1008
Smith and Jones P transposition a multigene process Identification of a regulatory product 147915-7927
Sprague Jr Bell R M Cronan 1 A mutant of Ecoli auxotrophic for organic phosphates evidence two defects in phosphate Mol Gen Genet 14371-77
Starlinger and Michaelis 1968 Suppression the sequential aO[)ealame of the galactose enzymes in a transferase amber mutant coli Mol Genet 102367-369
Starlinger 1977 IS elements in microorganisms MicrobioL Immunol
Steffens K Schneider E Herkernhoff B Schmid Altendorf K portion of Escherichia coli A TP synthase resolution of from subunit b J BioI Chern 2625866-5869
Steffens A Deckers-hebestreit G and Altendorf K 1987b The and functional relationship of A TP synthases (FoFI) from Ecoii and the thermophilic bacterium PS3 J Biol 2626334-6338
Strominger J and M S Smith 1959 Uridine diphosphoacetylglucosamine pyrophosphorylase 1 BioI Chern 2341822-1827
Sundstrom Skold 1990 dhfrI trimethoprim resistance gene of can be found at in other genetic surroundings Antimicrob Agents Chemother 34642shy650
Sundstrom L Roy P H and Skold Site-specific of three cassettes in Tn7 J Bacteriol 1733025-3028
Surin B P and Downie JA 1988 of the Rhizobium leguminosarum nodLMN involved efficient host-specific nodulation Mol Microbiol 2 173-183
B P Dixon N and Rosenburg 1986 Purification PhoU protein a OClItnl
regulator of the pho regulon of Ecoli K-1 J Bacteriol 168631
B P D A Jans A L Fimmel Shaw GB Cox Rosenburg Structural gene for the phosphate-repressible phosphate binding of Escherichia coli
own promoter nucleotide of the phoS 1 Bacteriol
158
Suzuki I Chang W and Takeuchi 1994 Oxidation of organic compounds by Thiobacilli Symp Series 55060-67
Takeyama M S Y Noumi T Maeda Ishibashi S and Futai M 1988 Beta subunit of Ecoli amino acid replacement within a conserved sequence G-X-X-X-X-GshyK-TS) v~u binding proteins lett 218222-226
Thomson JA M Hendson and R M 1981 Mutagenesis by insertion of drug resistance Tn7 into a vibrio 11 J Bacteriol
Tichy R Grotenhuis J T c Janssen van Houten R Rulkens and Lettinga 1993 Application of the sulphur cycle bioremediation of soils polluted with heavy metals In Int Conf Contaminated 93 ed F Arendt G J Annokee R Bosman and W J van der Brink Kluwer Academic Publishers Dordrecht pp 1461-1462
G and Mayer An electron approach to the quaternary structure of mitochondrial Eur J Biochem 13237-45
J and Tschape streptothricin resistance transposons Tn1825 and Tn1826 and transposon Tn 7 Plasmidnuu
18246-249
Tso J Y Zalkin H van Cleemput M Yanofsky C and JM 1982 Nucleotide of Escherichia coli and deduced amino glutamine
phosphribosylpyrophosphate am idotransferase J BioI Chern
V Mesyanzhinova I V Koslov I A and Orlova 1984 Structure of studied by electron and image processing Lett 167285-289
P Barber C and transposons Tn5 and Tn7 in Xanthomonas campestris pv campestris MoL Gen 157
Ullrich J and van Putten P Identification of the Gonococcal glmU gene UVUUIJ
the enzyme N-acetylglucosamine 1-Phosphate Uridyltransferase involved in the synthesis UDP-GlcNAc J of Bact 1776902-6909
M 1992 Eight bacterial proteins includin]g UDP-N -acetylglucosamine acyltransferase three vother of Escherichia coli of a six-residue n tVI
theme FEMS MicrobioL 97249-254
van der Steen J J D Doddema J and de Uidoging van zware metalen afvalstromen met behulp van thiobacilli (Removal metals from waste streams
using thiobacilli) Ministry of Housing Physical and Enviromental (VROM) Directoraat-Generaal Milieubeheer Rapport 199210 Hague
Vermoote P 1988 Universite Paris VI Paris Hrllnro
159
Vignais P V Lunard Issartel J P and Dupuis A 1985 Interaction n l1f middotn
oligomycin sensitivity protein (OSCP) beef heart mitochondrial FeATPase 2 Identification of interacting Fl subunits cross-linking Biochem J
Vik S and Dao NN Prediction of transmembrane topology of Fo proteins from Ecoli ATP synthase variational hydrophobic moment analyses Biochim Biophys Acta 1140 199-207
von Meyenburg B B Jcentrgensen J Nielsen and F Hansen 1982 Promoters of the atp operon for the membrane-bound ATP synthase of Ecoli mapped by TnlO insertion mutations MoL Gen Genet 188240-248
von Meyenberg F G Hansen 1980 The origin of replication oriC the Ecoli chromosome near oriC and construction of oriC mutants ICN-UCLA Symp MoLCellBiol 191 159
Waddell S and Craig N 1989 Tn7 transposition recognition of the attTn7 Proc Natl Sci USA 863958-3962
Waddell C S and Craig N L 1988 transposition two transposition pathways directed by five Tn7-encoded genes Develop 21
J E Gay N J Saraste M and A N 1984 DNA sequence the Escherichia coli unc operon Completion of the sequence of a kilobase segment containing
oriC unc and phoS J224799-815
and Collinson 1 1994 the role the stalk in the coupling mechanism of FEBS 34639-43
Walker 1 E Gay N J and Tybulewicz V L 1986 of transposon Tn7 into the Escherichia glmS transcriptional terminator Biochem 1 243 111-1
Wanner Land McSharry 1982 Phosphate-controlled gene in Escherichia coli using Mudl-directed lacZ fusions J Mol BioI 158347-363
Weng M and Zalkin H 1987 Structural role for a region the CTP synthetase glutamide amide transfer domain J Bacteriol 1693023-3028
Wheatcroft R and S and nucleotide sequence of Rhizobiwn meliloti insertion between the transposase encoded by ISRm3 and those encoded by Staphylococcus aureus and Thiobacillus ferrooxidans IST2 J 1732530-2538
160
Whitby M C and Lloyd R 1995 Branch of three-strand recombination intennediates by RecG a possible pathway for securing middotIULl initiated by duplex DNA 1 143303-3310
Wilkens and Capaldi R A Assymmetry and changes in examined by cryoelectronmicroscopy BioI Chern Hoppe-Seyler 37543-51
and Malamy M 1974 The loss of the PhoS periplasmic protein leads to a the specificity a constitutive phosphate transport system in Escherichia coli Biochem Res Commun 60226-233
WiUsky G Rand Malamy M 1980 of two separable inorganic phosphate transport systems in Escherichia coli J BacterioL 144356-365
P J and and C F 1973 enzyme of metabolism and Stadtman R eds) pp 343-363 Academic Press New York
Winterbum P J and Phelps F 197L control of hexosamine biosynthesis by glucosamine synthetase Biochem 1 121711
Wolf-Watz H and Norquist A 1979 Deoxyribonucleic acid and outer membrane protein to outer membrane protein involves a protein J 14043-49
Wolf-Watz H and M 1979 Deoxyribonucleic acid and outer membrane strains for oriC elevated levels deoxyribonucleic acid-binding protein and
11 for specific UU6 of the oriC region to outer membrane J Bacteriol 14050-58
Wolf-Watz H 1984 Affinity of two different chromosome to the outer membrane of Ecoli J Bacteriol 157968-970
Wu C and Te Wu 197 L Isolation and characterization of a glucosamine-requiring mutant of Escherichia 12 defective in glucoamine-6-phosphate synthetase 1 Bacteriol 105455-466
Yamada M Makino Shinagawa Nakata A 1990 Regulation of the phosphate regulon of Escherichia coli properties the pooR mutants and subcellular localization of protein Mol Genet 220366-372
Yates J R and Holmes D S 1987 Two families of repeatcXl DNA sequences Thiobacillus 1 Bacteriol 169 1861-1870
Yates J R R P and D S 1988 an insertion sequence from Thiobacillus errooxidans Proc Natl 857284-7287
161
Youvan D c Elder 1 T Sandlin D E Zsebo K Alder D P Panopoulos N 1 Marrs B L and Hearst 1 E 1982 R-prime site-directed transposon Tn7 mutagenesis of the photosynthetic apparatus in Rhodopseudomonas capsulata 1 Mol BioI 162 17-41
Zalkin H and Mei B 1990 Amino terminal deletions define a glutamide transfer domain in glutamine phosphoribosylpyrophosphate ami do transferase and other purF-type amidotransferases 1 Bacteriol 1723512-3514
Zalkin H and Weng M L 1987 Structural role for a conserved region III the CTP synthetase glutamide amide transfer domain 1 Bacteriol 169 (7)3023-3028
Zhang Y and Fillingame R H 1994 Essential aspartate in subunit c of FIF0 ATP synthase Effect of position 61 substitutions in helix-2 on function of Asp24 in helix-I 1 BioI Chern 2695473-5479
Subc10ning and single strand sequencing of DNA fragments covering a region of about 7 kb
beyond the 35 kb BamHI-BamHI fragment were carried out and the sequences searched
against the GenBank and EMBL databases Sequences homologous to the TnsBCD proteins
of Tn7 were found The TnsD-like protein of the Tn7-like element (registered as Tn5468) was
found to be shuffled truncated and rearranged Homologous sequence to the TnsE and the
antibiotic resistance markers of Tn7 were not found Instead single strand sequencing of a
further 35 kb revealed sequences which suggested the T ferrooxidans spo operon had been
encountered High amino acid sequence homology to two of the four genes of the spo operon
from Ecoli and Hinjluenzae namely spoT encoding guanosine-35-bis (diphosphate) 3shy
pyrophosphohydrolase (EC 3172) and recG encoding ATP-dependent DNA helicase RecG
(EC 361) was found This suggests that Tn5468 is incomplete and appears to terminate with
the reshuffled TnsD-like protein The orientation of the spoT and recG genes with respect to
each other was found to be different in T ferrooxidans compared to those of Ecoli and
Hinjluenzae
11 Bioleaching 1
112 Organism-substrate raction 1
113 Leaching reactions 2
114 Industrial applicat 3
12 Thiobacillus ferrooxidans 4
13 Region around the Ecoli unc operon 5
131 Region immediately Ie of the unc operon 5
132 The unc operon 8
1321 Fe subun 8
1322 subun 10
133 Region proximate right of the unc operon (EcoURF 1) 13
1331 Metabolic link between glmU and glmS 14
134 Glucosamine synthetase (GlmS) 15
135 The pho 18
1351 Phosphate uptake in Ecoli 18
1352 The Pst system 19
1353 Pst operon 20
1354 Modulation of Pho uptake 21
136 Transposable elements 25
1361 Transposons and Insertion sequences (IS) 27
1362 Tn7 27
13621 Insertion of Tn7 into Ecoli chromosome 29
13622 The Tn7 transposition mechanism 32
137 Region downstream unc operons
of Ecoli and Tferrooxidans 35
LIST OF FIGURES
Fig
Fig
Fig
Fig
Fig
Fig
Fig
Fig
la
Ib
Ie
Id
Ie
If
Ig
Ih
6
11
14
22
23
28
30
33
1
CHAPTER ONE
INTRODUCTION
11 BIOLEACHING
The elements iron and sulphur circulate in the biosphere through specific paths from the
environment to organism and back to the environment Certain paths involve only
microorganisms and it is here that biological reactions of relevance in leaching of metals from
mineral ores occur (Sand et al 1993 Liu et aI 1993) These organisms have evolved an
unusual mode of existence and it is known that their oxidative reactions have assisted
mankind over the centuries Of major importance are the biological oxidation of iron
elemental sulphur and mineral sulphides Metals can be dissolved from insoluble minerals
directly by the metabolism of these microorganisms or indrectly by the products of their
metabolism Many metals may be leached from the corresponding sulphides and it is this
process that has been utilized in the commercial leaching operations using microorganisms
112 Or2anismSubstrate interaction
Some microorganisms are capable of direct oxidative attack on mineral sulphides Scanning
electron micrographs have revealed that numerous bacteria attach themselves to the surface
of sulphide minerals in solution when supplemented with nutrients (Benneth and Tributsch
1978) Direct observation has indicated that bacteria dissolve a sulphide surface of the crystal
by means of cell contact (Buckley and Woods 1987) Microbial cells have also been shown
to attach to metal hydroxides (Kennedy et ai 1976) Silverman (1967) concluded that at
least two roles were performed by the bacteria in the solubilization of minerals One role
involved the ferric-ferrous cycle (indirect mechanism) whereas the other involved the physical
2
contact of the microrganism with the insoluble sulfide crystals and was independent of the
the ferric-ferrous cycle Insoluble sulphide minerals can be degraded by microrganisms in the
absence of ferric iron under conditions that preclude any likely involvement of a ferrous-ferric
cycle (Lizama and Sackey 1993) Although many aspects of the direct attack by bacteria on
mineral sulphides remain unknown it is apparent that specific iron and sulphide oxidizers
must play a part (Mustin et aI 1993 Suzuki et al 1994) Microbial involvement is
influenced by the chemical nature of both the aqueous and solid crystal phases (Mustin et al
1993) The extent of surface corrosion varies from crystal to crystal and is related to the
orientation of the mineral (Benneth and Tributsch 1978 Claassen 1993)
113 Leachinamp reactions
Attachment of the leaching bacteria to surfaces of pyrite (FeS2) and chalcopyrite (CuFeS2) is
pSIS1 construct was further subcloned to plasm ids IS30 IS40 pSlS50
pS183S p81841 and p81 (Fig Some of subclones were extensively mapped
although not all sites are shown in 22 exact positions of the ends of
T jerrooxidans plasmids pTfatpl and pTfatp2 on the cosmid p8I81 were identified
21)
Tfeooxidans
that the cosmid was
unrearranged DNA digests of cosmid pSISI and T jerrooxidans chromosomal DNA were
with pSI The of the bands gave a positive hybridization signal were
identical each of the three different restriction enzyme digests of p8I8l and chromosomal
In order to confirm of pSI81 and to
49
kb kb ~
(A)
11g 50Sts5
middot 2S4
17 bull tU (probe)
- tosect 0S1
(e)
kb
+- 10 kb
- 46 kb
28---+ 26-+
17---
Fig 23 Hybrdization of cosmid pSISI and T jerrooxitians (A TCC 33020) chromosomal
DNA by the KpnI-SalI fragment of pSIS52 (probe) (a) Autoradiographic image of the
restriction digests Lane x contains the probe lane 13 and 5 contain pSISI restricted with BamHl HindIII and Bgffi respectively Lanes 2 4 and 6 contain T errooxitians chromosomal DNA also restricted with same enzymes in similar order (b) The sizes of restricted pSISI
and T jerrooxitians chromosome hybridized by the probe The lanes correspond to those in Fig 23a A DNA digested with Pst served as the molecular weight nlUkermiddot
50
DNA (Fig BamHI digests SHklC at 26 and 2S kb 1 2) HindIII
at 10 kb 3 and 4) and BgnI at 46 (lanes 5 and 6) The signals in
the 181 was because the purified KpnI-San probe from p81 had a small quantity
ofcontaminating vector DNA which has regions ofhomology to the cosmid
vector The observation that the band sizes of hybridizing
for both pS181 and the T ferrooxidans ATCC 33020 same
digest that the region from BgnI site at to HindIII site at 16
kb on -VJjUIU 1 represents unrearranged chromosomal DNA from T ferrooxidans
ATCC there were no hybridization signals in to those predicted
the map with the 2 4 and 6 one can that are no
multiple copies of the probe (a Tn7-like segment) in chromosome of
and Lferrooxidans
of plasmid pSI was found to fall entirely within a Tn7-like trallSDOS()n nrpn
on chromosome 33020 4) A Southern hybridizationUUAcpoundUU
was out to determine whether Tn7-like element is on other
ofT ferrooxidans of T and Lferrooxidans results of this
experiment are shown 24 Lanes D E and F 24 represent strains
19377 DSM and Lferrooxidans DSM 2705 digested to
with BgnI enzyme A hybridization was obtained for
the three strains (lane A) ATCC 19859 (lane B) and UUHUltn
51
(A) AAB CD E F kb
140
115 shy
284 -244 = 199 -
_ I
116 -109 -
08
os -
(8)) A B c o E F
46 kb -----
Fig 24 (a) Autoradiographic image of chromosomal DNA of T ferrooxidans strains ATCC 33020 19859 23270 (lanes A B C) Tthiooxidans strains ATCC 19377 and DSM 504 (D and E) and Lferrooxidans strain DSM 2705 (lane F) all restricted with Bgffi A Pst molecular weight marker was used for sizing (b) Hybridization of the 17 kb KpnI-San piece of p81852 (probe) to the chromosomal DNA of the organisms mentioned 1 Fig 24a The lanes in Fig 24a correspond to those in Fig 24b
(lane C) uuvu (Fig 24) The same size BgllI fragments (46 kb) were
lanes A Band C This result
different countries and grown on two different
they Tn7-like transposon in apparently the same location
no hybridization signal was obtained for T thiooxidans
1 504 or Lferrooxidans DSM 2705 (lanes D E and F of
T thiooxidans have been found to be very closely related based on 1
rRNA et 1992) Since all Tferrooxidans and no Tthiooxidans
~~AA~~ have it implies either T ferrooxidans and
diverged before Tn7-like transposon or Tthiooxidans does not have
an attTn7 attachment the Tn7-like element Though strains
T ferrooxidans were 10catH)uS as apart as the USA and Japan
it is difficult to estimate acquired the Tn7-like transposon as
bacteria get around the world are horizontally transmitted It
remains to be is a general property
of all Lferrooxidans T ferrooxidans strains
harbour this Tn7-like element their
CHAPTER 3
5431 Summary
