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Vol. 176, No. 21JOURNAL OF BACrERIOLOGY, Nov. 1994, p.
6518-65270021-9193/94/$04.00+0Copyright X) 1994, American Society
for Microbiology
Cloning, Nucleotide Sequence, and Expression of theBacillus
subtilis ion Gene
SABINE RIETHDORF,* UWE VOLKERt ULF GERTH, ANEIT WINKLER,4SUSANNE
ENGELMANN, AND MICHAEL HECKER
Institut fur Mikrobiologie und Molekularbiologie,
Emst-Moritz-Amdt-Universitat,17487 Greifswald, Germany
Received 6 June 1994/Accepted 22 August 1994
The kon gene of Escherichia coli encodes the ATP-dependent
serine protease La and belongs to the family ofc32-dependent heat
shock genes. In this paper, we report the cloning and
characterization of the Ion gene fromthe gram-positive bacterium
Bacillus subtilis. The nucleotide sequence of the Ion locus, which
is localizedupstream of the heDL4XCDBL operon, was determined. The
Ion gene codes for an 87-kDa protein consisting of774 amino acid
residues. A comparison of the deduced amino acid sequence with
previously described ion geneproducts from E. coli, Bacillus
brevis, and Myxococcus xanthus revealed strong homologies among all
knownbacterial Lon proteins. Like the E. coli ion gene, the B.
subtilis Ion gene is induced by heat shock. Furthermore,the amount
of Ion-specific mRNA is increased after salt, ethanol, and
oxidative stress as well as after treatmentwith puromycin. The
potential promoter region does not show similarities to promoters
recognized by o+32 ofE. coli but contains sequences which resemble
promoters recognized by the vegetative RNA polymerase EoA ofB.
subtilis. A second gene designated orJX is suggested to be
transcribed together with ion and encodes a proteinwith 195 amino
acid residues and a calculated molecular weight of 22,000.
ATP-dependent proteases are involved in the regulation ofthe
level of a number of proteins with short half-lives, such asSulA
and RcsA of Escherichia coli (25, 26). In addition tobiologically
active proteins, many damaged and abnormalproteins resulting from
misfolding, premature termination, ordenaturation are subjected to
ATP-dependent proteolytic deg-radation. During various stresses,
increasing amounts of mis-folded and damaged proteins may
accumulate. Hence, theATP-dependent proteases are also important
during stress. InE. coli, two forms of energy-dependent proteolytic
systems, theLon (La) (11, 14) and Clp (32, 36) proteases, have been
fairlywell characterized. Lon (12, 22, 46) and ClpP (37) belong
tothe family of heat shock proteins. The ATP-dependent
serineprotease La is encoded by the ion gene of E. coli. Mutations
inion result in a pleiotropic phenotype of E. coli cells, with
anincreased sensitivity to UV light, mucoidy, filamentousgrowth,
and defects in the lysogenicity of some bacterio-phages and in the
degradation of regulatory or abnormalproteins (26).Although
extracellular proteases have been a matter of
extensive investigation in various bacilli because of their
indus-trial application, the knowledge of the structure and
functionof ATP-dependent proteases in Bacillus species is still
verylimited. Protein extracts of Bacillus subtilis cross-reacted
withantibodies raised against the protease La of E. coli (4).
Thegene coding for the Lon protease of Bacillus brevis was
clonedand sequenced. However, in contrast to E. coli, the gene is
notinduced by heat, and the insertion mutation does not cause
anyobvious phenotype (34).We are interested in the response of B.
subtilis to various
* Corresponding author. Phone: 03834/77271, ext. 210. Fax:
03834/883353.
t Present address: Department of Microbiology, The University
ofTexas Health Science Center at San Antonio, San Antonio,
TX78284-7758.
i Present address: Forschungsinstitut fuir Molekulare
Pharmakolo-gie, 10315 Berlin, Germany.
stresses. According to their regulation, the heat shock
proteinsof B. subtifis can be arranged into at least two groups
(29).Gene products of the groESL and the dnaK operons
representmembers of the first group. The heat induction of
theseproteins requires the vegetative sigma factor orA of B.
subtifis(9) and a conserved palindromic structure (CIRCE)
justdownstream of the start point of transcription (50, 67, 70).
Thealternative sigma factor orB of B. subtilis controls the heat
shockinduction of the second group (6, 7, 8, 62). Preliminary
dataindicate the existence of a third mode of induction of
heatshock genes in B. subtilis. We suggest that neither aB nor
thepalindromic structure is involved in the heat shock induction
ofthese genes (39, 62).
In this report, the cloning and characterization of the
heat-inducible ion gene of B. subtilis are described. The
identificationand cloning of the B. subtilis ion gene are
independentlyreported by Schmidt et al. in an accompanying paper
(51). Thedata indicate that the Ion gene product, along with ClpP
(62),and ClpC (39), belongs to the oB-independent heat
shockproteins of B. subtilis, which are induced by heat and
otherstress conditions. The ion operon contains a second
openreading frame, orfX, which codes for a 22-kDa protein.
