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University of Groningen
Nonribosomal peptide synthesis in Bacillus subtilisDuitman,
Erwin Hans
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Nonribosomal peptide synthesis in Bacillus subtilis. s.n.
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Transformation tools
45
Chapter three
Molecular tools to facilitate transformation ofBacillus
subtilis
Abstract
Here we describe two methods that facilitate transformation of
Bacillus subtilis. Thefirst method strongly increases competence
development and hence transformationefficiencies of B. subtilis
ATCC6633, B. subtilis ATCC21332, and probably other B.subtilis
strains that are difficult to transform, because of low
competencedevelopment. For this purpose we made use of the low-copy
plasmid pGSP12, whichcontains the comK gene under control of its
own promoter. Introduction of pGSP12in B. subtilis ATCC6633 and B.
subtilis ATCC21332 increased the transformationefficiency of these
strains 60 to 200 fold and also enables transformation of
thesestrains when cultured in rich media. The second method
facilitates Campbell-typegenomic integrations in B. subtilis of DNA
ligation mixtures without the prior need ofsubcloning in E. coli.
In this method Polyethyleneglycol 8000 (PEG8000) is used for
theproduction of large linear multimeric ligation products that
appeared to be efficientsubstrates for Campbell-type integrations
in competent B. subtilis cells.
Introduction
Bacillus subtilis exhibits a number ofinteresting features such
as synthesis andsecretion of degradative enzymes,production of
peptide antibiotics, sporulationand development of
competence(Sonenshein et al., 1993). The latter makesthis bacterium
easily accessible to geneticanalysis, and is one of the main
reasonswhy B. subtilis became a popularprokaryotic model organism.
Competent B.subtilis cells are able to take up exogenousDNA, and
subsequently, to integrate thisDNA into their genomes via
homologousrecombination (Dubnau, 1991).Competence in B. subtilis
developsoptimally in minimal medium at thebeginning of the
stationary growth phase,when the culture has reached a critical
celldensity. A complex signal transductioncascade integrates the
various external- and
internal signals, and carefully regulatesexpression of ComK, the
pivotalcompetence transcription factor necessaryfor the expression
of the genes encodingthe proteins involved in DNA uptake
andrecombination (Grossman, 1995; vanSinderen and Venema, 1994; van
Sinderenet al.,1995). In cultures of the commonlyused laboratory
strain, B. subtilis 168 (Kunstet al. 1997), about 10 % of the total
cellpopulation develops competence underoptimal conditions. This
relatively highpercentage of competence development isone of the
main reasons why this strain, orderivatives of this strain, became
such apopular laboratory strain. Most other B.subtilis strains,
many of which are producersof important industrial products, have
beenselected on other criteria and develop verylow or no competence
at all. The failure ofsuch strains to develop sufficient levels
ofcompetence may be caused at any level of
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Chapter 3
46
Table 3.1: Strains and plasmids.Relevant genotype/characteristi
Source or reference
Strains:B. subtilis ATCC21332 low competence development,
surfactin+. Mulligan et al., 1984.
B. subtilis ATCC6633 low competence development,
surfactin+,mycosubtilin+.
Garrido et al., 1982.
B. subtilis 168 high competence development, trpC2. Kunst et
al., 1997.B. subtilis 8G5 high competence development, trpC2,
tyr,
his, nic, ura, rib, met, ade. Bron and Venema, 1972.
B. subtilis BD1807 Cmr, ∆abrB Perego et al., 1988.B. subtilis
BV02J07 Kmr, sfp+ This workE. coli MC1061 forms multimeric DNA, F+,
araD139 ∆(ara-
leu)7696, ∆(lac)X74, galU, galK,hsdR2, mcrA, mcrB, rspL.
Wertman et al., 1986.
Plasmids:pGSP12 derivative of pHP12, Em r, contains comK
under the control of its ownpromotor.
Van Sinderen et al., 1995.
pKM1 Km r Kiel et al., 1987pLGW300 integration vector, Km r,
contains a
promotorless spo0V-lacZ fusion.Van Sinderen et al., 1990.
the complex regulation cascade underlyingcompetence development.
Alternativetransformation methods for B. subtilis, suchas
electroporation and protoplasttransformation, have been
developed(Chang and Cohen, 1979; Chassy et al.,1988). However,
these methods arerelatively inefficient and can not be used
formutating specific genes via homologousrecombination, since the
proteins requiredfor this process are only produced
duringcompetence development (Haijema et al.,1995).