54Introduction
56Materials and methods
56L Bacterial and plasmid vectors
Media and solutions
56333 DNA
57334 gel electrophoresis
Competent preparation
57336 Transformation of DNA into
58337 Recombinant DNA techniques
58ExonucleaseIII shortening
339 DNA sequencing
633310 Complementation studies
64Results discussion
1 DNA analysis
64Analysis of ORF-l
72343 Glucosamine gene
77344 Complementation of by T ferrooxidans glmS
57cosmid pSIS1 pSlS20
58Plasmidmap of p81816r
Fig Plasmidmap of lS16f
60Fig 34 Restriction digest p81S1Sr and pSIS16f with
63DNA kb BamHI-BamHI
Fig Codon and bias plot of the DNA sequence of pSlS16
Fig 37 Alignment of C-terminal sequences of and Bsubtilis
38 Alignment of amino sequences organisms with high
homology to that of T ferrooxidans 71
Fig Phylogenetic relationship organisms high glmS
sequence homology to Tferrooxidans
31 Summary
A 35 kb BamHI-BamHI p81820 was cloned into pUCBM20 and pUCBM21 to
produce constructs p818 and p81816r respectively This was completely
sequenced both rrelct1()ns and shown to cover the entire gmS (184 kb) Fig 31
and 34 The derived sequence of the T jerrooxidans synthetase was
compared to similar nrvnC ~ other organisms and found to have high sequence
homology was to the glucosamine the best studied
eubacterium EcoU Both constructs p81816f p81 the entire
glmS gene of T jerrooxidans also complemented an Ecoli glmS mutant for growth on medium
lacking N-acetyl glucosamine
32 =-====
Cell wall is vital to every microorganism In most procaryotes this process starts
with amino which are made from rructClsemiddotmiddotn-[)ll()S by the transfer of amide group
to a hexose sugar to form an This reaction is by
glucosamine synthetase the product of the gmS part of the catalysis
to the 40-residue N-terminal glutamine-binding domain (Denisot et al 1991)
participation of Cys 1 to generate a glutamyl thiol ester and nascent ammonia (Buchanan
368-residue C-terminal domain is responsible for the second part of the ClUUU
glucosamine 6-phosphate This been shown to require the ltgtttrltgtt1iltn
of Clpro-R hydrogen of a putative rrulctoseam 6-phosphate to fonn a cis-enolamine
intennediate which upon reprotonation to the gives rise to the product (Golinelli-
Pimpaneau et al 1989)
bacterial enzyme mn two can be separated by limited chymotryptic
proteolysis (Denisot et 199 binding domain encompassing residues 1 to
240 has the same capacity to hydrolyse (and the corresponding p-nitroanilide
derivative) into glutamate as amino acid porI11
binding domain is highly cOllserveu among members of the F-type
ases Enzymes in this include amidophosphophoribosyl et al
1982) asparagine synthetase (Andrulis et al 1987) glucosamine-6-P synmellase (Walker et
aI 1984) and the NodM protein of Rhizobium leguminosarum (Surin and 1988) The
368-residue carboxyl-tenninal domain retains the ability to bind fructose-6-phosphate
The complete DNA sequence of T ferrooxidans glmS a third of the
glmU gene (preceding glmS) sequences downstream of glmS are reported in this
chapter This contains a comparison of the VVA r glmS gene
and Mycobacterium (422 ) An interesting observation was that the T ferrooxidans
glmS gene had comparatively high homology sequences to the Rhizobium leguminosarum and
Rhizobium meliloti M the amino to was 44 and 436
respectively A consensus Dalgarno upstream of the start codon
(ATG) is in 35 No Ecoli a70-type n r~ consensus sequence was
detected in the bp preceding the start codon on intergenic distance
between the two and the absence of any promoter consensus sequence Plumbridge et
al (1993) that the Ecoli glmU and glmS were co-transcribed In the case
the glmU-glmS --n the T errooxidans the to be similar The sequences
homology to six other amidotransferases (Fig 38) which are
(named after the purF-encoded l phosphoribosylpyrophosphate
amidotransferase) and Weng (1987)
The glutamine amide transfer domain of approximately 194 amino acid residues is at the
of protein chain Zalkin and Mei (1989) using site-directed to
the 9 invariant amino acids in the glutamine amide transfer domain of
phosphoribosylpyrophosphated indicated in their a
catalytic triad is involved glutamine amide transfer function of
73
Comparison of the ammo acid sequence alignment of ghtcosamine-6-phosphate
amidotranferases (GFAT) of Rhizobium meliloti (R_m) Rhizobium leguminnosarum (RJ)
Ecoli (E_c) Hinjluenzae Mycobacterium leprae (M_I) Bsubtilis (B_s) and
Scerevisiae (S_c) to that of Tferrooxidans Amino acids are identified by their single
codes The asterisks () represent homologous amino acids of Tferrooxidans glmS to
at least two of the other Consensus ammo acids to all eight organisms are
highlighted (bold and underlined)
1 50 R m i bullbullbullbullbullbull hkps riag s tf bullbullbullbull R~l i bullbullbullbullbullbull hqps rvaep trod bullbullbullbull a
a bullbullbullbullbullbull aa 1r 1 d bullbullbull a a bullbullbullbullbullbull aa inh g bullbullbullbullbullbull sktIv pmilq 1 1g bullbullbull a MCGIVi V D Gli RI IYRGYPSAi AI D 1 gvmar s 1gsaks Ikek a bullbullbullbull qfcny Iversrgi tvq t dgd bullbull ead
51 100 R m hrae 19ktrIk bull bullbull bullbull s t ateR-l tqrae 19rkIk bullbullbull s t atrE-c htIrI qmaqae bull bull h bullbull h gt sv aae in qicI kads stbull bullbull k bullbull i gt T f- Ivsvr aetav bullbullbull r bullbull gq qg c
DL R R G V L AV E L G VGI lTRW E H ntvrrar Isesv1a mv bull sa nIgi rtr gihvfkekr adrvd bullbullbull bullbull nve aka g sy1stfiykqi saketk qnnrdvtv she reqv
101 150 R m ft g skka aevtkqt R 1 ft g ak agaqt E-c v hv hpr krtv aae in sg tf hrI ksrvlq T f- m i h q harah eatt
S E Eamp L A GY l SE TDTEVIAHL ratg eiavv av
qsaIg lqdvk vqv cqrs inkke cky
151 200 bullbull ltk bullbull dgcm hmcve fe a v n bull Iak bullbull dgrrm hmrvk fe st bull bull nweI bullbull kqgtr Iripqr gtvrh 1 s bull bull ewem bullbullbull tds kkvqt gvrh hlvs bull bull hh bullbull at rrvgdr iisg evm
V YR T DLF A A L AV S D PETV AR G M 1 eagvgs lvrrqh tvfana gvrs B s tef rkttIks iInn rvknk 5 c Ihlyntnlqn ~lt klvlees glckschy neitk
74
201 R m ph middot middot middot R 1 ah- middot middot middot middot middot middot E c 1 middot middot middot middot middot middot middot Hae in 1 middot middot middot middot middot T f - c11v middot middotmiddot middot middot middot middot middot M 1 tli middot middot middot middot middot middot middot middot middot B s 11 middot middot middot middot middot S c lvkse kk1kvdfvdv
251 R m middot middot middot middot middot middot middot middot middot middot middot middot middot middot
middot middot middot middot R-1 middot middot middot middot middot middot E c middot middot middot middot middot middot middot middot Hae in middot middot middot middot middot middot middot middot middot middot middot middot T f shy middot middot middot middot middot middot middot middot middot middot middot middot 1M middot middot middot middot middot middot middot middot middot middot middot middot middot middot B s middot middot middot middot middot middot middot middot middot middot middot middot middot middot middot S c feagsqnan1 1piaanefn1
301 R m fndtn wgkt R-1 fneti wgkt E c rf er Hae in srf er T f- rl mlqq
PVTR V YE DGDVA R M 1 ehqaeg qdqavad B s qneyem kmvdd S c khkkl dlhydg
351 R m nhpe v R-1 nhe v E c i nk Hae in k tli T f- lp hr
~ YRHlrM~ ~I EQP AVA M 1 geyl av B-s tpl tdvvrnr S-c pd stf
401 Rm slasa lk R-1 slasca lk E c hql nssr Hae in hq nar-T f f1s hytrq
RVL A SiIS A VG W M 1 ka slakt B s g kqS c lim scatrai
T ferrooxidans (Tf) Scerevisiae (Saee) Mleprae (Myele) and Bsublilis (Baesu)
tr1 ()-ltashyc 8 ~ er (11
-l l
CIgt
~ r (11-CIgt (11
-
$ -0 0shy
a c
omiddot s
S 0 0shyC iii omiddot $
ttl ~ () tI)
I-ltashyt ()
0
~ smiddot (11
81
CHAPTER 4
8341 Summary
8442 Introduction
8643 Materials and methods
431 Bacterial strains and plasmids 86
432 DNA constructs subclones and shortenings 86
864321 Contruct p81852
874322 Construct p81809
874323 Plasmid p8I809 and its shortenings
874324 p8I8II
8844 Results and discussion
441 Analysis of the sequences downstream the glmS gene 88
442 TnsBC homology 96
99443 Homology to the TnsD protein
118444 Analysis of DNA downstream of the region with TnsD homology
LIST OF FIGURES
87Fig 41 Alignment of Tn7-like sequence of T jerrooxidans to Ecoli Tn7
Fig 42 DNA sequences at the proximal end of Tn5468 and Ecoli glmS gene 88
Fig 43 Comparison of the inverted repeats of Tn7 and Tn5468 89
Fig 44 BLAST results of the sequence downtream of T jerrooxidans glmS 90
Fig 45 Alignment of the amino acid sequence of Tn5468 TnsA-like protein 92 to TnsA of Tn7
82
Fig 46 Restriction map of the region of cosmid p8181 with amino acid 95 sequence homology to TnsABC and part of TnsD of Tn7
Fig 47 Restriction map of cosmid p8181Llli with its subclonesmiddot and 96 shortenings
98 Fig 48 BLAST results and DNA sequence of p81852r
100 Fig 49 BLAST results and DNA sequence of p81852f
102 Fig 410 BLAST results and DNA sequence of p81809
Fig 411 Diagrammatic representation of the rearrangement of Tn5468 TnsDshy 106 like protein
107Fig 412 Restriction digests on p818lLlli
108 Fig 413 BLAST results and DNA sequence of p818lOEM
109 Fig 414 BLAST results on joined sequences of p818104 and p818103
111 Fig 415 BLAST results and DNA sequence of p818102
112 Fig 416 BLAST results and nucleotide sequence of p818101
Fig 417 BLAST results and DNA sequence of the combined sequence of 113p818l1f and p81810r
Fig 418 Results of the BLAST search on p81811r and the DNA sequence 117obtained from p81811r
Fig 419 Comparison of the spa operons of Ecali Hinjluenzae and 121 probable structure of T ferraaxidans spa operon
83
CHAPTER FOUR
TN7-LlKE TRANSPOSON OF TFERROOXIDANS
41 Summary
Various constructs and subclones including exonuclease ill shortenings were produced to gain
access to DNA fragments downstream of the T ferrooxidans glmS gene (Chapter 3) and
sequenced Homology searches were performed against sequences in GenBank and EMBL
databases in an attempt to find out how much of a Tn7-like transposon and its antibiotic
markers were present in the T ferrooxidans chromosome (downstream the glmS gene)
Sequences with high homology to the TnsA TnsB TnsC and TnsD proteins of Tn7 were
found covering a region of about 7 kb from the T ferrooxidans glmS gene
Further sequencing (plusmn 45 kb) beyond where TnsD protein homology had been found
revealed no sequences homologous to TnsE protein of Tn7 nor to any of the antibiotic
resistance markers associated with Tn7 However DNA sequences very homologous to Ecoli
ATP-dependentDNA helicase (RecG) protein (EC 361) and guanosine-35-bis (diphosphate)
3-pyrophosphohydrolase (EC 3172) stringent response protein of Ecoli HinJluenzae and
Vibrio sp were found about 15 kb and 4 kb respectively downstream of where homology to
the TnsD protein had been detected
84
42 Transposable insertion sequences in Thiobacillus ferrooxidizns
The presence of two families (family 1 and 2) of repetitive DNA sequences in the genome
of T ferrooxidans has been described previously (Yates and Holmes 1987) One member of
the family was shown to be a 15 kb insertion sequence (IST2) containing open reading
frames (ORFs) (Yates et al 1988) Sequence comparisons have shown that the putative
transposase encoded by IST2 has homology with the proteins encoded by IS256 and ISRm3
present in Staphylococcus aureus and Rhizobium meliloti respectively (Wheatcroft and
Laberge 1991) Restriction enzyme analysis and Southern hybridization of the genome of
T ferrooxidans is consistent with the concept that IST2 can transpose within Tferrooxidans
(Holmes and Haq 1990) Additionally it has been suggested that transposition of family 1
insertion sequences (lST1) might be involved in the phenotypic switching between iron and
sulphur oxidizing modes of growth including the reversible loss of the capacity of
T ferrooxidans to oxidise iron (Schrader and Holmes 1988)
The DNA sequence of IST2 has been determined and exhibits structural features of a typical
insertion sequence such as target-site duplications ORFs and imperfectly matched inverted
repeats (Yates et al 1988) A transposon-like element Tn5467 has been detected in
T ferrooxidans plasmid pTF-FC2 (Rawlings et al 1995) This transposon-like element is
bordered by 38 bp inverted repeat sequences which has sequence identity in 37 of 38 and in
38 of 38 to the tnpA distal and tnpA proximal inverted repeats of Tn21 respectively
Additionally Kusano et al (1991) showed that of the five potential ORFs containing merR
genes in T ferrooxidans strain E-15 ORFs 1 to 3 had significant homology to TnsA from
transposon Tn7
85
Analysis of the sequence at the tenninus of the 35 kb BamHI-BamHI fragment of p81820
revealed an ORF with very high homology to TnsA protein of the transposon Tn7 (Fig 44)
Further studies were carried out to detennine how much of the Tn7 transposition genes were
present in the region further downstream of the T ferroxidans glmS gene Tn7 possesses
trimethoprim streptomycin spectinomycin and streptothricin antibiotic resistance markers
Since T ferrooxidans is not exposed to a hospital environment it was of particular interest to
find out whether similar genes were present in the Tn7-like transposon In the Ecoli
chromosome the Tn7 insertion occurs between the glmS and the pho genes (pstS pstC pstA
pstB and phoU) It was also of interest to detennine whether atp-glm-pst operon order holds
for the T ferrooxidans chromosome It was these questions that motivated the study of this
region of the chromosome
43 OJII
strains and plasm ids used were the same as in Chapter Media and solutions (as
in Chapter 3) can in Appendix Plasmid DNA preparations agarose gel
electrophoresis competent cell preparations transformations and
were all carned out as Chapter 3 Probe preparation Southern blotting hybridization and
detection were as in Chapter 2 The same procedures of nucleotide
DNA sequence described 2 and 3 were
4321 ~lllu~~~
Plasmid p8I852 is a KpnI-SalI subclone p8I820 in the IngtUMnt vector kb) and
was described Chapter 2 where it was used to prepare the probe for Southern hybridization
DNA sequencing was from both (p81852r) and from the Sail sites
(p8I852f)
4322 ==---==
Construct p81809 was made by p8I820 The resulting fragment
(about 42 kb) was then ligated to a Bluescript vector KS+ with the same
enzymes) DNA sequencing was out from end of the construct fA
87
4323 ~mlJtLru1lbJmalpoundsectM~lllgsect
The 40 kb EeoRI-ClaI fragment p8181Llli into Bluescript was called p8181O
Exonuclease III shortening of the fragment was based on the method of Heinikoff (1984) The
vector was protected with and ClaI was as the susceptible for exonuclease III
Another construct p818IOEM was by digesting p81810 and pUCBM20 with MluI and
EeoRI and ligating the approximately 1 kb fragment to the vector
4324 p81811
A 25 ApaI-ClaI digest of p8181Llli was cloned into Bluescript vector KS+ and
sequenced both ends
88
44 Results and discussion
of glmS gene termini (approximately 170
downstream) between
comparison of the nucleotide
coli is shown in Fig 41 This region
site of Tn7 insertion within chromosome of E coli and the imperfect gtpound1
repeat sequences of was marked homology at both the andVI
gene but this homology decreased substantially
beyond the stop codon
amino acid sequence
been associated with duplication at
the insertion at attTn7 by (Lichtenstein and as
CCGGG in and underlined in Figs41
CCGGG and are almost equidistant from their respelt~tle
codons The and the Tn7-like transposon BU- there is
some homology transposons in the regions which includes
repeats
11 inverted
appears to be random thereafter 1) The inverted
repeats of to the inverted repeats of element of
(Fig 43) eight repeats
(four traJrlsposcm was registered with Stanford University
California as
A search on the (GenBank and EMBL) with
of 41 showed high homology to the Tn sA protein 44) Comparison
acid sequence of the TnsA-like protein Tn5468 to the predicted of the
89
Fig 41
Alignment of t DNA sequence of the Tn7-li e
Tferrooxi to E i Tn7 sequence at e of
insertion transcriptional t ion s e of
glmS The ent homology shown low occurs within
the repeats (underl of both the
Tn7-li transposon Tn7 (Fig 43) Homology
becomes before the end of t corresponding
impe s
T V E 10 20 30 40 50 60
Ec Tn 7
Tf7shy(like)
70 80 90 100 110 120
Tf7shy(1
130 140 150 160 170 180 Ec Tn 7 ~~~=-
Tf7shy(like)
90
Fig 42 DNA sequences at the 3 end of the Ecoll glmS and the tnsA proximal of Tn7 to the DNA the 3end of the Tferrooxidans gene and end of Tn7-like (a) DNA sequence determined in the Ecoll strain GD92Tn7 in which Tn been inserted into the glmS transcriptional terminator Walker at ai 1986 Nucleotide 38 onwards are the of the left end of Tn DNA