MATERIALS AND METHODS
Bacterial strains, plasmids, and growth conditions. Thebacterial
strains and plasmids used in this study are describedin Table 1. E.
coli DH5aL and JM110 were routinely grown in acomplex medium and
used as hosts for DNA manipulation. B.subtilis strains were
cultivated under vigorous agitation at 37'Cin LB or in a synthetic
medium described earlier (55). Cells ofB. subtilis were exposed to
different stress conditions asdescribed by Volker et al. (62). Heat
shock was achieved bytransferring exponentially growing cells from
37 to 48, 50, or52'C. The other stress conditions were provoked by
exposingexponentially growing cells to either 4% (wt/vol) NaCl,
5%(vol/vol) ethanol, 0.02% (wt/wt) H202, or 20 Rg of puromycinper
ml. The samples were taken during exponential growth
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CHARACTERIZATION OF THE B. SUBTILIS LON GENE 6519
TABLE 1. Bacterial strains and plasmids
Strain or plasmid Genotype or descriptiona Reference(s) or
source
E. coliDH5ot F' 480dlacZAM15 A(lacZYA-argF)U169 deoR recAl end4l
hsdR17 (rK- mK+) supE44 27
thi-1 gyrA96JM110 leu F' (traD36 proAB+ lacIq lacZAM15)
68BL21(DE3) hsdSgal(XcIts857 indl Sam7 ninS lacUV5-T7 genel) 54
B. subtilisIS58 trpC2 lys-3 5ML6 trpC2 sigB::AHindIII-EcoRV::cat
33BGWLON1 trpC2 lys-3 lon::pJH101 This studyBD224 trpC2 recE4
17
PlasmidspIB130 Cloning vector, Apr International Biotech-
nology Inc.pBluescriptIIKS/' Cloning vector, Apr
StratagenepBluescriptIISK+I' Cloning vector, Apr StratagenepSPT19
Cloning vector, Apr Boehringer MannheimpJH101 Insertion vector,
Apr, Tcr, Cmr 19pBL2637 pBL2 containing a 280-bp Sau3A fragment of
chromosomal DNA from B. subtilis 64pIBI2637 pIBI30 containing the
280-bp Sau3A fragment of pBL2637 This studypBSLON pJH101 digested
with EcoRV-AVal and ligated with the 290-bp XbaI-AVaI fragment This
study
of pIBI2637pBSLON1 Obtained after plasmid rescue from
EcoRI-digested chromosomal DNA of B. subtilis This study
BGWLON1pBSLON8 Obtained after plasmid rescue from NcoI-digested
chromosomal DNA of This study
B. subtilis BGWLON1pBSLON9 pBluescriptIISK+ containing the
4.038-kb EcoRV-BglII fragment of pBSLON1 This studypSPTLON pSPT19
containing the 300-bp EcoRI-XbaI fragment of pIBI2637 This
studypBSLON10 pBluescriptlIKS- containing the 770-bp PvuI-BamHI
fragment of pBSLON1 This studypBSLON11 pBluescriptIlKS- containing
the 467-bp NdeI fragment of pBSLON8 This studypWH703 Kmr,
promoterless xylE and cat-86 18, 64pWHLON1 pWH703 containing the
800-bp EcoRI-HincII fragment of pBSLON10 This studypWHLON2 pWH703
containing the 497-bp EcoRI-HincII fragment of pBSLON11 This studya
Abbreviations: Apr, Cmr, Kmr, and Tcr, resistance to ampicillin,
chloramphenicol, kanamycin, and tetracylin, respectively.
prior to and after the shift at the times indicated in Fig. 4
and5. The time of the shift was set at zero.
Construction of an insertion mutation in the ion gene of
B.subtilis. The 280-bp Sau3A fragment of pBL2637 (64) contain-ing
an internal fragment located near the N-terminus-encodingpart of
the ion gene of B. subtilis was ligated with theBamHI-digested
vector pIBI30, resulting in plasmid pIBI2637.This plasmid was
linearized with XbaI, the cohesive ends werefilled in with the
Klenow fragment of DNA polymerase I, andthe plasmid was cut with
AvaI. The 290-bp fragment wasinserted into the EcoRV-AvaI-digested
integration vectorpJH101. The resulting plasmid, pBSLON, was used
to trans-form competent cells of B. subtilis IS58.
Chloramphenicol-resistant colonies were selected on agar plates
containing 5 jugof chloramphenicol per ml. The integration of the
plasmid wasverified by Southern blot analysis using a
nonradioactive DNAlabeling and detection kit (Boehringer
Mannheim).
Cloning of the ion gene and DNA sequence
determination.Chromosomal DNA from B. subtilis BGWLON1, in which
theIon gene is disrupted by the integration of plasmid pBSLON,was
digested with EcoRI and NcoI, religated, and introducedinto E. coli
DH5ot, generating plasmids pBSLON1 andpBSLON8 (Fig. 1). For
sequencing, a 4.038-kb EcoRV-BglIIfragment from pBSLON1 was
subcloned into the vector pBlue-scriptIISK+ digested with EcoRV and
BamHI, yielding plas-mid pBSLON9. The sequence was determined by
primerwalking using plasmids pBSLON1 and pBSLON8 or from a setof
deletional plasmids of pBSLON9 generated by exonuclease
III/nuclease S1 digestion. Both strands were sequenced by
thedideoxy-chain termination method of Sanger et al. (49),
withplasmid DNA as the template.
Expression of orJX in E. coli. To express the orfX gene,plasmid
pBSLON10 was transformed into E. coli BL21(DE3)(54), which carries
the gene for the T7 RNA polymerase underthe control of an IPTG
(isopropyl-,3-D-thiogalactopyranoside)-inducible lacUV5 promoter.