In addition to transcriptionalactivation of several
competence-relatedgenes, ComK is also required for theexpression of
its own gene, and it has beenshown that introduction of comK on the
lowcopy plasmid pGSP12 resulted inoverproduction of ComK,
presumably due toan effective autostimulatory response (vanSinderen
et al., 1995). This overproductionresulted in competence
development, whichwas partially insensitive to medium- andgrowth
phase control. We assumed that theincreased expression of comK
bypassesseveral essential regulatory steps in thecompetence signal
transduction cascade
and consequently, that introduction ofpGSP12 might stimulate
development ofcompetence in B. subtilis strains whichnormally
exhibit low levels of competence.In this study we show that
thetransformation efficiencies of the poorlytransformable B.
subtilis strains ATCC6633and ATCC21332, both producers of
severallipopeptides with antiviral and antifungalactivities
(Garrido et al., 1982; Mulligan etal., 1984), was strongly
stimulated by theintroduction of pGSP12.
Campbell-type integrations, oftenused for interruption of genes
orconstruction of reporter gene fusions,require circular DNA.
Binding of DNA to thecell envelope of competent B. subtilis cellsis
accompanied by DNA cleavage.Therefore, Campbell-type single
cross-overswith linearized monomeric molecules willresult in
chromosome breakage, as nocircular molecules can be formed
afteruptake of the DNA, and integration will onlyresult from rare
double cross-over events(Dubnau and Cirigliano, 1972; Venema etal.,
1965). This complication can beovercome by using multimeric DNA,
whichafter uptake, circularizes on the basis of
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Transformation tools
47
redundant sequence homologies. Usually,certain E. coli strains
are used asintermediate host for the production of highamounts of
multimeric DNA. However, adisadvantage of the use of E. coli is
thatplasmids containing B. subtilis DNAfragments are often unstable
in thisbacterium. Multimeric DNA can also beobtained by means of
enzymatic ligation invitro, yet the efficiency of
Campbell-typeintegrations of ligation products is extremelypoor due
to the low percentage ofmultimeric DNA that is formed during
theligation reaction. Addition of highconcentrations of
polyethyleneglycol 8000(PEG8000) to the reaction mixture
causesmacromolecular crowding, which stronglyenhances blunt- and
cohesive-end ligationsand, most importantly, preventsintramolecular
circularization, thusfacilitating the formation of large
linearmultimeric products are formed during theligation reaction
(Pfeiffer and Zimmerman,1983; Zimmerman and Pfeiffer, 1983). Herewe
show that PEG8000-stimulated formationof multimeric DNA enables the
direct use ofligation products as efficient substrates
forCampbell-type integrations in B. subtilis.
Materials and methods
General materials and methods. Strainsand plasmids used in this
study are listed inTable 3.1. Molecular cloning and PCRprocedures
were carried out using standardmolecular biology techniques
described bySambrook et al. (Sambrook et al., 1989).TY-medium for
growth of E. coli and B.subtilis was prepared as described byBiswal
et al. (Biswal et al., 1967). Minimalmedium for growth of B.
subtilis wasprepared as described by Spizizen(Spizizen, 1958).
Plasmid DNA and B.subtilis chromosomal DNA were purifiedaccording
to protocols of Birnboim et al. and
Venema et al. (Birnboim et al., 1979;Venema et al., 1965),
respectively.Enzymes and chemicals were obtained fromRoche
diagnostics GmbH (Mannheim,Germany), Sigma (St. Louis, USA)
andMerck KGaA (Darmstadt, Germany).Oligonucleotides used for PCR
wereobtained from Gibco BRL (Paisley, UK).Transformation in the
presence ofpGSP12. Transformation of competent B.subtilis was
essentially performed asdescribed by Spizizen (Spizizen,
1958).Poorly competent B. subtilis strains werefirst transformed
with pGSP12, a low-copyplasmid containing comK, using
protoplasttransformation, and transformants wereselected on
regeneration-agar platescontaining 5 µg/ml erythromycin (Chang
andCohen, 1979). B. subtilis cells containingpGSP12 were grown to
competence asfollows. An overnight culture was grown inminimal
medium with 2.5 µg/mlerythromycin at 37ºC under continuousshaking
at 300 rpm. After 100-fold dilution ofthe overnight culture in the
same medium,growth was continued and followed in time.Two hours
after the transition fromexponential to stationary growth, 1 µg of
B.subtilis BD1807 chromosomal DNA,containing a chloramphenicol
resistancemarker at the abrB locus, was added to 0.5ml of cells.