determined in T strain ATCC 33020 with the of 7-like at the termination site of the glmS
gene Also shown are the 22 bp of the Tn7-like transposon as well as the region where homology the protein begins A good Shine
sequence is shown immediately upstream of what appears to be the TTG initiation codon for the transposon
(a)
_ -35 PLE -10 I tr T~ruR~1 truvmnWCIGACUur ~~GCfuA
100 no 110 110 110
4 M S S bull S I Q I amp I I I I bull I I Q I I 1 I Y I W L ~Tnrcmuamaurmcrmrarr~GGQ1lIGTuaDACf~1lIGcrA
DO 200 amp10 220 DG Zlaquot UO ZIG riO
T Q IT I I 1 I I I I I Y sir r I I T I ILL I D L I ilGIIilJAUlAmrC~GlWllCCCAcarr~GGAWtCmCamp1tlnmcrArClGACTAGNJ
Fig 45 (a) Alignment of the amino acid sequence of Tn5468 (which had homology to TnsA of Tn7 Fig 44) to the amino acid sequence of ORF2 (between the merR genes of Tferrooxidans strain E-1S) ORF2 had been found to have significant homology to Tn sA of Tn7 (Kusano et ai 1991) (b) Alignment of the amino acids translation of Tn5468 (above) to Tn sA of Tn7 (c) Alignment of the amino acid sequence of TnsA of Tn7 to the hypothetical ORF2 protein of Tferrooxidans strain E-1S
(al
Percent Similarity 60000 Percent Identity 44444
Tf Tn5468 1 LARQRYGVDEDRVARFQKEGRGQGRGADYHPWLTIQDVPSQGRSHRLKGI SO 11 1111111 I 1111 1111 I I 111
Fig 49 (a) Results of the BLAST search on the inverted and complemented nucleotide sequence of 1852f (from the Sall site) Results indicate amino acid sequence to the TnsC of Tn7 (protein E is the same as TnsC protein) (b) The nucleotide sequence and open reading frame of p81852f
High Prob producing Segment Pairs Frame Score P(N)
IB255431QQECE7 protein E - Escher +1 176 15e-20 gplX044921lSTN7EUR 1 Tn7 E with +1 176 15e-20 splP058461 ECOLI TRANSPOSON TN7 TRANSPOSITION PR +1 176 64e-20 gplU410111 4 D20248 gene product [Caenorhab +3 59 17e-06
IB255431QQECE7 ical protein E - Escherichia coli transposon Tn7 (fragment)
417 (al BLAST results obtained from combined sequence of (inverted and complemented) and 1810r good sequence to Ecoli and Hinfluenzae DNA RecG (EC 361) was found (b) The combined sequence and open frame which was homologous to RecG
444 Analysis of DNA downstream of region with TnsD homology
The location of the of p81811 ApaI-CLaI construct is shown in Fig 47 The single strand
sequence from the C LaI site was joined to p8181Or and searched using BLAST against the
GenBank and EMBL databases Good homology to Ecoli and Hinjluezae A TP-dependent
DNA helicase recombinase proteins (EC 361) was obtained (Fig 417) The BLAST search
with the sequence from the ApaI end showed high sequence homology to the stringent
response protein guanosine-35-bis (diphosphate) 3-pyrophosphohydrolase (EC 3172) of
Ecoli Hinjluenzae and Scoelicolor (Fig 418) In both Ecoli and Hinjluenzae the two
proteins RecG helicase recombinase and ppGpp stringent response protein constitute part of
the spo operon
445 Spo operon
A brief description will be given on the spo operons of Ecoli and Hinjluenzae which appear
to differ in their arrangements In Ecoli spoT gene encodes guanosine-35-bis
pyrophosphohydrolase (ppGpp) which is synthesized during stringent response to amino acid
starvation It is also known to be responsible for cellular ppGpp degradation (Gentry and
Cashel 1995) The RecG protein is required for normal levels of recombination and DNA
repair RecG protein is a junction specific DNA helicase that acts post-synaptically to drive
branch migration of Holliday junction intermediate made by RecA during the strand
exchange stage of recombination (Whitby and Lloyd 1995)
The spoS (also called rpoZ) encodes the omega subunit of RNA polymerase which is found
associated with core and holoenzyme of RNA polymerase The physiological function of the
omega subunit is unknown Nevertheless it binds stoichiometrically to RNA polymerase
119
Fig 418 BLAST search nucleotide sequence of 1811r I restrict ) inverted and complement high sequence homology to st in s 3 5 1 -bis ( e) 3 I ase (EC 31 7 2) [(ppGpp)shyase] -3-pyropho ase) of Ecoli Hinfluenzae (b) The nucleot and the open frame with n
Add 4 ml 52 mix by inversion at room temp for 5 mins
Add 4 ml 53 Mix by to homogenous suspension
Spin at 15 K 40 mins at 4
remove to fresh tube
Equilibrate column with 2 ml N2
Load supernatant 2 to 4 ml amounts
Wash column 2 X 4 of N3 Elute the DNA the first bed
each) add 07
volumes of isopropanol
Spin at 4 Wash with 70 Ethanol Resuspended pellet in n rv 100 AU of TE and scan
volume of about 8 to 10 drops) To the eluent (two
to
140
SEQUITHERM CYCLE SEQUENCING
Alf-express Cy5 end labelled promer method
only DNA transformed into end- Ecoli
The label is sensitive to light do all with fluorescent lights off
3-5 kb
3-7 kb
7-10 kb
Thaw all reagents from kit at well before use and keep on
1) Label 200 PCR tubes on or little cap flap
(The heated lid removes from the top of the tubes)
2) Add 3 Jtl of termination mixes to labelled tubes
3) On ice with off using 12 ml Eppendorf DNA up to
125 lll with MilliQ water
Add 1 ltl
Add lll of lOX
polymeraseAdd 1 ltl
Mix well Aliquot 38 lll from the eppendorf to Spin down
Push caps on nrnnp1
141
Hybaid thermal Cycler
Program
93 DC for 5 mins 1 cycle
93degC for 30 sees
55 DC for 30 secs
70 DC for 60 secs 30 cycle 93 DC_ 30s 55 DC-30s
70degC for 5 mins 1 cycle
Primer must be min 20 bp long and min 50 GC content if the annealing step is to be
omitted Incubate at 95 DC for 5 mins to denature before running Spin down Load 3 U)
SEQUITHERM CYCLE SEQUENCING
Ordinary method
Use only DNA transformed into end- Ecoli strain
3-5 kb 3J
3-7 kb 44
7-10 kb 6J
Thaw all reagents from kit at RT mix well before use and keep on ice
1) Label 200 PCR tubes on side or little cap flap
(The heated lid removes markings from the top of the tubes)
2) Add 3 AU of termination mixes to labelled tubes
3) On ice using l2 ml Eppendorf tubes make DNA up to 125 41 with MilliQ water
142
Add I Jtl of Primer
Add 25 ttl of lOX sequencing buffer
Add 1 Jtl Sequitherm DNA polymerase
Mix well spin Aliquot 38 ttl from the eppendorf to each termination tube Spin down
Push caps on properly
Hybaid thermal Cycler
Program
93 DC for 5 mins 1 cycle
93 DC for 30 secs
55 DC for 30 secs
70 DC for 60 secs 30 cycles
93 DC_ 30s 55 DC-30s 70 DC for 5 mins 1 cycle
Primer must be minimum of 20 bp long and min 50 GC content if the annealing step is
to be omitted Incubate at 95 DC for 5 mins to denature before running
Spin down Load 3 ttl for short and medium gels 21t1 for long gel runs
143
REFERENCES
144
REFERENCES
Abrahams J P Lutter R Todd R J van Raaij M J Leslie A G W and Walker J E 1993 Inherent asymmetry of the structure of F1-ATPase from bovine heart mitochondria at 65 Aresolution EMBO J 121775-1780
Abrahams J P Leslie A G W Lutter R and Walker 1 E 1994 Structure at 28 A resolution of FeATPase from bovine heart mitochondria Nature 370621-628
Adzumah K and Mizuuchi K 1988 Target immunity of Mu transposition reflects a differential distribution of Mu B protein Cell 53257-266
Akey C W Crepeau R H Dunn S D McCarty R E and Edelstein S J 1983 Electron microscopy of single molecules and crystals of F1-ATPases EMBO J 21409-1415
Allet B 1979 Mu insertion duplicates a 5 bp sequence at the host inserted site Cell 16 123shy129
Amzel L M and Pedersen P L 1983 Proton ATP-ases structure and mechanism Ann Rev Biochem 52801-824
Andersson L Mac Neela J and Wolfenden R 1985 Use of secondary isotope effects and varying pH to investigate mode of binding of inhibitory amino aldehydes by leucine aminopeptidase Biochem 24330-333
Andrews G F Dugan R P and Stevens C J 1992 Combining physical and bacterial treatment for removing pyritic sulfur from coal In Processing and Utilization of High-sulfur coals IV Dugan P R D Quigley and Y Attia (eds) p 515 Elsevier New York
Andrulis I L Chen J and Ray P N 1987 Isolation of human cDNAs for asparagine synthetase and expression in Jansen rat sarcoama cells Mol Cell BioI 72435-2443
Andruszkiewicz R Milewsky S Zieniawa T and Borowski E 1990 Anticandidal properties of N3 -(4-methoxy-fumaroyl)-L-2 3-diamino propanoic acid oligopeptides 1 Med Chern 33132-135
Arciszewska L K Drake D and Craig NL 1989 Transposon Tn7 cis-acting sequences in transposition and transposition immunity J Mol BioI 20735-42
Bachmann B J 1983 Linkage map of Escherichia coli K-12 edition 7 Microbiol Rev 47180-230
145
B Plumbridge 1 A Cochet D Souza J M M M and Calcagno M 1988 Cordinated regulation of amino J
Bacteriol 1754951-4956
0 B Vermoote P V and Le Goffic F 1993 synthetase from Ecoli lUllL- mechanism and inhibition by N3-fumaroyl-2 3-diaminopropionic derivatives Biochem 27 2282-2287
Vermoote P Haumont PY syrlthl~ta~e from Escherichia coli purification site location Biochemistry 26 1940-1948
Badet B Inagaki K Soda K Walsh dependent inhibition of Bacillus stearothermophilus alanine racemase by phosphonate isomers by isomerization to non covalent enzyme-(1-aminoethyl) complexes Biochem 253275-3282
Badet-Denisot M A and Badet of glucoseamine-6shyphosphate synthetase by diethylpyrocarbonates histidine requirement for enzymatic activity Arch Biochem Biophys
Bainton R Gamas P and transposition in vitro proceeds through an excised transposon intermediate (JpnIPtlltpi1 111 breaks in DNA Cell 65805-816
Barry G 1986 Permanent insertion of v~u into the chromosomes of soil bacteria BioTechnology 4446-449
Barth P T Datta N Grinter N S 1976 Transposition a deoxyribonucleic acid sequence trimethoprim and streptomycin resistances from R483 to other replicons 1 Bacteriol 125800-810
Bates C J Adams W and R R 1968 Control of the formation of uri dine diphospho-N-acetyl and glycoprotein synthesis in rat liver J Chern 2411705-17
Benjamin N 1989 Intramolecular transposition of TnlD Cell 59373shy383
Bennet R L and Malamy M resistant mutants of Escgerichia coli and phosphate Commun 40496-503
Benneth1 1978 Bacterial leaching patterns on pyrite crystal J Bacteriol
146
Berg D E Davies 1 Allet B and Rochaix 1 D 1975 Transposition of R factor genes to bacteriophage lambda Proc Natl Acad Sci USA 723268-3632
Berg D E and Drummond M1978 Absence of DNA sequences homologous to transposable element Tn5 (Kan) in the chromosome of Ecoli K-12 J Bcateriol 136419-422
Birkenhager R Hoppert M Deckers-Hebestriet G Mayer F and Altendorf K 1995 The Fo complex of the Ecoli ATP synthase investigation by electron imaging and immunoelectron microscopy Eur J Biochem 23058-67
Bjqgtrbaek C Foersom V and Michelsen O 1990 The transmembrane topology of the a subunit from ATPase in Escherichia coli analysed by PhoA protein fusions FEBS lett 26031-34
Boekemia E J Berden JA and van Heel MG 1986 sructure of mitochondrial F1-ATPase studied by electron microscopy and image processing Biochim Biophys Acta 851353-360
Bolton E Glynn P and OGara F 1984 Site specific transposition of Tn7 into a Rhizobiwn meliloti megaplasmid Mol Gen Genet 193153-157
Boos W 1974 Bacterial transport Annu Rev Biochem 43123-146
Boursaux-Eude C Girons I S and Zuerner R 1995 IS1500 an IS3-like element from Leptospira interrogans Microbiol 1412165-2173
Boyer P D 1993 The binding change mechanism for ATP synthase-some probabilities and possibilities Biochim Biophys Acta 1140215-250
Brachet P Eisen H and Rambach A 1970 Mutations in coliphage lambda affecting the expression of replicative functions 0 and P Mol Gen Genet 108266-276
Brink J Boekemia E 1 and van Bruggen E F 1987 The structure of NADH ubiquitone oxidoreductase fron beef heart mitochondria Crystals containing an octameric arrangement of iron-sulphur protein fragments Eur J Biochem 166287-294
Brock T D and Gustafson J 1976 Ferric iron reduction by sulphur- and iron-oxidizing bacteria Appl Environ Microbiol 32 567-571
Brown L D Dennehy M E and Rawlings D E 1994 The FI genes of the FIFo ATP synthase from the acidophilic bacterium Thiobacillus jerrooxidans complement Escherichia coli FI unc mutants FEMS Microbiol Lett 12219-25
Buchanan 1 1 Adv The Amidotransferases Enzymol 3991-183
Buckley Science
Caruso M Pseudomonas nOVHrn
oxidation of pyrite Applied
1 1982 Interactions Mol Gen Genet
phage 1
Chmara H by Biophysica
synthetase from bacteria antibiotic tetaine Biochimica et
Microbiol Lett 11197-206 controls on the oxidation of refractory Barberton
genetic elements and evolution Nature 263731shyCohen S N 1976 738
Corbet C M and 1 Ingledew 1987 Is oxidation by Thiobacillus ferrooxidans
Couillard D and ~~b 118(5)808-81
Metallurgical residue V UlllLltHIH of metals from sewage sludge J
Cox G B a reassessment of the
F and Hatch L 1986 The mechanism of ATP synthase of the b and a subunits Biochim Acta 84962-69
Craig N L 1995 Unity in Science 270253-254
Craig N and Gamas P 1 Purification and characterization of a transposition protein that binds ATP DNA Nuc Acids Res
1 2873-97 C A 1990 in response to environmental stress Advances in
alUIUH~ on the activity subunit b-specific polyc1onal
G Simoni and Altendorf K 1992 Influence vlJnu~ of the ATP synthase of t-S(nel~lCn
1 BioI Chern 26712364shy
M A Le Goffic F and 1991 Glucosamine- 6P two proteins on limited proteolysis ltVU Biophys 288225-230
148
Dobrogosz W J 1968 _l in Escherichia coli and its to catabolite repression J
Doublet P 1 van Heijenoort and 1993 The murI gene of is an essential gene that encodes klUltUllU~v racemase activity J Bacteriol 1752970-2979
P J van Heijenoort 1992 Identification of the Ecoli which is required for the D-glutamic acid a specific component peptidoglycan 1 Bacteriol
Garren A Garen and Torri ani 1961 Genetic control of repression UCUlllV phosphatase in Ecoli 1 Mol BioI
D J and Boeke 1 D 1990 A 1-1-0- structure is required for Ty1 transposition Genes Develop 4324-330
1982 Transposition of Tn7 occurs at a on Caulobacter crescentus 10SIClmle 1 Bacteriol 151 1056-1 058
H 1990 Molecular IHovUUU)11 -transporting 345-391 In T 12 Bacterial
Press Ltd London
transport and coupling by the synthase insights mechanism of function J Bioenerg 24485-491
1992b Subunit c of FIFo ATP role m transduction Biochim Biophys Acta 1101 v--rJ
Eliopoulos E E Jackson P 1 Keen IN HUIUVI L Thompson P and 1 Structure of a 16 kDa integral AU-UUl that identity to
of the vacuolar H+ -ATPase Protein 15
Fling M 1 C 1985 Nucleotide sequence of encoding the amino glycoside-modifying enzyme 3(9)-O-nucleotidyltransferase Res 137095-7106
Fling M and C l-1UUJIU nucleotide sequence of dihydrofolate rhnrpri by Tn7 Nucleic Acids Res 115
Flores Llchtenstem C P 1990 DNA sequence anlysis of tnsA for the Tn7 transposition Nucleic Acids 18901 911
149
149
Foster D L and Fillingame R H 1982 Stoichiometry of subunits in the H+-ATPase complex of Escherichia coli J BioI Chern 2572009-2015
Fraga D Hermolin J Oldenburg M Miller MJ and Fillingame R H 1994 Arginine 41 of subunit c of Escherichia coli H+-A TP synthase is essential in binding and coupling of F to Fo J BioI Chern 2697532-7537
Friedl P Hoppe J Gunsalus R P Michelsen 0 von Meyenburg K and Schairer H U 1983 Membrane integration and function of the three Fo subunits of the A TP-synthase of Escherichia coli K12 EMBO 1 299-103
Frisa P S and Sonneborn D R 1982 Developmentally regulated interconversion between end product inhibitable and non-inhibitable forms of a first pathway-specific enzyme activity can be mimicked in vitro by dephosphorylation reactions Proc Natl Acad Sci USA 796289-6293
Fujiwara T and Mizuuchi K 1988 Retroviral DNA integration Structure of an integration intermediate Cell 54497-504
Galas D J and Chandler M 1989 Bacterial insertion sequences In Mobile DNA Berg D E and Howe M M (eds) Washington D C American Society for Microbiology pp 109shy162
Gay N J Tybulewicz V L1 Walker JE 1986 Insertion of transposon Tn7 into the Ecoli gmS transcriptional terminator Biochem J 234 111-117
Gentry D R and Cashel M 1995 Cellular localization of the Escherichia coli SpoT protein J Bacteriol 177 3890-3893
Gentry D R Xiao H Burgess R R and Cashel M 1991 The omega subunit of Ecoli K-12 RNA polymerase is not required for stringent RNA control in vivo J Bacteriol 173 3901-3903
Girvin M E and Fillingame R H 1993 Helical structure and folding of subunit c of FIFo ATP synthase IH NMR resonace assignments and NOE analysis Biochemistry 3212167shy12177
Gogol E P Lucken U Bork T and Capaldi R A 1989 Molecular architecture of Escherichia coli F Adenosinetriphosphatase Biochemistry 284709-4716
Gogol E P Aggeler R Sagermann M and Capaldi R A 1989 Cryoelectronic Microscopy of Escherichia coli FI Adenosinetriphosphatase Decorated with Monoclonal Antibodies to Individual Subunits of the complex Biochemistry 284717-4724