For the construction of pBSLON10,the vector pBluescriptlIKS- was
digested with EcoRV andligated with the 770-bp PvuI-BamHI fragment
from pBSLON1previously treated with the Klenow fragment of DNA
poly-merase I. The orientation of the cloned fragment was tested
byEcoRI-SphI digestion. In pBSLON10, the DNA strand codingfor orJX
can be transcribed by the T7 RNA polymerase.
Plasmid-harboring cells were cultivated in a synthetic me-dium
(43) containing 100 jLg of ampicillin per ml. At an opticaldensity
of 0.8 at 500 nm, IPTG was added to a final concen-tration of 1 mM,
and 15 min later the culture was treated with200 jig of rifampin
per ml. The bacteria were pulse-labeled for3 min with 10 tICi of
L-[35S]methionine per ml both before and15 min after the addition
of rifampin and lysed by boiling for3 min. Proteins of the cell
extracts were fractionated on asodium dodecyl sulfate (SDS)-10 to
20% polyacrylamide gra-dient gel. Radioactively labeled proteins
were detected byautoradiography (Fig. 3). To analyze the N-terminal
proteinsequence, the bacteria were collected 2 h after the addition
ofrifampin and lysed by boiling, and the proteins were trans-ferred
from the gel onto a polyvinylidene difluoride membrane
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6520 RIETHDORF ET AL.
or
catIifPBSLON
amp
lon2637EcoR I
Ava ClapXbaI
xiion EcoRl
CaNRI NcoI
PBSLON8 L
chromosomol DNA
ton
integration of pBSLON
Bgll EcoRI
orfX orfY hemAXCDBLton
pBSLON 1
0 500 1000 bpsFIG. 1. Schematic representation of the
chromosomal rearrangement after integration of plasmid pBSLON into
the chromosomal Ion gene of
B. subtilis. Plasmids pBSLON1 and pBSLON8 were obtained after
plasmid rescue using the restriction sites EcoRI and NcoI as
described inMaterials and Methods.
by electroblotting. The protein of interest was sequenced on
anApplied Biosystems A473a protein sequencer.
Promoter cloning and measurement of catechol 2,3-oxygen-ase
activity. The promoter probe vector pWH703 (64) is aderivative of
plasmid pPL703 (18) and contains the promoter-less catechol
2,3-oxygenase gene (xylE) from Pseudomonasputida and the
promoterless cat-86 gene. For the constructionof pBSLON11, the
vector pBluescriptlIKS- was digested withEcoRV and ligated with the
467-bp NdeI fragment frompBSLON8 which was previously treated with
the Klenow frag-ment of DNA polymerase I. The direction of the
cloned frag-ment in pBSLON11 was determined by sequencing.
TheEcoRI-HincII fragments from pBSLON10 (800 bp) andpBSLON11 (497
bp) were inserted into the EcoRI-SmaI-digested vector pWH703,
resulting in plasmids pWHLON1and pWHLON2. Transformation of
protoplasts of B. subtilisBD224 was carried out as described by
Chang and Cohen (10),and transformants were selected after
regeneration of theprotoplasts on complex agar medium supplemented
with 150jug of kanamycin per ml. Transformants containing a
promoterupstream of the xylE gene developed a yellow color
aftercolonies were sprayed with catechol. These cells were able
togrow on plates with 10 jug of chloramphenicol per ml.
Catechol2,3-oxygenase activity was measured as described by Volker
etal. (64).
Analysis of transcription. Total RNA of the B. subtilisstrains
was isolated from exponentially growing or stressedcells by the
acid phenol method of Majumdar et al. (41), withsome modifications
(62). Serial dilutions of total RNA weretransferred onto a
positively charged nylon membrane by slotblotting and hybridized
with digoxigenin-labeled RNA probesas instructed by the
manufacturer (Boehringer Mannheim).The chemiluminograms were
quantified with a personal den-sitometer from Molecular Dynamics.
Induction ratios werecalculated by setting the value of the control
to 1. Hybridiza-tion specific for Ion mRNA was conducted with a
digoxigenin-
labeled RNA probe synthesized in vitro with T7 RNA poly-merase
(noncoding strand) from linearized plasmid pSPTLON. This plasmid is
derived from pSPT19 (BoehringerMannheim) and contains the 300-bp
EcoRI-XbaI fragment ofpIBI2637. Hybridization specific for orX mRNA
was carriedout by digoxigenin-labeling of the SphlI-linearized
plasmid pBSLON10 by T3 polymerase. The RNAs synthesized in vitro
fromthe coding strand were used as negative controls for
thehybridization and did not yield any specific hybridization
signal(data not shown).
Synthetic oligonucleotides complementary to the
N-termi-nus-encoding region of the lon gene (PLON,
5'-GAAATATCCTGCTGAGTGGC-3') and orX (PORF, 5'-CCCGGCACATCCACA-3')
were labeled with [y-32P]ATP and used asprimers for the primer
extension analysis as described byWetzstein et al. (67).
General methods. Plasmid isolation, restriction enzymeanalysis,
transformation of E. coli, exonuclease III digestionand filling in
of the recessed 3' termini, and 5'-3' exonucleasedigestion by the
Klenow fragment of DNA polymerase I wereperformed as described by
Sambrook et al. (48). ChromosomalDNA from B. subtilis was isolated
as described by Meade et al.(42). Transformation of natural
competent B. subtilis cells wascarried out by the two-step protocol
of Hoch (30).Computer analysis of sequence data. The sequence
data
manipulations were performed with the Genetics ComputerGroup
sequence analysis software package.
Nucleotide sequence accession number. The nucleotide se-quence
data reported in this paper appear in the EMBL andGenBank
nucleotide sequence databases under accessionnumber X76424.
RESULTS
Isolation of a strain with an insertion mutation in
Ion.Experiments designed to clone promoter-containing DNA
NcoI
Le~
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CHARACTERIZATION OF THE B. SUBTILIS LON GENE 6521
fragments of B. subtilis yielded a set of 15 fragments
whichcaused heat induction of the reporter gene bglM, coding for
the0-1,3-1,4-endoglucanase of Bacillus macerans (64). The
nucle-otide sequence of the promoter-containing fragment of
plas-mid pBL2637 exhibited 66.5% identity in a 282-bp overlap
withthe N-terminus-encoding region of the B. brevis Ion gene
(34).The 280-bp Sau3A fragment of pBL2637 which is internal tothe
Ion-like coding sequence was used to construct the inte-grational
vector pBSLON as described in Materials and Meth-ods. B. subtilis
IS58 was transformed with pBSLON andselected for chloramphenicol
resistance. Since pBSLON can-not replicate in B. subtilis, the
chloramphenicol-resistant trans-formants should carry the plasmid
integrated into the chromo-somal Ion gene by a Campbell-like
recombination, creating adisruption of the ion gene. Neither
mucoidy nor filamentationduring growth could be detected in the
mutant strains. Fur-thermore, no significant differences in the
growth parametersof the mutant strain B. subtilis BGWLON1 and the
wild-typestrain B. subtilisIS58 were observed at a temperature of
37, 48,50, or 520C.
Nucleotide sequence of the B. subtilis Ion gene. The ion genewas
cloned as described in Materials and Methods, the nucle-otide
sequence was determined from plasmids pBSLON1,pBSLON8, and pBSLON9
in both orientations (Fig. 1). Figure2 displays the sequence of a
3,853-bp NdeI-HindIII fragment.The sequence downstream of the NsiI
site (Fig. 2) is identicalto the sequence reported for the
hemAXCDBL operon byHansson et al. (28) (Fig. 1). This finding is
consistent with theresults of Schmidt et al. (51), who mapped the
Ion gene of B.subtilis at position 245° of the genetic map (3).
Computeranalysis of the sequence revealed the presence of two
openreading frames (ORFs), which seemed to be transcribed in
thesame direction as the hem operon. The first ORF starts with
anATG codon at position 333 and might encode a proteinconsisting of
774 amino acids with a predicted molecular massof 86,600 Da and an
isoelectric point of 6.13. The ATG codonat position 333 is preceded
by a potential ribosome binding sitecomplementary to the 3' end of
the 16S rRNA extending fromnucleotides 318 to 324 (Fig. 2). A
comparison of orfl withdatabase entries established a high degree
of identity withpreviously published sequences of bacterial Ion
genes (seebelow). Therefore, orfl was designated Ion.The
C-terminus-encoding part of an unknown ORF was
identified upstream of the ion gene. This ORF is followed by
apotential factor-independent terminator structure (Go = -9.9kcal
(1 kcal = 4.184 kJ)/mol) extending from nucleotides 151to
175.Another ORF with a length of 585 bp is located immediately
downstream of Ion (Fig. 2). This ORF was designated orfX.The
translation initiation codon AUG of the oijX gene ispreceded by a
Shine-Dalgarno sequence and overlaps the stopcodon of the ion gene
by one nucleotide (Fig. 2). A potentialterminator (Go = -16.6
kcal/mol) was identified downstreamof orfX from bases 3232 to 3259
(Fig. 2). These data suggestthat ion and orX might comprise an
operon.
Analysis of the sequence revealed the presence of a thirdORF
(designated orfY) between orJX and hemA but oriented inthe opposite
direction, with the translation initiation codon atposition 3760
(Fig. 1 and 2). It has the capacity to code for aprotein with 164
amino acid residues. No homologies to otherknown genes could be
found. The significance of this ORFremains to be elucidated.Amino
acid sequences of the B. subtilis Ion and orJX gene
products. The alignment of the deduced amino acid sequencewith
known bacterial Lon protein sequences revealed overallidentities
with B. brevis Lon (34) of 69%, E. coli La (2, 20, 56)
of 55%, Myxococcus xanthus LonV (57) of 55%, and M. xanthusLonD
(21, 58) of 49%. This high degree of identity encom-passes almost
the full length of the four sequences except theN-terminal region.
The potential ATP-binding sites derivedfrom the proposed consensus
sequences (GPPGVKT andQ-MKKAG--NPVFLL [12]), and the regions around
thesesequences are strongly conserved and were found at the
samepositions within the four proteins (12, 21, 34, 57, 58).