150
Heinikoff S 1984
Golinelli-Pimpaneaux B and Badet involvement of Lys603 from Ecoli glucosamoine-6-phosphate synthase substrate fructose-6-phosphate 1 Biochem 201175-182
Gottesman M M and Rosner 1 L of a detenninant of uvu_bullu
resistance by coliphage lambda Sci USA 725041-5045
Grunstein M and Hogness D
Colony hybridization a method for isolation cloned DNAs that contain a 1Jbullu Proc NatL Acad Sci USA 723961-3965
Hauer B and Shapiro 1 A 1984 Control of Tn7 transposition Mol Gen Genet 194
Hedges R W and Jacob Transposition of ampicillin resistance from to other replicons MoL 1-40
Hennolin 1 Gallant 1 psi subunit in the Fo sector of the H+ -ATPase of Ecoli J Bioi
R H 1983 Topology organization and function
Hennolin J and R 1989 Assembly of Fo sector of synthase Interdepenndence of subunit insertion into the membrane 1 2817
van Montagu M Holsters Zambryski P Beuckeleer M Willmitzer and Schell 1 M 1980 interaction of
DNA plant cells Proc Soc Lond 210351-365
P 1972 Insertion mutations control region of Physical characterization of the mutants Mol Gen Genet
115266-276
Holmes applications pI
UI Haq 1990 Adaptation of Thiobacillus vu)nuu for industrial In J Salley R G McCready Wichlacz (ed)
1989 CANMET Ottawa Canada
Holtje J V and U Schwarz 1985 Biosynthesis and growth murein sacculus p 77shy119 InN (ed) Molecular cytology of Escherichia Academic Press Inc New
Acad Sci USA
Heffron E Reubens C and which mediates ampicillin
Translocation of a plasmid DNA sequence nature and specificity of Natl
digestion with exonuclease III creates fl tr breakpoints 1-369
Hernalsteens J M Agrobacterium
Hirsch H the galactose nnnn fJu
151
Holzenburg A Jones P c Franklin T Padi J B and Finbow M E 1993 Evidence for a common structure 1 Biochem 21321-30
Hoppe 1 and Sebald W 1986 Topological pathway of the protons through Fo is provided by amino acid the lipid phase Biochimie (Paris) 68427-434
S Otsubo H Davidson N and 1975 Electron microscope heteroduplex of sequence relations among plasmids identification and mapping of the
insertion sequences IS] and IS2 in F and R plasmids J Bacteriol 22762-775
Inoue c Sugawara K and 1991 regulatory gene in Thiobacillus ferrooxidans is spaced apart from Mol Microbiol 52707-2718
Ish-Horowicz D and Burke and cosmid cloning Nucleic Acids Res 92989-2998
Johnston B Clennell M and D 1995 Structure and function of Tn5467 a Tn2l-like transposon located on T jerrooxidans broad host range plasmid Appl Envir Micro
Kahmann R and Kamp Nucleotide sequences of the attachment bacteriophage Mu DNA Nature 280247-250
Kahn K and Schaefer M R Characterization of trans po son 5469 from cyanobacterium Fremyella diplosiphon J 1777026-7032
Karavaiko G Golovacheva S Pivovarova T A Tzaplina I A and Vartanjan 1988 Thermophilic ltPM Sulfobacillus page 29-41 In Biohydrometallurgyshy87 Norris P and Science and Technology Letters Kew 1
Kennedy J and Humpphreys J D 1976 Microbial cells living immobilised on Nature 261242-244
Koonin 1993 SpoU protein of Escherichia coli belongs to a new family of Nucleic Acids Res 19
Kopecko J and site specific recA mdependtm recombination between bacterial Pt of palindromes at the N ad Acad Sci USA
on L-glutamine D-fructose ud()transtiera~e 1 BioI
152
Krumholz L R Esser U and Simoni RD 1989 Nucleotide sequence of the unc operon of Vibrio alginolyticus Nucleic Acids Res 177993-7994
Kubo K and Craig N 1990 Bacterial transposon Tn7 utilizes two classes of target sites 1 Bacteriol 1722774-2778
Kucharczyk N Denisot M A Le Goffic F and Badet B 1990 Glucosarnine-6-phosphate synthase from Ecoli detennination of the mechanism of inactivation by N3 fumaroyl-L-2-3 diarninoproprionic derivatives Biochemistry 293668-3676
Kusano M Takeshima T Inoue C and Sugawara K 1991 Evidence for two sets of structural genes coding for Ribulose biphosphate carboxylase in Thiobacillus ferrooxidans 1 Bacteriol 1737313-7323
Lane Dl A P Harrison lnr D Stahl B Pace SJ Giovannoni GJ Olsen and N R Pace 1992 Evolutionary relationship among sulphur- and iron-oxidizing eubacteria 1 Bacteriol 174267-278
Lee C H Bhagwhat A and Heffron F 1983 Identification of a transposon TnJ sequence required for transposition immunity Natl Acad Sci USA 806765-6769
Lewis M 1 Chang 1 A and Somoni R D 1990 A topological analysis of subunit a from Escherichia coli F1Fo-ATP synthase predicts eight transmembrane segments 1 BioI Chern 265 10541-10550
Lichtenstein C P and Brenner S 1982 Unique insertion site of Tn7 in the Ecoli chromosome Nature (London) 297601-603
Lichtenstein C P and Brenner S 1981 Site-specific properties of transposition to the Ecoli chromosome Mol Gen Genet 183380-387
Liu X Petersson S and Sandstrom A Mesophilic versus moderate thennophilic bioleaching Biohydrometallurgy Technologies Vol 1 A E Tonna 1 E Wey and Lakshmanan V 1 (ed) pp 29-38
Lizarna H M and Sankey B M 1993 Oxidation of H2S by Thiobacillus thiooxidans is inhibited by substrate and methane BiohydrometaHurgy Technologies Vol II A E Tonna 1 E Wey and C L Brierley (ed) pp 339-364
Lundgren D G and Silver M 1980 Ore leaching by bacteria Ann Rev Microbiol 34263shy268
Makino K Amemura M Shinagawa H Kobayashi A and Nakata A 1985 Sequence of the genes involved in phosphate transport and regulation of the phosphate regulon in Escherichia coli 1 Mol BioI 184231-240
Makino K Shinagawa Amemura M Kimura S Nakata A and Ishihama 1988 Euuv1J of the phosphate regulon of Ecoli Activation of pstS by the PhoB
protein in vitro 1 Mol BioI 20385-95
Makino K Shinagawa H Amemura M Yamada M and 1990 Signal transduction in the phosphate regulon of the Escherichia coli involves phosphotransfer nprlrJp~middotn PhoR and PhoB proteins J Mol BioI 21055
Malamy 1966 Frameshift mutations in the nnlnn of Escherichia coli Cold Harb Symp Quant BioI 31189-201
Malamy M H 1972 Electron microscopy insertions in the lac operon of Escherichia coli MoL Gen
Malamy M H and Bennett R L 1970 mutants of Ecoli and phosphate transport Biochem Biophys Res Commun
Maniatis T Fritsch EF Sambrook J 1982 nn A laboratory manual Cold Spring Harbor Laboratory Press Cold
McKnight G L S L Mudri SH Mathewest R S Marshall P O Sheppard and P J OHara 1990 Molecular cloning synthesis and bacterial expression of human glutaminefructose-6-phosphate UUAUU u J Chem 26725208-25212
Medveczky N and H Rosenburg 1970 phosphate-binding protein of Escherichia Biochim Biophys Acta 211 158-168
Mei B and Zalkin H 1989 A Cysteine-Histidine-Aspartate catalytic triad is involved in Glutamide Amide Transfer Function purF-type Glutamine amodotransferases 1 BioI Chem 264 16613-16619
Mengin-Lecreulx D and J van 1993 Identification of glmU gene encoding Nshyacetylglucosamine in Escherichia coli J Bacteriol 1756150shy6157
Michaelis M J and Criddle M J 1970 Mitochondrial DNA and mutants in Saccharomyces cerevisiae Biochem Genet 5487-495
Michaelis Starlinger P 1969 Two insertions in the jO v
operon having different homologous DNA sequences MoL Gen Genet 10437 377
Miller J H Calos Transposable elements Cell 20579-595
154
Miller J Oldenburg M and Fillingame R 1990 The essential carboxyl group of in subunit c the FIFo ATP synthase can be and H( +)-translocating function retained Proc NatL Acad 874900-4904
Mitcell P 1966 Chemiosmotic coupling in - and phosphorylation BioL Rev 41455-502 (1966)
Mizuuchi K 1984 Mechanism of transposition of bacteriophage Mu Polarity of the strand reaction at the initiation of the transposon Cell 39395-404
C Ph de Donato Berthelin J Surface oxidized species a key factor in the study of bioleaching processes Biohydrometallurgical In A J
Weyand V I Lakshmanan ed 1993 Vol 1 175-184
A Ishino Shinagawa H Makino K and M 1987 Nucleotide of the iap gene responsible for alkaline phosphatase isoenzyme conversion in Ecoli
identification of product 1 1695429-5433
K 1989 The tsr gene-coding prevents thiopeptin from inhibiting ppGpp synthesis in Streptomyces lividans FEMS Microbiol Lett
Ogasawara N and Yoshikawa H 1992 and their organIzatIon in the repnc()n of the bacterial chromosome Mol Microbiol 6(5)629-634
Ohtsubo Davidson N and 1975 microscope heteroduplex studies of sequence relations among plasmids identification and mapping of the insertion sequences 1 and IS2 in F and R plasmids 1 122746-775
Olson G J 1994 Microbial oxidation gold ores and gold bioleaching FEMS un Lett 119 1-6
Orle K A N L 1991 Identification of transposition prroteins by the bacterial transposon [corrected republished originally printed in Gene 1990 Nov 30 96(1) Gene 10425-31
Ouellette M Roy P Homology of ORFs from and from to site specific recombinases 1987 Nucl Acids 10055-10059
Murein synthesis p663-671ln Neidhardt J L Ingraham B Louw M~ M Schaechter H E Umbarger (ed) Escherichia coli and Salmonella
ryphimurium cellular and bioI voL 1 American Society Microbiology Washington
Perlin D S and Senior A Functional and cross-reactivity of antibody to purified subunit b (uncF protein) of Escherichia coli proton-ATPase Arch Biochem Biophys 236603-611
155
Plumbridge J A O Cochet Souza J M Altramirano M M Calcagno M L and Badet B 1993 Coordinated regulation of amino sugar-synthesizing and -degrading enzymes in Escherichia coli K-12 J Bacteriol 175(16)4951-4956
Pretorius I M Rawlings D E and Woods D R 1986 Identification and cloning of Thiobacillus jerrooxidans structural Nif genes in Escherichia coli Gene 4559-65
Qadri M I Flores C c Davis J and Lichtenstein C 1989 Genetic analysis of attTn7 the transposon Tn7 attachment site in Escherichia coli using a novel M13-based transduction assay 1 Mol BioI 20785-98
Radstrom P Skold 0 Swedberg J Roy P H and Sundstrom L 1994 Tn5090 of plasmid R751 which carries integron is related to Tn7 Mu and retroelements J Bacteriol 1763257-3268
Raetz C R H 1987 Structure and biosynthesis of lipid A in Escherichia coli pp 498-503 In F C Niedhardt J L Ingraham K B Low B Magasanik M Schaechter and H E Umbarger (ed) Escherichia coli and Salmonella typhimurium cellular and molecular biology vol 1 American Society for Microbiology Washington DC
Rao N N and Torriani A 1990 Molecular aspects of phosphate transport in Escherichia coli Mol Microbiol 4(7) 1083-1090
Rawlings DE D R Woods and NP Mjoli 1991 The cloning and structre of genes from the autotrophic biomining bacterium Thiobacillusjerrooxidans p 215-237 In PJ Greenaway (ed) Advances in gene technology vol 2 JAI Press London
Richet E and O Raibaud 1989 MalT the regulatory protein of the Escherichia coli maltose system is an A TP-dependent transcriptional activator EMBO 1 8981-987
Rogers M Ekaterrinaki N Nimmo E and Sherratt D 1986 Analysis of Tn7 transposition Mol Gen Genet 205550-556
Ronson C W Nixon B T Albright L M anf Ausubel F M 1987 Rhizobium meliloti ntrA (rpoN) gene is required for diverse metabolic functions J Bacteriol 1692424-2431
Rosenburg H Gerdes R G and Chegwidden K 1977 Two systems for the uptake of phosphate in Ecoli JBacterioL 131505-511
Rosenburg H 1987 In ion transport in Prokaryotes Rosen B P and Silver S (eds) New York Academic Press pp 205-248
Ross D G Swan 1 and N Klecker 1979 Physical structures of TnlO-promoted deletions and inversions role of 1400 base pairs inverted repetitions Cell 16721-731
156
Saedler H and P 19670 mutation in the galactose operon in Ecoli II Physiological characterization MoL Gen Genet 100 190-202
Saedler P 1968 00 and strong polar mutations in the Genet 102353-363
Sambrook J Fritsch Maniatis 1989 Molecular cloning a laboratory manual Cold Harbor Laboratory Cold Spring Harbor New York
Sand W Gehrke T Hallmann Rohde K Sobotke B and Wintzien S 1993 Bioleaching metal sulphides The importance of Leptospirillum ferrooxidans Biohydromemetallurgical Technologies Voll pp 15-25
Sanger Nicklen and Coulson A DNA sequencing with chain-tennination inhibitors Natl Acad USA 745463-5467
Schneider E and Altendorf K All the three subunits are required for an active proton channel (Fo) of Escherichia coli synthase (FIFo) ~-
518
Schneider E and Allendorf K 1987 Bacterial adenosine 5-triphosphate synthase purification and reconstitution complexes and biochemical and functional characterization of subunits Microbio Rev 51477-497
Schrader 1 A and D S Holmes 1988 Phenotypic switching Thiobacillus ferrooxidans J BacterioL 1703915-3023
Scordilis G E H and Lassie 1987 Identification of transposable elements which activates gene expression in Pseudomonas cepacia J Bacteriol 1698-13
Sekine Eisaki N and Ohtsubo E 1996 Identification and characterization of the lS3 molecules generated by breaks 1 BioL Chern 271197-202
Senior A E 1990 The proton-trans locating of Escherichia coli Annu Rev Biophys Chern 194-41
Shapiro 1 and Adhya S 1969 The jLU operon of K-12 n A deletion analysis of the structure polarity 62249-264
J A 1969 Mutations caused by insertion genenc material the galactose operon Escherichia 1 MoL BioI 4093-105
Silvennan M P 1967 Mechanism of bacterial pyrite oxidation 1 Bacteriol 941046-1051
157
C C ElIson and Levinson 1983 Identification of the typel trimethoprim resistance reductase specified by the R-plasmid R43 comparison with procaryotic and eucaryotic dihydrofolate reductase 1 Bacteriol 155 100 1-1008
Smith and Jones P transposition a multigene process Identification of a regulatory product 147915-7927
Sprague Jr Bell R M Cronan 1 A mutant of Ecoli auxotrophic for organic phosphates evidence two defects in phosphate Mol Gen Genet 14371-77
Starlinger and Michaelis 1968 Suppression the sequential aO[)ealame of the galactose enzymes in a transferase amber mutant coli Mol Genet 102367-369
Starlinger 1977 IS elements in microorganisms MicrobioL Immunol
Steffens K Schneider E Herkernhoff B Schmid Altendorf K portion of Escherichia coli A TP synthase resolution of from subunit b J BioI Chern 2625866-5869
Steffens A Deckers-hebestreit G and Altendorf K 1987b The and functional relationship of A TP synthases (FoFI) from Ecoii and the thermophilic bacterium PS3 J Biol 2626334-6338
Strominger J and M S Smith 1959 Uridine diphosphoacetylglucosamine pyrophosphorylase 1 BioI Chern 2341822-1827
Sundstrom Skold 1990 dhfrI trimethoprim resistance gene of can be found at in other genetic surroundings Antimicrob Agents Chemother 34642shy650
Sundstrom L Roy P H and Skold Site-specific of three cassettes in Tn7 J Bacteriol 1733025-3028
Surin B P and Downie JA 1988 of the Rhizobium leguminosarum nodLMN involved efficient host-specific nodulation Mol Microbiol 2 173-183
B P Dixon N and Rosenburg 1986 Purification PhoU protein a OClItnl
regulator of the pho regulon of Ecoli K-1 J Bacteriol 168631
B P D A Jans A L Fimmel Shaw GB Cox Rosenburg Structural gene for the phosphate-repressible phosphate binding of Escherichia coli
own promoter nucleotide of the phoS 1 Bacteriol
158
Suzuki I Chang W and Takeuchi 1994 Oxidation of organic compounds by Thiobacilli Symp Series 55060-67
Takeyama M S Y Noumi T Maeda Ishibashi S and Futai M 1988 Beta subunit of Ecoli amino acid replacement within a conserved sequence G-X-X-X-X-GshyK-TS) v~u binding proteins lett 218222-226
Thomson JA M Hendson and R M 1981 Mutagenesis by insertion of drug resistance Tn7 into a vibrio 11 J Bacteriol
Tichy R Grotenhuis J T c Janssen van Houten R Rulkens and Lettinga 1993 Application of the sulphur cycle bioremediation of soils polluted with heavy metals In Int Conf Contaminated 93 ed F Arendt G J Annokee R Bosman and W J van der Brink Kluwer Academic Publishers Dordrecht pp 1461-1462
G and Mayer An electron approach to the quaternary structure of mitochondrial Eur J Biochem 13237-45
J and Tschape streptothricin resistance transposons Tn1825 and Tn1826 and transposon Tn 7 Plasmidnuu
18246-249
Tso J Y Zalkin H van Cleemput M Yanofsky C and JM 1982 Nucleotide of Escherichia coli and deduced amino glutamine
phosphribosylpyrophosphate am idotransferase J BioI Chern
V Mesyanzhinova I V Koslov I A and Orlova 1984 Structure of studied by electron and image processing Lett 167285-289
P Barber C and transposons Tn5 and Tn7 in Xanthomonas campestris pv campestris MoL Gen 157
Ullrich J and van Putten P Identification of the Gonococcal glmU gene UVUUIJ
the enzyme N-acetylglucosamine 1-Phosphate Uridyltransferase involved in the synthesis UDP-GlcNAc J of Bact 1776902-6909
M 1992 Eight bacterial proteins includin]g UDP-N -acetylglucosamine acyltransferase three vother of Escherichia coli of a six-residue n tVI
theme FEMS MicrobioL 97249-254
van der Steen J J D Doddema J and de Uidoging van zware metalen afvalstromen met behulp van thiobacilli (Removal metals from waste streams