Theserine residue at the position 677 (Fig. 2) corresponds to
thepreviously described active serine site at position 679 of E.
coliprotease La and is also conserved in the five sequences (1,
20,69).The nucleotide sequence of B. subtifis orX exhibits 62%
identity in a 488-bp overlap with a partially sequenced
putativeofX gene in B. brevis (34) and significant identity with an
ORFdownstream of the poLA gene of E. coli encoding the proteinYihA,
the function of which is unknown (35, 47). Comparisonof the derived
amino acid sequences by multiple sequencealignment established 59%
identity in a 119-amino-acid over-lap to B. brevis OrfX and 37.5%
identity in a 184-amino-acidoverlap to E. coli YihA. Schmidt et al.
(51) indicate for B.brevis an error at position 2744 in the
sequence published byIto et al. (34). The sequence has not been
redetermined, butthe insertion of one base pair at this position
would generatean ORF with 64t% identity in a 162-amino-acid overlap
withthe corresponding OrfX of B. subtilis. Furthermore, the
cor-rected version of OrfX of B. brevis displays a strong
homologywith the actual N terminus of the B. subtilis OrfX (see
below).
Expression of the orJX gene in E. coli. orX might encode
aprotein with a calculated molecular weight of 22,000. To testthis
assumption, we expressed the orX gene of B. subtilis in E.coli. For
this purpose, plasmid pBSLON10, containing the T7promoter in front
of the orfX gene, was transformed into E. coliBL21(DE3). Expression
of the T7 RNA polymerase wasinduced by the addition of IPTG in the
presence of rifampin,which prevented the initiation of
transcription by the RNApolymerase of E. coii. Figure 3 displays an
autoradiogram of aseparation of protein extracts on an
SDS-polyacrylamide gelafter labeling of the proteins with
L-[35S]methionine as de-scribed in Materials and Methods. A major
band with anestimated molecular weight of 22,000 to 23,000 was
detected inextracts from a culture of BL21(DE3) containing
plasmidpBSLON10 when the cells were treated with rifampin after
theaddition of IPTG (Fig. 3, lane 4). The same protein wasproduced
to a lesser extent when these cells were treated withIPTG only
(Fig. 3, lane 3). No protein band was found inextracts of BL21(DE3)
containing plasmid pBluescriptIIKS-either after the addition of
IPTG alone (Fig. 3, lane 1) or afterthe addition of IPTG and
rifampin together (Fig. 3, lane 2).The N-terminal sequence of the
major protein -MKVTK-
SEIVIS- confirmed the expected translational start point
de-duced from the nucleotide sequence (Fig. 2).
Regulation of transcription of Ion and oryX. The Ion gene ofE.
coli is induced by heat shock and other stresses. Therefore,we
analyzed the influence of heat stress on the transcription ofion in
B. subtiiis. Slot blot filters with total RNA of B. subtilisIS58
isolated before and after the stress were hybridized
withdigoxigenin-labeled RNA probes obtained by in vitro
tran-scription of the linearized plasmid pSPTLON with T7
RNApolymerase. A four- to sixfold increase in the amount
ofIon-specific mRNA could be measured within 3 to 6 min aftera
shift from 37 to either 48,50, or 520C (Fig. 4 and
unpublishedresults). A similar induction was found after salt
stress in acomplex medium (Fig. 4) but surprisingly not in a
syntheticmedium (data not shown). The ion mRNA level was
alsoenhanced after ethanol or oxidative stress (Fig. 4) and
after
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Nd-I5&AVATCAGCAGGCCATCTTA~C^ATAGCAGTGCGCCAGATTATTGCAGTGAaGACGTTCCAAGAAGTGCTTGATGAAAmTTTGTAATCCGCCAACG
GAAaCAAAAaCC
Y B N Q Q A I L K Q I D G I S I I A V K T F Q B V L D 8 I L V N
P P T B Q K P P
TTCATATCGAMaTCAaTAAAGAATCTCCTTAAAC
ACT~~TaAGlmTaGAACTTCmTTAAAAaCaG~CH I B I N K B 8 V -35 -10 81
mU
AGC~~~tGA~~pCIS~~aTCTAAAGTCACAGT~~~CACATGGCAGAAGAATTAAAACGCAGCATCC
-35 -10 82 SD M A S S L K R S I P1 _>
Su3A.NdoICGCTCIAmTATT CGGTTCAGGCTCTTGAACAGGCAATGATGC C=AtTATTTTL
L P L R C L L V Y P T M V L H L D V G R D R S V QA L B Q AL M H D H
M I F L
TAGCCACTCCAGCAGAATCGATAAmCAAATTATT -lTA TT GA
CCACGCACGTCTGTGTGTGA T Q Q D 8 I D 8 P G 8 D B I P T V G T Y T K I
K Q M L K L P N G T I R V L V
TGGAGGOCGCGTAATACTT_=AAGC^GATAGC8 G L K R A H I V K Y N 8 H E D
Y T 8 V D I Q L I H B D D 8 K D T 8 D E A L M R
.Sau3A *coRVGGACTTTGCTAGACCACTT5 %ACAT^AAAAAmrCTAAAAAAATCTWWC1
OTGCACClCT L L D H F D Q Y I K I 8 K K I S A 8 T Y A A V T D I B B
P G R M A D I V A S H
ATCTG=CCC_______ATAATL P L K L K D K Q D I L B T A D V K D R L N
K V I D F I N N B K 8 V L S I B K K
I G Q R V K R S8 8 R T Q K 8 Y Y L R B Q M K A I QK L G D K G K
T G B VQ
AGACGCTGAOAAAATCGMA^AG C _C G G lCICCGTCAAGTTCTGC
GGAAAGCTCGS.TCCT L T D K I B B A G M P D H V K B T A L K B L N R Y
E K I P S O S a SS S V I R
GCAACTATATCGCTGCTlrCTCTTCC w 1 AAG~AGAA
CTCTTGGACGAAGAGCACCACGGGCTTGAAAAAGTAAAAGN Y I DS L V a L PI T D E T
D D K L D L KL A G R L L D E AH H G L B K V K B
AA
GaT~Ga~CZCCCAGAAGCT.ACaAAAATCCCTGaAAGGCCCGATTCTCISTGTTAGGACCTCCAGGTG;TCGGAAAAACGTCTTTAGCCAAATCAATTGCAAAAAR
I LS Y L A V Q 1 L T K S L K G P I L C L A G P P G V G K TS L A K 8
I A K 8
GCTTGGGACGCAATCGTCAGTCTCWACGAGGAGTTCWGOGTGAATCAGAGATACGCGCACCGACCTATGTCGGAGCAATGCCTGGACGTATTATTCAAGGGATGAAAAL
G R K F V R I L G G V R D B 8 B I R G H R R T Y V G A L P G R I I Q
G M K K
AAGCGGGCAAGCTG AATCCGGTCTT
TCATTGATTTAGGGAGCCCATCATCCGCTATGCTTGAAGTGCTTGATCCAGAGCAAAACAGCAGCTa
C K L N P V F L L D AI D K M S LD F R G D P 8 8 a M L TV L D P E Q
N S 8 F
TCAGTGATIAALT G LLLGAGAAACCTTTGFSYVRSAT BSLT D H Y I 8 8 T F D L
S K V L F I a T A N N L A T I P G P L R D R M E I I N I A
CAGGCTACACAGAAATAGAAAAACTTGAAATTGTAAAGGATCACTTGCTTCCAGCAAATCAAAGAACACGGGCTGAAGAAAAaGCAATC-TTCAGCTGCGTGATCAGGCGATTCTTGATAG
Y T 8 IA I L E I V K D H L L P K Q I LE H G L K K SN L Q L R D Q A
I L D I
TTATTTTT C C G A T TG a C ~ S l G A TI R Y Y VR SA G V R L SR Q
L A A I C R K A A K A I V A L8 R K R I T V 0 B
AGAAGAACCTTCAAGRTTrTSATCOGAAAaCGCATTTTCAGATATGGACAAGCTGAAACAGAGGATCAAGrrGGTCSACTGACAGGGCTTGCG;TATACAACCGTTGGCGAGATACGCTTTK
N L D F I GP R I F R Y G Q A BF I DS V G V V S G L A Y A T V G G D
S L B
CGATTGAAGTATCGCTTTCACCGGGAAAAGGGAAATTAATCCTGCAGAAACTCGGGATGTTATGAGAGAGTCTGCTCAGGCTGCArrCAGCTATGTGCGATCCAAAAaCAGAAGAACI
8 V SL 8 P G K G K L I L T G K L L D V 0R 18 A Q A A F S Y V R S K
T P B L
TTGGCATTGAACCTGACTTTCATGAGAAGTTATTCTATACATASTTACCAGGTG
MCCCAAAAGATGCTCCCTCAGCCGG;TATTACGATGACGCGTGTTCTG=TG I 8 P D F H B K
Y D I H I H V P G A VP P A G I TL A T A L V P A L
.PvuITAACOGGACGGG
BlTrCGCGTGAAGTCGGCATGACTGGGAATAGcrSCGCGCCGCTTWOCTSiTq~=TAAAGGAAAAAGCGCTTGGCGCACATAGAGCGOGATTAAS
G R A V 8 R E V G M T GA I T L R G R V L P I G G L KP K A L G A H R
A G L T
CGACCASSATTCCGCCTAAAGATATAAAAGA _TAAGATATCCGGAAAGCGTCAGGGAGGGG
CTGACATT ATATTGCTCCCCTTAG CACG 7TGGAlCATGCCTTAGT I I A I P S V RD G
L T F I L A -H L D E V L B H A L V
TAiGVAAAATGAAAITCACAAAGTCAGAAATCGTGATCAGTGCAGTAAAACCGAACAGTACCCTGAAG'CC
TCCATGCCGGAAGATCGAACCTAGAAAG E K K I
SD M K V T K S8E I V I 8 A V K P B Q Y P B G G L P e I A L A G R
8 N V G Korffl --->
ATCGTCTTTTATCAATCTTAATCAATQCGCAAAAATCTTGCGAGAACGTCATCA
AAGCCGGAACCAAACGCTTAATTCTACATTAT G8 8 F I N 8 L I N R K N L A R T S
8 K P G K T Q S L N F Y I I N D B L H F V D V
GCCGGGCTACGGTTGCAAAGTGTCAAAG;T 1C CTA
TATCACGACACGCGAGGAATTAAAAGCTGTGIGCAGATCGTTGATTTGCGP G Y G F A K V 8
K 8 8 R B A It G R M I B T Y I T T R B B L K A V V Q I V D L R
8phI
H A P 8 N D D V Q M Y B F L K Y Y G I P V I V I A T K A D R I P
K G K V D K H A
C r Wl M C
CCWAAGACG~aCTGATCT~rTTTCTTCC~rUCCINGCCGCACAAAXAATGATAAACCOCTAK V V
R Q S L N I D P B D B L I L F 8 8 B T K K G K D B A N G A I K K M I
N R
BjHIGAgaCTACC 1
CGTTTTATCAATAATAACAATAAACTGATTCCQATAAAGACAAACGGCCAATACCTCCTTCAGAACCTTOA~ATT
>>>>
-
CHARACTERIZATION OF THE B. SUBTILIS LON GENE 6523
FIG. 2. Nucleotide sequence of the B. subtilis Ion locus and
deduced amino acid sequences of its gene products. Putative
Shine-Dalgarnosequences, the potential -35 and -10 regions of the
ion promoters, and restriction enzyme sites used in this study are
underlined. The two possiblestart points of transcription, S1 and
S2, of the Ion operon are indicated. Factor-independent terminators
are labeled by arrows under thenucleotides. Termination codons are
indicated by asterisks. The underlined potential promoter region
and the Shine-Dalgarno sequence of orfYare derived from the
nucleotide sequence. The putative active serine site is printed
boldface and underlined.