using thiobacilli) Ministry of Housing Physical and Enviromental (VROM) Directoraat-Generaal Milieubeheer Rapport 199210 Hague
Vermoote P 1988 Universite Paris VI Paris Hrllnro
159
Vignais P V Lunard Issartel J P and Dupuis A 1985 Interaction n l1f middotn
oligomycin sensitivity protein (OSCP) beef heart mitochondrial FeATPase 2 Identification of interacting Fl subunits cross-linking Biochem J
Vik S and Dao NN Prediction of transmembrane topology of Fo proteins from Ecoli ATP synthase variational hydrophobic moment analyses Biochim Biophys Acta 1140 199-207
von Meyenburg B B Jcentrgensen J Nielsen and F Hansen 1982 Promoters of the atp operon for the membrane-bound ATP synthase of Ecoli mapped by TnlO insertion mutations MoL Gen Genet 188240-248
von Meyenberg F G Hansen 1980 The origin of replication oriC the Ecoli chromosome near oriC and construction of oriC mutants ICN-UCLA Symp MoLCellBiol 191 159
Waddell S and Craig N 1989 Tn7 transposition recognition of the attTn7 Proc Natl Sci USA 863958-3962
Waddell C S and Craig N L 1988 transposition two transposition pathways directed by five Tn7-encoded genes Develop 21
J E Gay N J Saraste M and A N 1984 DNA sequence the Escherichia coli unc operon Completion of the sequence of a kilobase segment containing
oriC unc and phoS J224799-815
and Collinson 1 1994 the role the stalk in the coupling mechanism of FEBS 34639-43
Walker 1 E Gay N J and Tybulewicz V L 1986 of transposon Tn7 into the Escherichia glmS transcriptional terminator Biochem 1 243 111-1
Wanner Land McSharry 1982 Phosphate-controlled gene in Escherichia coli using Mudl-directed lacZ fusions J Mol BioI 158347-363
Weng M and Zalkin H 1987 Structural role for a region the CTP synthetase glutamide amide transfer domain J Bacteriol 1693023-3028
Wheatcroft R and S and nucleotide sequence of Rhizobiwn meliloti insertion between the transposase encoded by ISRm3 and those encoded by Staphylococcus aureus and Thiobacillus ferrooxidans IST2 J 1732530-2538
160
Whitby M C and Lloyd R 1995 Branch of three-strand recombination intennediates by RecG a possible pathway for securing middotIULl initiated by duplex DNA 1 143303-3310
Wilkens and Capaldi R A Assymmetry and changes in examined by cryoelectronmicroscopy BioI Chern Hoppe-Seyler 37543-51
and Malamy M 1974 The loss of the PhoS periplasmic protein leads to a the specificity a constitutive phosphate transport system in Escherichia coli Biochem Res Commun 60226-233
WiUsky G Rand Malamy M 1980 of two separable inorganic phosphate transport systems in Escherichia coli J BacterioL 144356-365
P J and and C F 1973 enzyme of metabolism and Stadtman R eds) pp 343-363 Academic Press New York
Winterbum P J and Phelps F 197L control of hexosamine biosynthesis by glucosamine synthetase Biochem 1 121711
Wolf-Watz H and Norquist A 1979 Deoxyribonucleic acid and outer membrane protein to outer membrane protein involves a protein J 14043-49
Wolf-Watz H and M 1979 Deoxyribonucleic acid and outer membrane strains for oriC elevated levels deoxyribonucleic acid-binding protein and
11 for specific UU6 of the oriC region to outer membrane J Bacteriol 14050-58
Wolf-Watz H 1984 Affinity of two different chromosome to the outer membrane of Ecoli J Bacteriol 157968-970
Wu C and Te Wu 197 L Isolation and characterization of a glucosamine-requiring mutant of Escherichia 12 defective in glucoamine-6-phosphate synthetase 1 Bacteriol 105455-466
Yamada M Makino Shinagawa Nakata A 1990 Regulation of the phosphate regulon of Escherichia coli properties the pooR mutants and subcellular localization of protein Mol Genet 220366-372
Yates J R and Holmes D S 1987 Two families of repeatcXl DNA sequences Thiobacillus 1 Bacteriol 169 1861-1870
Yates J R R P and D S 1988 an insertion sequence from Thiobacillus errooxidans Proc Natl 857284-7287
161
Youvan D c Elder 1 T Sandlin D E Zsebo K Alder D P Panopoulos N 1 Marrs B L and Hearst 1 E 1982 R-prime site-directed transposon Tn7 mutagenesis of the photosynthetic apparatus in Rhodopseudomonas capsulata 1 Mol BioI 162 17-41
Zalkin H and Mei B 1990 Amino terminal deletions define a glutamide transfer domain in glutamine phosphoribosylpyrophosphate ami do transferase and other purF-type amidotransferases 1 Bacteriol 1723512-3514
Zalkin H and Weng M L 1987 Structural role for a conserved region III the CTP synthetase glutamide amide transfer domain 1 Bacteriol 169 (7)3023-3028
Zhang Y and Fillingame R H 1994 Essential aspartate in subunit c of FIF0 ATP synthase Effect of position 61 substitutions in helix-2 on function of Asp24 in helix-I 1 BioI Chern 2695473-5479
11 Bioleaching 1
112 Organism-substrate raction 1
113 Leaching reactions 2
114 Industrial applicat 3
12 Thiobacillus ferrooxidans 4
13 Region around the Ecoli unc operon 5
131 Region immediately Ie of the unc operon 5
132 The unc operon 8
1321 Fe subun 8
1322 subun 10
133 Region proximate right of the unc operon (EcoURF 1) 13
1331 Metabolic link between glmU and glmS 14
134 Glucosamine synthetase (GlmS) 15
135 The pho 18
1351 Phosphate uptake in Ecoli 18
1352 The Pst system 19
1353 Pst operon 20
1354 Modulation of Pho uptake 21
136 Transposable elements 25
1361 Transposons and Insertion sequences (IS) 27
1362 Tn7 27
13621 Insertion of Tn7 into Ecoli chromosome 29
13622 The Tn7 transposition mechanism 32
137 Region downstream unc operons
of Ecoli and Tferrooxidans 35
LIST OF FIGURES
Fig
Fig
Fig
Fig
Fig
Fig
Fig
Fig
la
Ib
Ie
Id
Ie
If
Ig
Ih
6
11
14
22
23
28
30
33
1
CHAPTER ONE
INTRODUCTION
11 BIOLEACHING
The elements iron and sulphur circulate in the biosphere through specific paths from the
environment to organism and back to the environment Certain paths involve only
microorganisms and it is here that biological reactions of relevance in leaching of metals from
mineral ores occur (Sand et al 1993 Liu et aI 1993) These organisms have evolved an
unusual mode of existence and it is known that their oxidative reactions have assisted
mankind over the centuries Of major importance are the biological oxidation of iron
elemental sulphur and mineral sulphides Metals can be dissolved from insoluble minerals
directly by the metabolism of these microorganisms or indrectly by the products of their
metabolism Many metals may be leached from the corresponding sulphides and it is this
process that has been utilized in the commercial leaching operations using microorganisms
112 Or2anismSubstrate interaction
Some microorganisms are capable of direct oxidative attack on mineral sulphides Scanning
electron micrographs have revealed that numerous bacteria attach themselves to the surface
of sulphide minerals in solution when supplemented with nutrients (Benneth and Tributsch
1978) Direct observation has indicated that bacteria dissolve a sulphide surface of the crystal
by means of cell contact (Buckley and Woods 1987) Microbial cells have also been shown
to attach to metal hydroxides (Kennedy et ai 1976) Silverman (1967) concluded that at
least two roles were performed by the bacteria in the solubilization of minerals One role
involved the ferric-ferrous cycle (indirect mechanism) whereas the other involved the physical
2
contact of the microrganism with the insoluble sulfide crystals and was independent of the
the ferric-ferrous cycle Insoluble sulphide minerals can be degraded by microrganisms in the
absence of ferric iron under conditions that preclude any likely involvement of a ferrous-ferric
cycle (Lizama and Sackey 1993) Although many aspects of the direct attack by bacteria on
mineral sulphides remain unknown it is apparent that specific iron and sulphide oxidizers
must play a part (Mustin et aI 1993 Suzuki et al 1994) Microbial involvement is
influenced by the chemical nature of both the aqueous and solid crystal phases (Mustin et al
1993) The extent of surface corrosion varies from crystal to crystal and is related to the
orientation of the mineral (Benneth and Tributsch 1978 Claassen 1993)
113 Leachinamp reactions
Attachment of the leaching bacteria to surfaces of pyrite (FeS2) and chalcopyrite (CuFeS2) is
pSIS1 construct was further subcloned to plasm ids IS30 IS40 pSlS50
pS183S p81841 and p81 (Fig Some of subclones were extensively mapped
although not all sites are shown in 22 exact positions of the ends of
T jerrooxidans plasmids pTfatpl and pTfatp2 on the cosmid p8I81 were identified
21)
Tfeooxidans
that the cosmid was
unrearranged DNA digests of cosmid pSISI and T jerrooxidans chromosomal DNA were
with pSI The of the bands gave a positive hybridization signal were
identical each of the three different restriction enzyme digests of p8I8l and chromosomal
In order to confirm of pSI81 and to
49
kb kb ~
(A)
11g 50Sts5
middot 2S4
17 bull tU (probe)
- tosect 0S1
(e)
kb
+- 10 kb
- 46 kb
28---+ 26-+
17---
Fig 23 Hybrdization of cosmid pSISI and T jerrooxitians (A TCC 33020) chromosomal
DNA by the KpnI-SalI fragment of pSIS52 (probe) (a) Autoradiographic image of the
restriction digests Lane x contains the probe lane 13 and 5 contain pSISI restricted with BamHl HindIII and Bgffi respectively Lanes 2 4 and 6 contain T errooxitians chromosomal DNA also restricted with same enzymes in similar order (b) The sizes of restricted pSISI
and T jerrooxitians chromosome hybridized by the probe The lanes correspond to those in Fig 23a A DNA digested with Pst served as the molecular weight nlUkermiddot
50
DNA (Fig BamHI digests SHklC at 26 and 2S kb 1 2) HindIII
at 10 kb 3 and 4) and BgnI at 46 (lanes 5 and 6) The signals in
the 181 was because the purified KpnI-San probe from p81 had a small quantity
ofcontaminating vector DNA which has regions ofhomology to the cosmid
vector The observation that the band sizes of hybridizing
for both pS181 and the T ferrooxidans ATCC 33020 same
digest that the region from BgnI site at to HindIII site at 16
kb on -VJjUIU 1 represents unrearranged chromosomal DNA from T ferrooxidans
ATCC there were no hybridization signals in to those predicted
the map with the 2 4 and 6 one can that are no
multiple copies of the probe (a Tn7-like segment) in chromosome of
and Lferrooxidans
of plasmid pSI was found to fall entirely within a Tn7-like trallSDOS()n nrpn
on chromosome 33020 4) A Southern hybridizationUUAcpoundUU
was out to determine whether Tn7-like element is on other
ofT ferrooxidans of T and Lferrooxidans results of this
experiment are shown 24 Lanes D E and F 24 represent strains
19377 DSM and Lferrooxidans DSM 2705 digested to
with BgnI enzyme A hybridization was obtained for
the three strains (lane A) ATCC 19859 (lane B) and UUHUltn
51
(A) AAB CD E F kb
140
115 shy
284 -244 = 199 -
_ I
116 -109 -
08
os -
(8)) A B c o E F
46 kb -----
Fig 24 (a) Autoradiographic image of chromosomal DNA of T ferrooxidans strains ATCC 33020 19859 23270 (lanes A B C) Tthiooxidans strains ATCC 19377 and DSM 504 (D and E) and Lferrooxidans strain DSM 2705 (lane F) all restricted with Bgffi A Pst molecular weight marker was used for sizing (b) Hybridization of the 17 kb KpnI-San piece of p81852 (probe) to the chromosomal DNA of the organisms mentioned 1 Fig 24a The lanes in Fig 24a correspond to those in Fig 24b
(lane C) uuvu (Fig 24) The same size BgllI fragments (46 kb) were
lanes A Band C This result
different countries and grown on two different
they Tn7-like transposon in apparently the same location
no hybridization signal was obtained for T thiooxidans
1 504 or Lferrooxidans DSM 2705 (lanes D E and F of
T thiooxidans have been found to be very closely related based on 1
rRNA et 1992) Since all Tferrooxidans and no Tthiooxidans
~~AA~~ have it implies either T ferrooxidans and
diverged before Tn7-like transposon or Tthiooxidans does not have
an attTn7 attachment the Tn7-like element Though strains
T ferrooxidans were 10catH)uS as apart as the USA and Japan
it is difficult to estimate acquired the Tn7-like transposon as
bacteria get around the world are horizontally transmitted It
remains to be is a general property
of all Lferrooxidans T ferrooxidans strains
harbour this Tn7-like element their
CHAPTER 3
5431 Summary
54Introduction
56Materials and methods
56L Bacterial and plasmid vectors
Media and solutions
56333 DNA
57334 gel electrophoresis
Competent preparation
57336 Transformation of DNA into
58337 Recombinant DNA techniques
58ExonucleaseIII shortening
339 DNA sequencing
633310 Complementation studies
64Results discussion
1 DNA analysis
64Analysis of ORF-l
72343 Glucosamine gene
77344 Complementation of by T ferrooxidans glmS
57cosmid pSIS1 pSlS20
58Plasmidmap of p81816r
Fig Plasmidmap of lS16f
60Fig 34 Restriction digest p81S1Sr and pSIS16f with
63DNA kb BamHI-BamHI
Fig Codon and bias plot of the DNA sequence of pSlS16
Fig 37 Alignment of C-terminal sequences of and Bsubtilis
38 Alignment of amino sequences organisms with high
homology to that of T ferrooxidans 71
Fig Phylogenetic relationship organisms high glmS
sequence homology to Tferrooxidans
31 Summary
A 35 kb BamHI-BamHI p81820 was cloned into pUCBM20 and pUCBM21 to
produce constructs p818 and p81816r respectively This was completely
sequenced both rrelct1()ns and shown to cover the entire gmS (184 kb) Fig 31
and 34 The derived sequence of the T jerrooxidans synthetase was
compared to similar nrvnC ~ other organisms and found to have high sequence
homology was to the glucosamine the best studied
eubacterium EcoU Both constructs p81816f p81 the entire
glmS gene of T jerrooxidans also complemented an Ecoli glmS mutant for growth on medium
lacking N-acetyl glucosamine
32 =-====
Cell wall is vital to every microorganism In most procaryotes this process starts
with amino which are made from rructClsemiddotmiddotn-[)ll()S by the transfer of amide group
to a hexose sugar to form an This reaction is by
glucosamine synthetase the product of the gmS part of the catalysis
to the 40-residue N-terminal glutamine-binding domain (Denisot et al 1991)
participation of Cys 1 to generate a glutamyl thiol ester and nascent ammonia (Buchanan
368-residue C-terminal domain is responsible for the second part of the ClUUU
glucosamine 6-phosphate This been shown to require the ltgtttrltgtt1iltn
of Clpro-R hydrogen of a putative rrulctoseam 6-phosphate to fonn a cis-enolamine
intennediate which upon reprotonation to the gives rise to the product (Golinelli-
Pimpaneau et al 1989)
bacterial enzyme mn two can be separated by limited chymotryptic
proteolysis (Denisot et 199 binding domain encompassing residues 1 to
240 has the same capacity to hydrolyse (and the corresponding p-nitroanilide
derivative) into glutamate as amino acid porI11
binding domain is highly cOllserveu among members of the F-type
ases Enzymes in this include amidophosphophoribosyl et al
1982) asparagine synthetase (Andrulis et al 1987) glucosamine-6-P synmellase (Walker et
aI 1984) and the NodM protein of Rhizobium leguminosarum (Surin and 1988) The
368-residue carboxyl-tenninal domain retains the ability to bind fructose-6-phosphate
The complete DNA sequence of T ferrooxidans glmS a third of the
glmU gene (preceding glmS) sequences downstream of glmS are reported in this
chapter This contains a comparison of the VVA r glmS gene
and Mycobacterium (422 ) An interesting observation was that the T ferrooxidans
glmS gene had comparatively high homology sequences to the Rhizobium leguminosarum and
Rhizobium meliloti M the amino to was 44 and 436
respectively A consensus Dalgarno upstream of the start codon
(ATG) is in 35 No Ecoli a70-type n r~ consensus sequence was
detected in the bp preceding the start codon on intergenic distance
between the two and the absence of any promoter consensus sequence Plumbridge et
al (1993) that the Ecoli glmU and glmS were co-transcribed In the case
the glmU-glmS --n the T errooxidans the to be similar The sequences
homology to six other amidotransferases (Fig 38) which are
(named after the purF-encoded l phosphoribosylpyrophosphate
amidotransferase) and Weng (1987)
The glutamine amide transfer domain of approximately 194 amino acid residues is at the
of protein chain Zalkin and Mei (1989) using site-directed to
the 9 invariant amino acids in the glutamine amide transfer domain of
phosphoribosylpyrophosphated indicated in their a
catalytic triad is involved glutamine amide transfer function of
73
Comparison of the ammo acid sequence alignment of ghtcosamine-6-phosphate
amidotranferases (GFAT) of Rhizobium meliloti (R_m) Rhizobium leguminnosarum (RJ)
Ecoli (E_c) Hinjluenzae Mycobacterium leprae (M_I) Bsubtilis (B_s) and
Scerevisiae (S_c) to that of Tferrooxidans Amino acids are identified by their single
codes The asterisks () represent homologous amino acids of Tferrooxidans glmS to
at least two of the other Consensus ammo acids to all eight organisms are
highlighted (bold and underlined)
1 50 R m i bullbullbullbullbullbull hkps riag s tf bullbullbullbull R~l i bullbullbullbullbullbull hqps rvaep trod bullbullbullbull a
a bullbullbullbullbullbull aa 1r 1 d bullbullbull a a bullbullbullbullbullbull aa inh g bullbullbullbullbullbull sktIv pmilq 1 1g bullbullbull a MCGIVi V D Gli RI IYRGYPSAi AI D 1 gvmar s 1gsaks Ikek a bullbullbullbull qfcny Iversrgi tvq t dgd bullbull ead
51 100 R m hrae 19ktrIk bull bullbull bullbull s t ateR-l tqrae 19rkIk bullbullbull s t atrE-c htIrI qmaqae bull bull h bullbull h gt sv aae in qicI kads stbull bullbull k bullbull i gt T f- Ivsvr aetav bullbullbull r bullbull gq qg c
DL R R G V L AV E L G VGI lTRW E H ntvrrar Isesv1a mv bull sa nIgi rtr gihvfkekr adrvd bullbullbull bullbull nve aka g sy1stfiykqi saketk qnnrdvtv she reqv
101 150 R m ft g skka aevtkqt R 1 ft g ak agaqt E-c v hv hpr krtv aae in sg tf hrI ksrvlq T f- m i h q harah eatt
S E Eamp L A GY l SE TDTEVIAHL ratg eiavv av
qsaIg lqdvk vqv cqrs inkke cky
151 200 bullbull ltk bullbull dgcm hmcve fe a v n bull Iak bullbull dgrrm hmrvk fe st bull bull nweI bullbull kqgtr Iripqr gtvrh 1 s bull bull ewem bullbullbull tds kkvqt gvrh hlvs bull bull hh bullbull at rrvgdr iisg evm
V YR T DLF A A L AV S D PETV AR G M 1 eagvgs lvrrqh tvfana gvrs B s tef rkttIks iInn rvknk 5 c Ihlyntnlqn ~lt klvlees glckschy neitk
74
201 R m ph middot middot middot R 1 ah- middot middot middot middot middot middot E c 1 middot middot middot middot middot middot middot Hae in 1 middot middot middot middot middot T f - c11v middot middotmiddot middot middot middot middot middot M 1 tli middot middot middot middot middot middot middot middot middot B s 11 middot middot middot middot middot S c lvkse kk1kvdfvdv
251 R m middot middot middot middot middot middot middot middot middot middot middot middot middot middot
middot middot middot middot R-1 middot middot middot middot middot middot E c middot middot middot middot middot middot middot middot Hae in middot middot middot middot middot middot middot middot middot middot middot middot T f shy middot middot middot middot middot middot middot middot middot middot middot middot 1M middot middot middot middot middot middot middot middot middot middot middot middot middot middot B s middot middot middot middot middot middot middot middot middot middot middot middot middot middot middot S c feagsqnan1 1piaanefn1
301 R m fndtn wgkt R-1 fneti wgkt E c rf er Hae in srf er T f- rl mlqq
PVTR V YE DGDVA R M 1 ehqaeg qdqavad B s qneyem kmvdd S c khkkl dlhydg
351 R m nhpe v R-1 nhe v E c i nk Hae in k tli T f- lp hr
~ YRHlrM~ ~I EQP AVA M 1 geyl av B-s tpl tdvvrnr S-c pd stf
401 Rm slasa lk R-1 slasca lk E c hql nssr Hae in hq nar-T f f1s hytrq
RVL A SiIS A VG W M 1 ka slakt B s g kqS c lim scatrai
T ferrooxidans (Tf) Scerevisiae (Saee) Mleprae (Myele) and Bsublilis (Baesu)
tr1 ()-ltashyc 8 ~ er (11
-l l
CIgt
~ r (11-CIgt (11
-
$ -0 0shy
a c
omiddot s
S 0 0shyC iii omiddot $
ttl ~ () tI)
I-ltashyt ()
0
~ smiddot (11
81
CHAPTER 4
8341 Summary
8442 Introduction
8643 Materials and methods
431 Bacterial strains and plasmids 86
432 DNA constructs subclones and shortenings 86
864321 Contruct p81852
874322 Construct p81809
874323 Plasmid p8I809 and its shortenings
874324 p8I8II
8844 Results and discussion
441 Analysis of the sequences downstream the glmS gene 88
442 TnsBC homology 96
99443 Homology to the TnsD protein
118444 Analysis of DNA downstream of the region with TnsD homology
LIST OF FIGURES
87Fig 41 Alignment of Tn7-like sequence of T jerrooxidans to Ecoli Tn7
Fig 42 DNA sequences at the proximal end of Tn5468 and Ecoli glmS gene 88
Fig 43 Comparison of the inverted repeats of Tn7 and Tn5468 89
Fig 44 BLAST results of the sequence downtream of T jerrooxidans glmS 90
Fig 45 Alignment of the amino acid sequence of Tn5468 TnsA-like protein 92 to TnsA of Tn7
82
Fig 46 Restriction map of the region of cosmid p8181 with amino acid 95 sequence homology to TnsABC and part of TnsD of Tn7
Fig 47 Restriction map of cosmid p8181Llli with its subclonesmiddot and 96 shortenings
98 Fig 48 BLAST results and DNA sequence of p81852r
100 Fig 49 BLAST results and DNA sequence of p81852f
102 Fig 410 BLAST results and DNA sequence of p81809
Fig 411 Diagrammatic representation of the rearrangement of Tn5468 TnsDshy 106 like protein
107Fig 412 Restriction digests on p818lLlli
108 Fig 413 BLAST results and DNA sequence of p818lOEM
109 Fig 414 BLAST results on joined sequences of p818104 and p818103
111 Fig 415 BLAST results and DNA sequence of p818102
112 Fig 416 BLAST results and nucleotide sequence of p818101
Fig 417 BLAST results and DNA sequence of the combined sequence of 113p818l1f and p81810r
Fig 418 Results of the BLAST search on p81811r and the DNA sequence 117obtained from p81811r
Fig 419 Comparison of the spa operons of Ecali Hinjluenzae and 121 probable structure of T ferraaxidans spa operon
83
CHAPTER FOUR
TN7-LlKE TRANSPOSON OF TFERROOXIDANS
41 Summary
Various constructs and subclones including exonuclease ill shortenings were produced to gain
access to DNA fragments downstream of the T ferrooxidans glmS gene (Chapter 3) and
sequenced Homology searches were performed against sequences in GenBank and EMBL
databases in an attempt to find out how much of a Tn7-like transposon and its antibiotic
markers were present in the T ferrooxidans chromosome (downstream the glmS gene)
Sequences with high homology to the TnsA TnsB TnsC and TnsD proteins of Tn7 were
found covering a region of about 7 kb from the T ferrooxidans glmS gene
Further sequencing (plusmn 45 kb) beyond where TnsD protein homology had been found
revealed no sequences homologous to TnsE protein of Tn7 nor to any of the antibiotic
resistance markers associated with Tn7 However DNA sequences very homologous to Ecoli
ATP-dependentDNA helicase (RecG) protein (EC 361) and guanosine-35-bis (diphosphate)
3-pyrophosphohydrolase (EC 3172) stringent response protein of Ecoli HinJluenzae and
Vibrio sp were found about 15 kb and 4 kb respectively downstream of where homology to
the TnsD protein had been detected
84
42 Transposable insertion sequences in Thiobacillus ferrooxidizns
The presence of two families (family 1 and 2) of repetitive DNA sequences in the genome
of T ferrooxidans has been described previously (Yates and Holmes 1987) One member of
the family was shown to be a 15 kb insertion sequence (IST2) containing open reading
frames (ORFs) (Yates et al 1988) Sequence comparisons have shown that the putative
transposase encoded by IST2 has homology with the proteins encoded by IS256 and ISRm3
present in Staphylococcus aureus and Rhizobium meliloti respectively (Wheatcroft and
Laberge 1991) Restriction enzyme analysis and Southern hybridization of the genome of
T ferrooxidans is consistent with the concept that IST2 can transpose within Tferrooxidans
(Holmes and Haq 1990) Additionally it has been suggested that transposition of family 1
insertion sequences (lST1) might be involved in the phenotypic switching between iron and
sulphur oxidizing modes of growth including the reversible loss of the capacity of
T ferrooxidans to oxidise iron (Schrader and Holmes 1988)
The DNA sequence of IST2 has been determined and exhibits structural features of a typical
insertion sequence such as target-site duplications ORFs and imperfectly matched inverted
repeats (Yates et al 1988) A transposon-like element Tn5467 has been detected in
T ferrooxidans plasmid pTF-FC2 (Rawlings et al 1995) This transposon-like element is
bordered by 38 bp inverted repeat sequences which has sequence identity in 37 of 38 and in
38 of 38 to the tnpA distal and tnpA proximal inverted repeats of Tn21 respectively
Additionally Kusano et al (1991) showed that of the five potential ORFs containing merR
genes in T ferrooxidans strain E-15 ORFs 1 to 3 had significant homology to TnsA from
transposon Tn7
85
Analysis of the sequence at the tenninus of the 35 kb BamHI-BamHI fragment of p81820
revealed an ORF with very high homology to TnsA protein of the transposon Tn7 (Fig 44)
Further studies were carried out to detennine how much of the Tn7 transposition genes were
present in the region further downstream of the T ferroxidans glmS gene Tn7 possesses
trimethoprim streptomycin spectinomycin and streptothricin antibiotic resistance markers
Since T ferrooxidans is not exposed to a hospital environment it was of particular interest to
find out whether similar genes were present in the Tn7-like transposon In the Ecoli
chromosome the Tn7 insertion occurs between the glmS and the pho genes (pstS pstC pstA
pstB and phoU) It was also of interest to detennine whether atp-glm-pst operon order holds
for the T ferrooxidans chromosome It was these questions that motivated the study of this
region of the chromosome
43 OJII
strains and plasm ids used were the same as in Chapter Media and solutions (as
in Chapter 3) can in Appendix Plasmid DNA preparations agarose gel
electrophoresis competent cell preparations transformations and
were all carned out as Chapter 3 Probe preparation Southern blotting hybridization and
detection were as in Chapter 2 The same procedures of nucleotide
DNA sequence described 2 and 3 were
4321 ~lllu~~~
Plasmid p8I852 is a KpnI-SalI subclone p8I820 in the IngtUMnt vector kb) and
was described Chapter 2 where it was used to prepare the probe for Southern hybridization
DNA sequencing was from both (p81852r) and from the Sail sites
(p8I852f)
4322 ==---==
Construct p81809 was made by p8I820 The resulting fragment
(about 42 kb) was then ligated to a Bluescript vector KS+ with the same
enzymes) DNA sequencing was out from end of the construct fA
87
4323 ~mlJtLru1lbJmalpoundsectM~lllgsect
The 40 kb EeoRI-ClaI fragment p8181Llli into Bluescript was called p8181O
Exonuclease III shortening of the fragment was based on the method of Heinikoff (1984) The
vector was protected with and ClaI was as the susceptible for exonuclease III
Another construct p818IOEM was by digesting p81810 and pUCBM20 with MluI and
EeoRI and ligating the approximately 1 kb fragment to the vector
4324 p81811
A 25 ApaI-ClaI digest of p8181Llli was cloned into Bluescript vector KS+ and
sequenced both ends
88
44 Results and discussion
of glmS gene termini (approximately 170
downstream) between
comparison of the nucleotide
coli is shown in Fig 41 This region
site of Tn7 insertion within chromosome of E coli and the imperfect gtpound1
repeat sequences of was marked homology at both the andVI
gene but this homology decreased substantially
beyond the stop codon
amino acid sequence
been associated with duplication at
the insertion at attTn7 by (Lichtenstein and as
CCGGG in and underlined in Figs41
CCGGG and are almost equidistant from their respelt~tle
codons The and the Tn7-like transposon BU- there is
some homology transposons in the regions which includes
repeats
11 inverted
appears to be random thereafter 1) The inverted
repeats of to the inverted repeats of element of
(Fig 43) eight repeats
(four traJrlsposcm was registered with Stanford University
California as
A search on the (GenBank and EMBL) with
of 41 showed high homology to the Tn sA protein 44) Comparison
acid sequence of the TnsA-like protein Tn5468 to the predicted of the
89
Fig 41
Alignment of t DNA sequence of the Tn7-li e
Tferrooxi to E i Tn7 sequence at e of
insertion transcriptional t ion s e of
glmS The ent homology shown low occurs within
the repeats (underl of both the
Tn7-li transposon Tn7 (Fig 43) Homology
becomes before the end of t corresponding
impe s
T V E 10 20 30 40 50 60
Ec Tn 7
Tf7shy(like)
70 80 90 100 110 120
Tf7shy(1
130 140 150 160 170 180 Ec Tn 7 ~~~=-
Tf7shy(like)
90
Fig 42 DNA sequences at the 3 end of the Ecoll glmS and the tnsA proximal of Tn7 to the DNA the 3end of the Tferrooxidans gene and end of Tn7-like (a) DNA sequence determined in the Ecoll strain GD92Tn7 in which Tn been inserted into the glmS transcriptional terminator Walker at ai 1986 Nucleotide 38 onwards are the of the left end of Tn DNA
determined in T strain ATCC 33020 with the of 7-like at the termination site of the glmS
gene Also shown are the 22 bp of the Tn7-like transposon as well as the region where homology the protein begins A good Shine
sequence is shown immediately upstream of what appears to be the TTG initiation codon for the transposon
(a)
_ -35 PLE -10 I tr T~ruR~1 truvmnWCIGACUur ~~GCfuA
100 no 110 110 110
4 M S S bull S I Q I amp I I I I bull I I Q I I 1 I Y I W L ~Tnrcmuamaurmcrmrarr~GGQ1lIGTuaDACf~1lIGcrA
DO 200 amp10 220 DG Zlaquot UO ZIG riO
T Q IT I I 1 I I I I I Y sir r I I T I ILL I D L I ilGIIilJAUlAmrC~GlWllCCCAcarr~GGAWtCmCamp1tlnmcrArClGACTAGNJ
Fig 45 (a) Alignment of the amino acid sequence of Tn5468 (which had homology to TnsA of Tn7 Fig 44) to the amino acid sequence of ORF2 (between the merR genes of Tferrooxidans strain E-1S) ORF2 had been found to have significant homology to Tn sA of Tn7 (Kusano et ai 1991) (b) Alignment of the amino acids translation of Tn5468 (above) to Tn sA of Tn7 (c) Alignment of the amino acid sequence of TnsA of Tn7 to the hypothetical ORF2 protein of Tferrooxidans strain E-1S
(al
Percent Similarity 60000 Percent Identity 44444
Tf Tn5468 1 LARQRYGVDEDRVARFQKEGRGQGRGADYHPWLTIQDVPSQGRSHRLKGI SO 11 1111111 I 1111 1111 I I 111
Fig 49 (a) Results of the BLAST search on the inverted and complemented nucleotide sequence of 1852f (from the Sall site) Results indicate amino acid sequence to the TnsC of Tn7 (protein E is the same as TnsC protein) (b) The nucleotide sequence and open reading frame of p81852f
High Prob producing Segment Pairs Frame Score P(N)
IB255431QQECE7 protein E - Escher +1 176 15e-20 gplX044921lSTN7EUR 1 Tn7 E with +1 176 15e-20 splP058461 ECOLI TRANSPOSON TN7 TRANSPOSITION PR +1 176 64e-20 gplU410111 4 D20248 gene product [Caenorhab +3 59 17e-06
IB255431QQECE7 ical protein E - Escherichia coli transposon Tn7 (fragment)
417 (al BLAST results obtained from combined sequence of (inverted and complemented) and 1810r good sequence to Ecoli and Hinfluenzae DNA RecG (EC 361) was found (b) The combined sequence and open frame which was homologous to RecG
444 Analysis of DNA downstream of region with TnsD homology
The location of the of p81811 ApaI-CLaI construct is shown in Fig 47 The single strand
sequence from the C LaI site was joined to p8181Or and searched using BLAST against the
GenBank and EMBL databases Good homology to Ecoli and Hinjluezae A TP-dependent
DNA helicase recombinase proteins (EC 361) was obtained (Fig 417) The BLAST search
with the sequence from the ApaI end showed high sequence homology to the stringent
response protein guanosine-35-bis (diphosphate) 3-pyrophosphohydrolase (EC 3172) of
Ecoli Hinjluenzae and Scoelicolor (Fig 418) In both Ecoli and Hinjluenzae the two
proteins RecG helicase recombinase and ppGpp stringent response protein constitute part of
the spo operon
445 Spo operon
A brief description will be given on the spo operons of Ecoli and Hinjluenzae which appear
to differ in their arrangements In Ecoli spoT gene encodes guanosine-35-bis
pyrophosphohydrolase (ppGpp) which is synthesized during stringent response to amino acid
starvation It is also known to be responsible for cellular ppGpp degradation (Gentry and
Cashel 1995) The RecG protein is required for normal levels of recombination and DNA
repair RecG protein is a junction specific DNA helicase that acts post-synaptically to drive
branch migration of Holliday junction intermediate made by RecA during the strand
exchange stage of recombination (Whitby and Lloyd 1995)
The spoS (also called rpoZ) encodes the omega subunit of RNA polymerase which is found
associated with core and holoenzyme of RNA polymerase The physiological function of the
omega subunit is unknown Nevertheless it binds stoichiometrically to RNA polymerase
119
Fig 418 BLAST search nucleotide sequence of 1811r I restrict ) inverted and complement high sequence homology to st in s 3 5 1 -bis ( e) 3 I ase (EC 31 7 2) [(ppGpp)shyase] -3-pyropho ase) of Ecoli Hinfluenzae (b) The nucleot and the open frame with n
Add 4 ml 52 mix by inversion at room temp for 5 mins
Add 4 ml 53 Mix by to homogenous suspension
Spin at 15 K 40 mins at 4
remove to fresh tube
Equilibrate column with 2 ml N2
Load supernatant 2 to 4 ml amounts
Wash column 2 X 4 of N3 Elute the DNA the first bed
each) add 07
volumes of isopropanol
Spin at 4 Wash with 70 Ethanol Resuspended pellet in n rv 100 AU of TE and scan