treatment with puromycin, leading to the production of abnor-mal
proteins in the cells (data not shown). This induction ofthe ion
gene was significant, because the level of xynA mRNAdid not change
in response to any of the conditions analyzed inthis study (data
not shown). xynA codes for the enzymexylanase of B. subtilis (40).
In a sigB mutant of B. subtilis, theion gene was induced at the
same rate, indicating that theinduction of the B. subtilis ion gene
may be independent of aB.The presence of a putative
factor-independent terminator
upstream of ion indicates that the potential promoter shouldbe
located between this terminator and the translation initia-tion
codon for Ion at position 333 (Fig. 2). Furthermore,catechol
2,3-oxygenase activity could be measured (about 4 X102 iiU/108
cells) in the chloramphenicol-resistant cells of B.subtilis BD224
containing plasmid pWHLON2, suggesting thatthe essential promoter
elements of the ion gene are locatedbetween the two NdeI sites in
front of the Ion gene (Fig. 2). Incontrast, no activity of this
enzyme (
-
6524 RIETHDORF ET AL.
heat salt ethanol H202
Ion atL ||| 62O
time (min) time (min) Urns (mn) time (min)
time (min) time (min) time (min) time (min)FIG. 4. Schematic
representation of the increase of the Ion and ofX mRNA levels
caused by different stresses. Bacteria were exposed to
different stresses as described in Materials and Methods. Serial
dilutions of total RNA prepared from B. subtilis IS58 before (0
min) and differenttimes (3, 6, 9, 12, 15, 20, and 30 min) after the
exposure to stress were bound to a positively charged nylon
membrane and hybridized with thedigoxigenin-labeled antisense RNA
probes specific for the corresponding genes. The hybridization
signals were quantified with a personaldensitometer as described in
Materials and Methods. The induction ratios of the mRNAs are
shown.
subtilis is induced by heat shock. The intensity of the
hybrid-ization signals after heat shock and the rate of heat
inductionwere stronger at the putative transcriptional start point
S2 thanat S1, leading to the suggestion of a preferable usage of
S2under conditions of heat stress.The first hints for the existence
of a B. subtilis ion gene were
obtained during the cloning of heat-sensitive promoters (64).The
fragment which triggered the heat induction of a fusionpromoter was
internal to ion and did not contain the promotersidentified in this
study. Whether sequences in the structuralgene participate in the
heat shock regulation of the B. subtilision gene should be
investigated.The induction by heat and ethanol stress of a B.
subtilis
protein cross-reactive to antibodies raised against La of E.
coliwas previously observed by Arnosti et al. (4). The
transcrip-tional analysis revealed that B. subtilis Ion is induced
not onlyby heat shock but also by ethanol, salt, or oxidative
stress (Fig.4) as well as after exposure of B. subtilis cells to
puromycin
A
(data not shown). However, the increase of lon-specific
mRNAafter the imposition of stress is lower than that observed
formost other general stress genes (39, 62). Such a rather
lowinduction ratio is in agreement with results obtained for E.
coli.Goff et al. (22) measured a two- to threefold-higher level
oftranscription of the ion gene after heat shock. Chuang
andcoworkers (13) also found only a two- to threefold increase
ofIon-specific mRNA after heat shock. Similar induction ratioswere
found for E. coli Ion during ethanol stress or afterpuromycin
treatment (23). But even a two- to threefoldincrease in ion
transcription will considerably enhance the rateof degradation of
abnormal proteins (23), whereas a stillhigher intracellular level
of the protease La can be stronglydeleterious (24). Because of the
toxic effects of the overpro-duced La protease in E. coli, the
artificial production of largeamounts of this protein requires an
inducible expressionsystem (56).