volume of about 8 to 10 drops) To the eluent (two
to
140
SEQUITHERM CYCLE SEQUENCING
Alf-express Cy5 end labelled promer method
only DNA transformed into end- Ecoli
The label is sensitive to light do all with fluorescent lights off
3-5 kb
3-7 kb
7-10 kb
Thaw all reagents from kit at well before use and keep on
1) Label 200 PCR tubes on or little cap flap
(The heated lid removes from the top of the tubes)
2) Add 3 Jtl of termination mixes to labelled tubes
3) On ice with off using 12 ml Eppendorf DNA up to
125 lll with MilliQ water
Add 1 ltl
Add lll of lOX
polymeraseAdd 1 ltl
Mix well Aliquot 38 lll from the eppendorf to Spin down
Push caps on nrnnp1
141
Hybaid thermal Cycler
Program
93 DC for 5 mins 1 cycle
93degC for 30 sees
55 DC for 30 secs
70 DC for 60 secs 30 cycle 93 DC_ 30s 55 DC-30s
70degC for 5 mins 1 cycle
Primer must be min 20 bp long and min 50 GC content if the annealing step is to be
omitted Incubate at 95 DC for 5 mins to denature before running Spin down Load 3 U)
SEQUITHERM CYCLE SEQUENCING
Ordinary method
Use only DNA transformed into end- Ecoli strain
3-5 kb 3J
3-7 kb 44
7-10 kb 6J
Thaw all reagents from kit at RT mix well before use and keep on ice
1) Label 200 PCR tubes on side or little cap flap
(The heated lid removes markings from the top of the tubes)
2) Add 3 AU of termination mixes to labelled tubes
3) On ice using l2 ml Eppendorf tubes make DNA up to 125 41 with MilliQ water
142
Add I Jtl of Primer
Add 25 ttl of lOX sequencing buffer
Add 1 Jtl Sequitherm DNA polymerase
Mix well spin Aliquot 38 ttl from the eppendorf to each termination tube Spin down
Push caps on properly
Hybaid thermal Cycler
Program
93 DC for 5 mins 1 cycle
93 DC for 30 secs
55 DC for 30 secs
70 DC for 60 secs 30 cycles
93 DC_ 30s 55 DC-30s 70 DC for 5 mins 1 cycle
Primer must be minimum of 20 bp long and min 50 GC content if the annealing step is
to be omitted Incubate at 95 DC for 5 mins to denature before running
Spin down Load 3 ttl for short and medium gels 21t1 for long gel runs
143
REFERENCES
144
REFERENCES
Abrahams J P Lutter R Todd R J van Raaij M J Leslie A G W and Walker J E 1993 Inherent asymmetry of the structure of F1-ATPase from bovine heart mitochondria at 65 Aresolution EMBO J 121775-1780
Abrahams J P Leslie A G W Lutter R and Walker 1 E 1994 Structure at 28 A resolution of FeATPase from bovine heart mitochondria Nature 370621-628
Adzumah K and Mizuuchi K 1988 Target immunity of Mu transposition reflects a differential distribution of Mu B protein Cell 53257-266
Akey C W Crepeau R H Dunn S D McCarty R E and Edelstein S J 1983 Electron microscopy of single molecules and crystals of F1-ATPases EMBO J 21409-1415
Allet B 1979 Mu insertion duplicates a 5 bp sequence at the host inserted site Cell 16 123shy129
Amzel L M and Pedersen P L 1983 Proton ATP-ases structure and mechanism Ann Rev Biochem 52801-824
Andersson L Mac Neela J and Wolfenden R 1985 Use of secondary isotope effects and varying pH to investigate mode of binding of inhibitory amino aldehydes by leucine aminopeptidase Biochem 24330-333
Andrews G F Dugan R P and Stevens C J 1992 Combining physical and bacterial treatment for removing pyritic sulfur from coal In Processing and Utilization of High-sulfur coals IV Dugan P R D Quigley and Y Attia (eds) p 515 Elsevier New York
Andrulis I L Chen J and Ray P N 1987 Isolation of human cDNAs for asparagine synthetase and expression in Jansen rat sarcoama cells Mol Cell BioI 72435-2443
Andruszkiewicz R Milewsky S Zieniawa T and Borowski E 1990 Anticandidal properties of N3 -(4-methoxy-fumaroyl)-L-2 3-diamino propanoic acid oligopeptides 1 Med Chern 33132-135
Arciszewska L K Drake D and Craig NL 1989 Transposon Tn7 cis-acting sequences in transposition and transposition immunity J Mol BioI 20735-42
Bachmann B J 1983 Linkage map of Escherichia coli K-12 edition 7 Microbiol Rev 47180-230
145
B Plumbridge 1 A Cochet D Souza J M M M and Calcagno M 1988 Cordinated regulation of amino J
Bacteriol 1754951-4956
0 B Vermoote P V and Le Goffic F 1993 synthetase from Ecoli lUllL- mechanism and inhibition by N3-fumaroyl-2 3-diaminopropionic derivatives Biochem 27 2282-2287
Vermoote P Haumont PY syrlthl~ta~e from Escherichia coli purification site location Biochemistry 26 1940-1948
Badet B Inagaki K Soda K Walsh dependent inhibition of Bacillus stearothermophilus alanine racemase by phosphonate isomers by isomerization to non covalent enzyme-(1-aminoethyl) complexes Biochem 253275-3282
Badet-Denisot M A and Badet of glucoseamine-6shyphosphate synthetase by diethylpyrocarbonates histidine requirement for enzymatic activity Arch Biochem Biophys
Bainton R Gamas P and transposition in vitro proceeds through an excised transposon intermediate (JpnIPtlltpi1 111 breaks in DNA Cell 65805-816
Barry G 1986 Permanent insertion of v~u into the chromosomes of soil bacteria BioTechnology 4446-449
Barth P T Datta N Grinter N S 1976 Transposition a deoxyribonucleic acid sequence trimethoprim and streptomycin resistances from R483 to other replicons 1 Bacteriol 125800-810
Bates C J Adams W and R R 1968 Control of the formation of uri dine diphospho-N-acetyl and glycoprotein synthesis in rat liver J Chern 2411705-17
Benjamin N 1989 Intramolecular transposition of TnlD Cell 59373shy383
Bennet R L and Malamy M resistant mutants of Escgerichia coli and phosphate Commun 40496-503
Benneth1 1978 Bacterial leaching patterns on pyrite crystal J Bacteriol
146
Berg D E Davies 1 Allet B and Rochaix 1 D 1975 Transposition of R factor genes to bacteriophage lambda Proc Natl Acad Sci USA 723268-3632
Berg D E and Drummond M1978 Absence of DNA sequences homologous to transposable element Tn5 (Kan) in the chromosome of Ecoli K-12 J Bcateriol 136419-422
Birkenhager R Hoppert M Deckers-Hebestriet G Mayer F and Altendorf K 1995 The Fo complex of the Ecoli ATP synthase investigation by electron imaging and immunoelectron microscopy Eur J Biochem 23058-67
Bjqgtrbaek C Foersom V and Michelsen O 1990 The transmembrane topology of the a subunit from ATPase in Escherichia coli analysed by PhoA protein fusions FEBS lett 26031-34
Boekemia E J Berden JA and van Heel MG 1986 sructure of mitochondrial F1-ATPase studied by electron microscopy and image processing Biochim Biophys Acta 851353-360
Bolton E Glynn P and OGara F 1984 Site specific transposition of Tn7 into a Rhizobiwn meliloti megaplasmid Mol Gen Genet 193153-157
Boos W 1974 Bacterial transport Annu Rev Biochem 43123-146
Boursaux-Eude C Girons I S and Zuerner R 1995 IS1500 an IS3-like element from Leptospira interrogans Microbiol 1412165-2173
Boyer P D 1993 The binding change mechanism for ATP synthase-some probabilities and possibilities Biochim Biophys Acta 1140215-250
Brachet P Eisen H and Rambach A 1970 Mutations in coliphage lambda affecting the expression of replicative functions 0 and P Mol Gen Genet 108266-276
Brink J Boekemia E 1 and van Bruggen E F 1987 The structure of NADH ubiquitone oxidoreductase fron beef heart mitochondria Crystals containing an octameric arrangement of iron-sulphur protein fragments Eur J Biochem 166287-294
Brock T D and Gustafson J 1976 Ferric iron reduction by sulphur- and iron-oxidizing bacteria Appl Environ Microbiol 32 567-571
Brown L D Dennehy M E and Rawlings D E 1994 The FI genes of the FIFo ATP synthase from the acidophilic bacterium Thiobacillus jerrooxidans complement Escherichia coli FI unc mutants FEMS Microbiol Lett 12219-25
Buchanan 1 1 Adv The Amidotransferases Enzymol 3991-183
Buckley Science
Caruso M Pseudomonas nOVHrn
oxidation of pyrite Applied
1 1982 Interactions Mol Gen Genet
phage 1
Chmara H by Biophysica
synthetase from bacteria antibiotic tetaine Biochimica et
Microbiol Lett 11197-206 controls on the oxidation of refractory Barberton
genetic elements and evolution Nature 263731shyCohen S N 1976 738
Corbet C M and 1 Ingledew 1987 Is oxidation by Thiobacillus ferrooxidans
Couillard D and ~~b 118(5)808-81
Metallurgical residue V UlllLltHIH of metals from sewage sludge J
Cox G B a reassessment of the
F and Hatch L 1986 The mechanism of ATP synthase of the b and a subunits Biochim Acta 84962-69
Craig N L 1995 Unity in Science 270253-254
Craig N and Gamas P 1 Purification and characterization of a transposition protein that binds ATP DNA Nuc Acids Res
1 2873-97 C A 1990 in response to environmental stress Advances in
alUIUH~ on the activity subunit b-specific polyc1onal
G Simoni and Altendorf K 1992 Influence vlJnu~ of the ATP synthase of t-S(nel~lCn
1 BioI Chern 26712364shy
M A Le Goffic F and 1991 Glucosamine- 6P two proteins on limited proteolysis ltVU Biophys 288225-230
148
Dobrogosz W J 1968 _l in Escherichia coli and its to catabolite repression J
Doublet P 1 van Heijenoort and 1993 The murI gene of is an essential gene that encodes klUltUllU~v racemase activity J Bacteriol 1752970-2979
P J van Heijenoort 1992 Identification of the Ecoli which is required for the D-glutamic acid a specific component peptidoglycan 1 Bacteriol
Garren A Garen and Torri ani 1961 Genetic control of repression UCUlllV phosphatase in Ecoli 1 Mol BioI
D J and Boeke 1 D 1990 A 1-1-0- structure is required for Ty1 transposition Genes Develop 4324-330
1982 Transposition of Tn7 occurs at a on Caulobacter crescentus 10SIClmle 1 Bacteriol 151 1056-1 058
H 1990 Molecular IHovUUU)11 -transporting 345-391 In T 12 Bacterial
Press Ltd London
transport and coupling by the synthase insights mechanism of function J Bioenerg 24485-491
1992b Subunit c of FIFo ATP role m transduction Biochim Biophys Acta 1101 v--rJ
Eliopoulos E E Jackson P 1 Keen IN HUIUVI L Thompson P and 1 Structure of a 16 kDa integral AU-UUl that identity to
of the vacuolar H+ -ATPase Protein 15
Fling M 1 C 1985 Nucleotide sequence of encoding the amino glycoside-modifying enzyme 3(9)-O-nucleotidyltransferase Res 137095-7106
Fling M and C l-1UUJIU nucleotide sequence of dihydrofolate rhnrpri by Tn7 Nucleic Acids Res 115
Flores Llchtenstem C P 1990 DNA sequence anlysis of tnsA for the Tn7 transposition Nucleic Acids 18901 911
149
149
Foster D L and Fillingame R H 1982 Stoichiometry of subunits in the H+-ATPase complex of Escherichia coli J BioI Chern 2572009-2015
Fraga D Hermolin J Oldenburg M Miller MJ and Fillingame R H 1994 Arginine 41 of subunit c of Escherichia coli H+-A TP synthase is essential in binding and coupling of F to Fo J BioI Chern 2697532-7537
Friedl P Hoppe J Gunsalus R P Michelsen 0 von Meyenburg K and Schairer H U 1983 Membrane integration and function of the three Fo subunits of the A TP-synthase of Escherichia coli K12 EMBO 1 299-103
Frisa P S and Sonneborn D R 1982 Developmentally regulated interconversion between end product inhibitable and non-inhibitable forms of a first pathway-specific enzyme activity can be mimicked in vitro by dephosphorylation reactions Proc Natl Acad Sci USA 796289-6293
Fujiwara T and Mizuuchi K 1988 Retroviral DNA integration Structure of an integration intermediate Cell 54497-504
Galas D J and Chandler M 1989 Bacterial insertion sequences In Mobile DNA Berg D E and Howe M M (eds) Washington D C American Society for Microbiology pp 109shy162
Gay N J Tybulewicz V L1 Walker JE 1986 Insertion of transposon Tn7 into the Ecoli gmS transcriptional terminator Biochem J 234 111-117
Gentry D R and Cashel M 1995 Cellular localization of the Escherichia coli SpoT protein J Bacteriol 177 3890-3893
Gentry D R Xiao H Burgess R R and Cashel M 1991 The omega subunit of Ecoli K-12 RNA polymerase is not required for stringent RNA control in vivo J Bacteriol 173 3901-3903
Girvin M E and Fillingame R H 1993 Helical structure and folding of subunit c of FIFo ATP synthase IH NMR resonace assignments and NOE analysis Biochemistry 3212167shy12177
Gogol E P Lucken U Bork T and Capaldi R A 1989 Molecular architecture of Escherichia coli F Adenosinetriphosphatase Biochemistry 284709-4716
Gogol E P Aggeler R Sagermann M and Capaldi R A 1989 Cryoelectronic Microscopy of Escherichia coli FI Adenosinetriphosphatase Decorated with Monoclonal Antibodies to Individual Subunits of the complex Biochemistry 284717-4724
150
Heinikoff S 1984
Golinelli-Pimpaneaux B and Badet involvement of Lys603 from Ecoli glucosamoine-6-phosphate synthase substrate fructose-6-phosphate 1 Biochem 201175-182
Gottesman M M and Rosner 1 L of a detenninant of uvu_bullu
resistance by coliphage lambda Sci USA 725041-5045
Grunstein M and Hogness D
Colony hybridization a method for isolation cloned DNAs that contain a 1Jbullu Proc NatL Acad Sci USA 723961-3965
Hauer B and Shapiro 1 A 1984 Control of Tn7 transposition Mol Gen Genet 194
Hedges R W and Jacob Transposition of ampicillin resistance from to other replicons MoL 1-40
Hennolin 1 Gallant 1 psi subunit in the Fo sector of the H+ -ATPase of Ecoli J Bioi
R H 1983 Topology organization and function
Hennolin J and R 1989 Assembly of Fo sector of synthase Interdepenndence of subunit insertion into the membrane 1 2817
van Montagu M Holsters Zambryski P Beuckeleer M Willmitzer and Schell 1 M 1980 interaction of
DNA plant cells Proc Soc Lond 210351-365
P 1972 Insertion mutations control region of Physical characterization of the mutants Mol Gen Genet
115266-276
Holmes applications pI
UI Haq 1990 Adaptation of Thiobacillus vu)nuu for industrial In J Salley R G McCready Wichlacz (ed)
1989 CANMET Ottawa Canada
Holtje J V and U Schwarz 1985 Biosynthesis and growth murein sacculus p 77shy119 InN (ed) Molecular cytology of Escherichia Academic Press Inc New
Acad Sci USA
Heffron E Reubens C and which mediates ampicillin
Translocation of a plasmid DNA sequence nature and specificity of Natl
digestion with exonuclease III creates fl tr breakpoints 1-369
Hernalsteens J M Agrobacterium
Hirsch H the galactose nnnn fJu
151
Holzenburg A Jones P c Franklin T Padi J B and Finbow M E 1993 Evidence for a common structure 1 Biochem 21321-30
Hoppe 1 and Sebald W 1986 Topological pathway of the protons through Fo is provided by amino acid the lipid phase Biochimie (Paris) 68427-434
S Otsubo H Davidson N and 1975 Electron microscope heteroduplex of sequence relations among plasmids identification and mapping of the
insertion sequences IS] and IS2 in F and R plasmids J Bacteriol 22762-775
Inoue c Sugawara K and 1991 regulatory gene in Thiobacillus ferrooxidans is spaced apart from Mol Microbiol 52707-2718
Ish-Horowicz D and Burke and cosmid cloning Nucleic Acids Res 92989-2998
Johnston B Clennell M and D 1995 Structure and function of Tn5467 a Tn2l-like transposon located on T jerrooxidans broad host range plasmid Appl Envir Micro
Kahmann R and Kamp Nucleotide sequences of the attachment bacteriophage Mu DNA Nature 280247-250
Kahn K and Schaefer M R Characterization of trans po son 5469 from cyanobacterium Fremyella diplosiphon J 1777026-7032
Karavaiko G Golovacheva S Pivovarova T A Tzaplina I A and Vartanjan 1988 Thermophilic ltPM Sulfobacillus page 29-41 In Biohydrometallurgyshy87 Norris P and Science and Technology Letters Kew 1
Kennedy J and Humpphreys J D 1976 Microbial cells living immobilised on Nature 261242-244
Koonin 1993 SpoU protein of Escherichia coli belongs to a new family of Nucleic Acids Res 19
Kopecko J and site specific recA mdependtm recombination between bacterial Pt of palindromes at the N ad Acad Sci USA
on L-glutamine D-fructose ud()transtiera~e 1 BioI
152
Krumholz L R Esser U and Simoni RD 1989 Nucleotide sequence of the unc operon of Vibrio alginolyticus Nucleic Acids Res 177993-7994
Kubo K and Craig N 1990 Bacterial transposon Tn7 utilizes two classes of target sites 1 Bacteriol 1722774-2778
Kucharczyk N Denisot M A Le Goffic F and Badet B 1990 Glucosarnine-6-phosphate synthase from Ecoli detennination of the mechanism of inactivation by N3 fumaroyl-L-2-3 diarninoproprionic derivatives Biochemistry 293668-3676
Kusano M Takeshima T Inoue C and Sugawara K 1991 Evidence for two sets of structural genes coding for Ribulose biphosphate carboxylase in Thiobacillus ferrooxidans 1 Bacteriol 1737313-7323
Lane Dl A P Harrison lnr D Stahl B Pace SJ Giovannoni GJ Olsen and N R Pace 1992 Evolutionary relationship among sulphur- and iron-oxidizing eubacteria 1 Bacteriol 174267-278
Lee C H Bhagwhat A and Heffron F 1983 Identification of a transposon TnJ sequence required for transposition immunity Natl Acad Sci USA 806765-6769
Lewis M 1 Chang 1 A and Somoni R D 1990 A topological analysis of subunit a from Escherichia coli F1Fo-ATP synthase predicts eight transmembrane segments 1 BioI Chern 265 10541-10550
Lichtenstein C P and Brenner S 1982 Unique insertion site of Tn7 in the Ecoli chromosome Nature (London) 297601-603
Lichtenstein C P and Brenner S 1981 Site-specific properties of transposition to the Ecoli chromosome Mol Gen Genet 183380-387
Liu X Petersson S and Sandstrom A Mesophilic versus moderate thennophilic bioleaching Biohydrometallurgy Technologies Vol 1 A E Tonna 1 E Wey and Lakshmanan V 1 (ed) pp 29-38
Lizarna H M and Sankey B M 1993 Oxidation of H2S by Thiobacillus thiooxidans is inhibited by substrate and methane BiohydrometaHurgy Technologies Vol II A E Tonna 1 E Wey and C L Brierley (ed) pp 339-364
Lundgren D G and Silver M 1980 Ore leaching by bacteria Ann Rev Microbiol 34263shy268
Makino K Amemura M Shinagawa H Kobayashi A and Nakata A 1985 Sequence of the genes involved in phosphate transport and regulation of the phosphate regulon in Escherichia coli 1 Mol BioI 184231-240
Makino K Shinagawa Amemura M Kimura S Nakata A and Ishihama 1988 Euuv1J of the phosphate regulon of Ecoli Activation of pstS by the PhoB
protein in vitro 1 Mol BioI 20385-95
Makino K Shinagawa H Amemura M Yamada M and 1990 Signal transduction in the phosphate regulon of the Escherichia coli involves phosphotransfer nprlrJp~middotn PhoR and PhoB proteins J Mol BioI 21055
Malamy 1966 Frameshift mutations in the nnlnn of Escherichia coli Cold Harb Symp Quant BioI 31189-201
Malamy M H 1972 Electron microscopy insertions in the lac operon of Escherichia coli MoL Gen
Malamy M H and Bennett R L 1970 mutants of Ecoli and phosphate transport Biochem Biophys Res Commun
Maniatis T Fritsch EF Sambrook J 1982 nn A laboratory manual Cold Spring Harbor Laboratory Press Cold
McKnight G L S L Mudri SH Mathewest R S Marshall P O Sheppard and P J OHara 1990 Molecular cloning synthesis and bacterial expression of human glutaminefructose-6-phosphate UUAUU u J Chem 26725208-25212
Medveczky N and H Rosenburg 1970 phosphate-binding protein of Escherichia Biochim Biophys Acta 211 158-168
Mei B and Zalkin H 1989 A Cysteine-Histidine-Aspartate catalytic triad is involved in Glutamide Amide Transfer Function purF-type Glutamine amodotransferases 1 BioI Chem 264 16613-16619
Mengin-Lecreulx D and J van 1993 Identification of glmU gene encoding Nshyacetylglucosamine in Escherichia coli J Bacteriol 1756150shy6157
Michaelis M J and Criddle M J 1970 Mitochondrial DNA and mutants in Saccharomyces cerevisiae Biochem Genet 5487-495
Michaelis Starlinger P 1969 Two insertions in the jO v
operon having different homologous DNA sequences MoL Gen Genet 10437 377
Miller J H Calos Transposable elements Cell 20579-595
154
Miller J Oldenburg M and Fillingame R 1990 The essential carboxyl group of in subunit c the FIFo ATP synthase can be and H( +)-translocating function retained Proc NatL Acad 874900-4904
Mitcell P 1966 Chemiosmotic coupling in - and phosphorylation BioL Rev 41455-502 (1966)
Mizuuchi K 1984 Mechanism of transposition of bacteriophage Mu Polarity of the strand reaction at the initiation of the transposon Cell 39395-404
C Ph de Donato Berthelin J Surface oxidized species a key factor in the study of bioleaching processes Biohydrometallurgical In A J
Weyand V I Lakshmanan ed 1993 Vol 1 175-184
A Ishino Shinagawa H Makino K and M 1987 Nucleotide of the iap gene responsible for alkaline phosphatase isoenzyme conversion in Ecoli
identification of product 1 1695429-5433
K 1989 The tsr gene-coding prevents thiopeptin from inhibiting ppGpp synthesis in Streptomyces lividans FEMS Microbiol Lett
Ogasawara N and Yoshikawa H 1992 and their organIzatIon in the repnc()n of the bacterial chromosome Mol Microbiol 6(5)629-634
Ohtsubo Davidson N and 1975 microscope heteroduplex studies of sequence relations among plasmids identification and mapping of the insertion sequences 1 and IS2 in F and R plasmids 1 122746-775
Olson G J 1994 Microbial oxidation gold ores and gold bioleaching FEMS un Lett 119 1-6
Orle K A N L 1991 Identification of transposition prroteins by the bacterial transposon [corrected republished originally printed in Gene 1990 Nov 30 96(1) Gene 10425-31
Ouellette M Roy P Homology of ORFs from and from to site specific recombinases 1987 Nucl Acids 10055-10059
Murein synthesis p663-671ln Neidhardt J L Ingraham B Louw M~ M Schaechter H E Umbarger (ed) Escherichia coli and Salmonella
ryphimurium cellular and bioI voL 1 American Society Microbiology Washington
Perlin D S and Senior A Functional and cross-reactivity of antibody to purified subunit b (uncF protein) of Escherichia coli proton-ATPase Arch Biochem Biophys 236603-611
155
Plumbridge J A O Cochet Souza J M Altramirano M M Calcagno M L and Badet B 1993 Coordinated regulation of amino sugar-synthesizing and -degrading enzymes in Escherichia coli K-12 J Bacteriol 175(16)4951-4956
Pretorius I M Rawlings D E and Woods D R 1986 Identification and cloning of Thiobacillus jerrooxidans structural Nif genes in Escherichia coli Gene 4559-65
Qadri M I Flores C c Davis J and Lichtenstein C 1989 Genetic analysis of attTn7 the transposon Tn7 attachment site in Escherichia coli using a novel M13-based transduction assay 1 Mol BioI 20785-98
Radstrom P Skold 0 Swedberg J Roy P H and Sundstrom L 1994 Tn5090 of plasmid R751 which carries integron is related to Tn7 Mu and retroelements J Bacteriol 1763257-3268
Raetz C R H 1987 Structure and biosynthesis of lipid A in Escherichia coli pp 498-503 In F C Niedhardt J L Ingraham K B Low B Magasanik M Schaechter and H E Umbarger (ed) Escherichia coli and Salmonella typhimurium cellular and molecular biology vol 1 American Society for Microbiology Washington DC
Rao N N and Torriani A 1990 Molecular aspects of phosphate transport in Escherichia coli Mol Microbiol 4(7) 1083-1090
Rawlings DE D R Woods and NP Mjoli 1991 The cloning and structre of genes from the autotrophic biomining bacterium Thiobacillusjerrooxidans p 215-237 In PJ Greenaway (ed) Advances in gene technology vol 2 JAI Press London
Richet E and O Raibaud 1989 MalT the regulatory protein of the Escherichia coli maltose system is an A TP-dependent transcriptional activator EMBO 1 8981-987
Rogers M Ekaterrinaki N Nimmo E and Sherratt D 1986 Analysis of Tn7 transposition Mol Gen Genet 205550-556
Ronson C W Nixon B T Albright L M anf Ausubel F M 1987 Rhizobium meliloti ntrA (rpoN) gene is required for diverse metabolic functions J Bacteriol 1692424-2431
Rosenburg H Gerdes R G and Chegwidden K 1977 Two systems for the uptake of phosphate in Ecoli JBacterioL 131505-511
Rosenburg H 1987 In ion transport in Prokaryotes Rosen B P and Silver S (eds) New York Academic Press pp 205-248
Ross D G Swan 1 and N Klecker 1979 Physical structures of TnlO-promoted deletions and inversions role of 1400 base pairs inverted repetitions Cell 16721-731
156
Saedler H and P 19670 mutation in the galactose operon in Ecoli II Physiological characterization MoL Gen Genet 100 190-202
Saedler P 1968 00 and strong polar mutations in the Genet 102353-363
Sambrook J Fritsch Maniatis 1989 Molecular cloning a laboratory manual Cold Harbor Laboratory Cold Spring Harbor New York
Sand W Gehrke T Hallmann Rohde K Sobotke B and Wintzien S 1993 Bioleaching metal sulphides The importance of Leptospirillum ferrooxidans Biohydromemetallurgical Technologies Voll pp 15-25
Sanger Nicklen and Coulson A DNA sequencing with chain-tennination inhibitors Natl Acad USA 745463-5467
Schneider E and Altendorf K All the three subunits are required for an active proton channel (Fo) of Escherichia coli synthase (FIFo) ~-
518
Schneider E and Allendorf K 1987 Bacterial adenosine 5-triphosphate synthase purification and reconstitution complexes and biochemical and functional characterization of subunits Microbio Rev 51477-497
Schrader 1 A and D S Holmes 1988 Phenotypic switching Thiobacillus ferrooxidans J BacterioL 1703915-3023
Scordilis G E H and Lassie 1987 Identification of transposable elements which activates gene expression in Pseudomonas cepacia J Bacteriol 1698-13
Sekine Eisaki N and Ohtsubo E 1996 Identification and characterization of the lS3 molecules generated by breaks 1 BioL Chern 271197-202
Senior A E 1990 The proton-trans locating of Escherichia coli Annu Rev Biophys Chern 194-41
Shapiro 1 and Adhya S 1969 The jLU operon of K-12 n A deletion analysis of the structure polarity 62249-264
J A 1969 Mutations caused by insertion genenc material the galactose operon Escherichia 1 MoL BioI 4093-105
Silvennan M P 1967 Mechanism of bacterial pyrite oxidation 1 Bacteriol 941046-1051
157
C C ElIson and Levinson 1983 Identification of the typel trimethoprim resistance reductase specified by the R-plasmid R43 comparison with procaryotic and eucaryotic dihydrofolate reductase 1 Bacteriol 155 100 1-1008
Smith and Jones P transposition a multigene process Identification of a regulatory product 147915-7927
Sprague Jr Bell R M Cronan 1 A mutant of Ecoli auxotrophic for organic phosphates evidence two defects in phosphate Mol Gen Genet 14371-77
Starlinger and Michaelis 1968 Suppression the sequential aO[)ealame of the galactose enzymes in a transferase amber mutant coli Mol Genet 102367-369
Starlinger 1977 IS elements in microorganisms MicrobioL Immunol
Steffens K Schneider E Herkernhoff B Schmid Altendorf K portion of Escherichia coli A TP synthase resolution of from subunit b J BioI Chern 2625866-5869
Steffens A Deckers-hebestreit G and Altendorf K 1987b The and functional relationship of A TP synthases (FoFI) from Ecoii and the thermophilic bacterium PS3 J Biol 2626334-6338
Strominger J and M S Smith 1959 Uridine diphosphoacetylglucosamine pyrophosphorylase 1 BioI Chern 2341822-1827
Sundstrom Skold 1990 dhfrI trimethoprim resistance gene of can be found at in other genetic surroundings Antimicrob Agents Chemother 34642shy650
Sundstrom L Roy P H and Skold Site-specific of three cassettes in Tn7 J Bacteriol 1733025-3028
Surin B P and Downie JA 1988 of the Rhizobium leguminosarum nodLMN involved efficient host-specific nodulation Mol Microbiol 2 173-183
B P Dixon N and Rosenburg 1986 Purification PhoU protein a OClItnl
regulator of the pho regulon of Ecoli K-1 J Bacteriol 168631
B P D A Jans A L Fimmel Shaw GB Cox Rosenburg Structural gene for the phosphate-repressible phosphate binding of Escherichia coli
own promoter nucleotide of the phoS 1 Bacteriol
158
Suzuki I Chang W and Takeuchi 1994 Oxidation of organic compounds by Thiobacilli Symp Series 55060-67
Takeyama M S Y Noumi T Maeda Ishibashi S and Futai M 1988 Beta subunit of Ecoli amino acid replacement within a conserved sequence G-X-X-X-X-GshyK-TS) v~u binding proteins lett 218222-226
Thomson JA M Hendson and R M 1981 Mutagenesis by insertion of drug resistance Tn7 into a vibrio 11 J Bacteriol
Tichy R Grotenhuis J T c Janssen van Houten R Rulkens and Lettinga 1993 Application of the sulphur cycle bioremediation of soils polluted with heavy metals In Int Conf Contaminated 93 ed F Arendt G J Annokee R Bosman and W J van der Brink Kluwer Academic Publishers Dordrecht pp 1461-1462
G and Mayer An electron approach to the quaternary structure of mitochondrial Eur J Biochem 13237-45
J and Tschape streptothricin resistance transposons Tn1825 and Tn1826 and transposon Tn 7 Plasmidnuu
18246-249
Tso J Y Zalkin H van Cleemput M Yanofsky C and JM 1982 Nucleotide of Escherichia coli and deduced amino glutamine
phosphribosylpyrophosphate am idotransferase J BioI Chern
V Mesyanzhinova I V Koslov I A and Orlova 1984 Structure of studied by electron and image processing Lett 167285-289
P Barber C and transposons Tn5 and Tn7 in Xanthomonas campestris pv campestris MoL Gen 157
Ullrich J and van Putten P Identification of the Gonococcal glmU gene UVUUIJ
the enzyme N-acetylglucosamine 1-Phosphate Uridyltransferase involved in the synthesis UDP-GlcNAc J of Bact 1776902-6909
M 1992 Eight bacterial proteins includin]g UDP-N -acetylglucosamine acyltransferase three vother of Escherichia coli of a six-residue n tVI
theme FEMS MicrobioL 97249-254
van der Steen J J D Doddema J and de Uidoging van zware metalen afvalstromen met behulp van thiobacilli (Removal metals from waste streams
using thiobacilli) Ministry of Housing Physical and Enviromental (VROM) Directoraat-Generaal Milieubeheer Rapport 199210 Hague
Vermoote P 1988 Universite Paris VI Paris Hrllnro
159
Vignais P V Lunard Issartel J P and Dupuis A 1985 Interaction n l1f middotn
oligomycin sensitivity protein (OSCP) beef heart mitochondrial FeATPase 2 Identification of interacting Fl subunits cross-linking Biochem J
Vik S and Dao NN Prediction of transmembrane topology of Fo proteins from Ecoli ATP synthase variational hydrophobic moment analyses Biochim Biophys Acta 1140 199-207
von Meyenburg B B Jcentrgensen J Nielsen and F Hansen 1982 Promoters of the atp operon for the membrane-bound ATP synthase of Ecoli mapped by TnlO insertion mutations MoL Gen Genet 188240-248
von Meyenberg F G Hansen 1980 The origin of replication oriC the Ecoli chromosome near oriC and construction of oriC mutants ICN-UCLA Symp MoLCellBiol 191 159
Waddell S and Craig N 1989 Tn7 transposition recognition of the attTn7 Proc Natl Sci USA 863958-3962
Waddell C S and Craig N L 1988 transposition two transposition pathways directed by five Tn7-encoded genes Develop 21
J E Gay N J Saraste M and A N 1984 DNA sequence the Escherichia coli unc operon Completion of the sequence of a kilobase segment containing
oriC unc and phoS J224799-815
and Collinson 1 1994 the role the stalk in the coupling mechanism of FEBS 34639-43
Walker 1 E Gay N J and Tybulewicz V L 1986 of transposon Tn7 into the Escherichia glmS transcriptional terminator Biochem 1 243 111-1
Wanner Land McSharry 1982 Phosphate-controlled gene in Escherichia coli using Mudl-directed lacZ fusions J Mol BioI 158347-363
Weng M and Zalkin H 1987 Structural role for a region the CTP synthetase glutamide amide transfer domain J Bacteriol 1693023-3028
Wheatcroft R and S and nucleotide sequence of Rhizobiwn meliloti insertion between the transposase encoded by ISRm3 and those encoded by Staphylococcus aureus and Thiobacillus ferrooxidans IST2 J 1732530-2538
160
Whitby M C and Lloyd R 1995 Branch of three-strand recombination intennediates by RecG a possible pathway for securing middotIULl initiated by duplex DNA 1 143303-3310
Wilkens and Capaldi R A Assymmetry and changes in examined by cryoelectronmicroscopy BioI Chern Hoppe-Seyler 37543-51
and Malamy M 1974 The loss of the PhoS periplasmic protein leads to a the specificity a constitutive phosphate transport system in Escherichia coli Biochem Res Commun 60226-233
WiUsky G Rand Malamy M 1980 of two separable inorganic phosphate transport systems in Escherichia coli J BacterioL 144356-365
P J and and C F 1973 enzyme of metabolism and Stadtman R eds) pp 343-363 Academic Press New York
Winterbum P J and Phelps F 197L control of hexosamine biosynthesis by glucosamine synthetase Biochem 1 121711
Wolf-Watz H and Norquist A 1979 Deoxyribonucleic acid and outer membrane protein to outer membrane protein involves a protein J 14043-49
Wolf-Watz H and M 1979 Deoxyribonucleic acid and outer membrane strains for oriC elevated levels deoxyribonucleic acid-binding protein and
11 for specific UU6 of the oriC region to outer membrane J Bacteriol 14050-58
Wolf-Watz H 1984 Affinity of two different chromosome to the outer membrane of Ecoli J Bacteriol 157968-970
Wu C and Te Wu 197 L Isolation and characterization of a glucosamine-requiring mutant of Escherichia 12 defective in glucoamine-6-phosphate synthetase 1 Bacteriol 105455-466
Yamada M Makino Shinagawa Nakata A 1990 Regulation of the phosphate regulon of Escherichia coli properties the pooR mutants and subcellular localization of protein Mol Genet 220366-372
Yates J R and Holmes D S 1987 Two families of repeatcXl DNA sequences Thiobacillus 1 Bacteriol 169 1861-1870
Yates J R R P and D S 1988 an insertion sequence from Thiobacillus errooxidans Proc Natl 857284-7287
161
Youvan D c Elder 1 T Sandlin D E Zsebo K Alder D P Panopoulos N 1 Marrs B L and Hearst 1 E 1982 R-prime site-directed transposon Tn7 mutagenesis of the photosynthetic apparatus in Rhodopseudomonas capsulata 1 Mol BioI 162 17-41
Zalkin H and Mei B 1990 Amino terminal deletions define a glutamide transfer domain in glutamine phosphoribosylpyrophosphate ami do transferase and other purF-type amidotransferases 1 Bacteriol 1723512-3514
Zalkin H and Weng M L 1987 Structural role for a conserved region III the CTP synthetase glutamide amide transfer domain 1 Bacteriol 169 (7)3023-3028
Zhang Y and Fillingame R H 1994 Essential aspartate in subunit c of FIF0 ATP synthase Effect of position 61 substitutions in helix-2 on function of Asp24 in helix-I 1 BioI Chern 2695473-5479