Since the potential promoters upstream of Ion display a high
BA C G T co Co 3 6 9
AcAGAFA
DNA~~~~~~~~~~~~~~~~~~sequencing
~~~~~~~~~~~~~~~~~~~A¢W§:V;7&___ g I ~~~~~~~~~~~~~~AS
7 t9, S _ , .~~~~~~~~~~~~~~~~~a, X,, r it Ad5! I 11:_T
FIG. 5. Mapping the 5' end of the Ion mRNA by primer extension
analysis before (37°C) and after (50'C) heat shock. RNA was
isolated fromB. subtilis IS58 before (co) and at different times
(3, 6, and 9 min) after heat shock. Equal amounts of total RNA (10
,ug) were used for the primerextension analysis. The potential
start points of transcription, S1 (A) and S2 (B), are marked with
asterisks. Lanes A, C, G. and T show the dideoxysequencing ladder
obtained with the same primer as used for primer extension. The
sequence displayed is complementary to that determined byDNA
sequencing.
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CHARACTERIZATION OF THE B. SUBTILIS LON GENE 6525
degree of similarity to promoters of housekeeping genes,
thequestion arose as to which mechanism controls the
stressinduction of the lon gene. According to our current
knowledge,the stress genes of B. subtilis belong to at least two
differentregulons (62). The well-known chaperones encoded by
thegroESL and dnaK operons of B. subtilis (50, 67) belong to
thefirst group. The vegetative form of RNA polymerase Eu43seems to
be involved in the induction of both operons by heatshock (50, 67).
In E. coli, on the other hand, the chaperonegenes are recognized by
Eu32 (45). Chaperone genes of mainlygram-positive bacteria possess
a 9-bp inverted repeat immedi-ately upstream of the coding region
which is required for anormal heat shock response by the dnaK and
groESL operons(70). Such an inverted repeat is not present in the
regionimmediately upstream of Ion in B. subtilis. Hence,
anothermechanism must be responsible for the stress induction
oflon.Recently, the alternative sigma factor cuB has been shown to
bea heat shock protein (6) which is responsible for the inductionof
several stress proteins in B. subtilis (6, 7, 62). The ion genedoes
not require this alternative sigma factor for its heat shockor
stress induction, since the level of ion mRNA and theinduction
ratio do not differ between a mutant with a deletionin sigB and the
wild-type strain.
Apparently the ion gene might be a member of yet anotherclass of
stress genes of B. subtilis. Similar to ion, clpP and clpCare
induced by various stresses independent of the alternativesigma
factor aB (39, 62). No CIRCE (70) was found in theregulatory region
of the clpC operon (38). Unfortunately, thesequence of the
regulatory region of clpP has not beenelucidated yet. Therefore,
the presence of the CIRCE cannotbe excluded. However, clpP and clpC
exhibit an inductionpattern distinctively different from that of
groESL or dnaK ofB. subtilis (39, 62, 63).An insertional mutation
in the ion gene of B. subtilis did not
lead to any obvious phenotypical changes. The mutation didnot
impair growth at 48 or 500C in comparison with the wildtype (data
not shown). Similar to B. brevis (34), features whichare
characteristic of E. coli Ion mutants such as mucoidy,filamentous
growth, and increased sensitivity to UV light (25,26) have not been
observed in the B. subtilis ion mutant. In E.coli, these effects
are due to a diminished rate of the degrada-tion of regulatory
proteins such as SulA (31, 44, 52) or RscA(53, 59). The same
pleiotropic phenotypic properties werefound in Salmonella
typhimurium lon mutants (16). M. xanthuscarries two lon-related
genes, lonV (57) and lonD or bsg4 (21,58). Whereas lonV is
essential for vegetative growth (57), noeffects of a lonD mutation
on vegetative growth could beobserved. However, lonD mutants can
neither aggregate norform spores (58). Schmidt et al. (51) provide
evidence for aninfluence of a mutation in the B. subtilis ion gene
on theaccumulation and activity of the alternative sigma factor
'G,which is produced predominantly in the forespore. In
anaccompanying report (51), it is proposed that the Lon
proteaseprevents inappropriate synthesis of aG and hence the
tran-scription of or -dependent genes.
Sequencing revealed the presence of an additional openreading
frame, orX, immediately downstream of the lon gene.Our results
suggest that this gene is transcribed together withlon. An inverted
repeat downstream of orJX could act as afactor-independent
transcriptional terminator of the putativeoperon. Slot blot
hybridizations revealed a similar increase oflon-and orJX-specific
mRNAs under various stress conditions.Furthermore, we could not
find an internal promoter upstreamof orX either by primer extension
or RNase protection assayor by subcloning a putative promoter
region into a promoterprobe vector. The ofX gene codes for a
protein of 195 amino
acids with a calculated molecular weight of 22,000. A productof
about 22 kDa was produced in E. coli by the expression oforX, and
the translation initiation codon postulated from thesequence data
was confirmed by N-terminal sequencing. Inter-estingly, OrfX of B.
subtilis, the homologous putative proteinfrom B. brevis, and the E.
coli YihA protein contain threeconserved G regions of a class of
GTP-binding proteins (51).
Initial experiments to analyze the role of B. subtilis OrfX
didnot yield any clear evidence for a function of this protein in
theregulation of the putative Ion operon itself (unpublished
data).
ACKNOWLEDGMENTS
We are very grateful to R. Losick and R. Schmidt for critical
readingof the manuscript and helpful discussions. We thank R.
Schmidt and K.Altendorf for N-terminal sequencing of the OrfX
protein, R. Losickfor providing the strain ML6, and R. Gloger, V.
Grapentin, and A.Harang for technical assistance.
This work was supported by grants from the Deutsche
Forschungs-gemeinschaft and the Fonds der Chemischen Industrie to
M.H.
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