After 20 minutes, 0.3 ml of TYmedium was added and growth
wascontinued for another 30 minutes, afterwhich the cells were
plated on TY-agarplates containing 5 µg/ml chloramphenicol.From a
number of chloramphenicol resistantcolonies chromosomal DNA was
isolated toverify the insertion of the chloramphenicolresistance
marker. The presence andposition of this marker was determined
bymeans of PCR using the following primers;Cm1: 5'- GAC AAT TGG AAG
AGA AAAGAG -3’, Cm2: 5'- CAT CAC AAA CAG AATGAT GTA C -3’, AbrB1:
5'- GGA AAC CCTCAC TGC GAA AGA AC -3’, and AbrB2: 5'-
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Chapter 3
48
GCT GTT ATT TCG GTA GTT TCC AAGAC -3’.Campbell-type integration
using DNAligation products. The plasmid used tomeasure the
efficiency of Campbell-typeintegration of ligation products,
pLGW300, isa general integration vector often used tointroduce
lacZ-reporter fusions viaCampbell-type integrations in B.
subtilis(van Sinderen et al., 1990). All ligationswere performed
overnight at roomtemperature using T4 ligase and bufferobtained
from Roche diagnostics GmbH(Mannheim, Germany), in a total volume
of30 µl. For PEG-ligations, PEG8000 wasadded to a final
concentration of 15%(Pfeiffer and Zimmerman, 1983;Zimmerman and
Pfeiffer, 1983). To monitorCampbell-type integration, a DNA
fragmentcontaining the dacC region of B. subtilisstrain168 was
ligated to pLGW300, whichwas linearized by digestion with EcoRI
andXhoI. The dacC region was obtained byPCR using the following
primers; DacC-up1:5'- ATA TCC TCG AGA AGG ATA AACACA ACC ATC TTC AC
-3’ (XhoIrecognition site is underlined), and DacC2:5'- ACA CCG AAT
TCC GGA AGC GTACTG TTT AAC -3’ (EcoRI recognition site
isunderlined). B. subtilis strain 168 and 8G5were transformed with
1 µg of DNA ligationmixture, and transformants were selectedon
TY-agar plates containing 5 µg/mlkanamycin. Whether integration
hadoccurred at the correct position and in theright orientation was
determined by PCRusing the following primers; DacC-up3: 5’-GTC CAG
CTT TTG CGT GAA C -3’, andLac2: 5’- CCA GCT GGC GAA AGG GGG
-3’.
To demonstrate that the efficiency ofPEG-ligations would enable
Campbell-typeintegration without the use of plasmidvectors and an
intermediate cloning hostlike E. coli, we attempted to integrate
just akanamycin resistance marker into a specific
site on the B. subtilis genome. For thispurpose, the 3'-part of
the wild-type sfpgene of B. subtilis ATCC21332 was ligatedto the
kanamycin resistance marker ofpKM1 (Kiel et al., 1987), in the
presence of15% PEG8000, and the ligation mixture wasused to
transform B. subtilis strain 168containing a mutation at the 3'-end
of sfp.The 3'-part of the sfp gene and thekanamycin resistance
marker were obtainedby PCR using the following primer sets;Sfp1:
5’- CGC GGA TCC AGT CAT AAGCAG GCA GTA TC -3’, Sfp2: 5’- CCG GAATTC
CCT CAC ATG TCA TCT ATT TG -3’,and Km1: 5’- CGC GGA TCC GTC GACCAT
ATT TA -3’, Km2: 5’- CCG GAA TTCGGG ACC CCT ATC TAG CGA AC -3’,
forthe 3'-part of sfp and the kanamycinresistance marker,
respectively (BamHI andEcoRI recognition sites are
underlined).Prior to ligation both PCR products weredigested with
BamHI and EcoRI to ensurethe desired orientation of both genes in
themultimeric ligation products. Transformantswere selected on
TY-agar plates containing5 µg/ml kanamycin, and surfactin
productionwas tested using a halo-assay on blood-agar plates as
described by Mulligan et al.(1984). The correct insertion of
thekanamycin resistance marker wasdetermined by means of PCR using
thefollowing primers; Km1, Km2, SFP3: 5'- TTGTTC TGC GCT GGA CAT TT
-3’, and sfp4:5'- TTT ATT TAT GGG ACG CCA AA -3’.
Results and discussion
Stimulating transformation byintroduction of plasmid-located
comK.Both B. subtilis ATCC6633 and ATCC21332produce lipopeptides
with antiviral andantifungal activities, such as surfactin
andmycosubtilin (Garrido et al., 1982; Mulliganet al., 1984). In
order to study theexpression and regulation of the operons
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Transformation tools
49
Figure 3.1. pGSP12-stimulated transformation of B. subtilis
ATCC6633 and ATCC21332. A) Growth curves ofB. subtilis ATCC6633
(▲/△ with pGSP12 and ■/□ without pGSP12) and B. subtilis ATCC21332
(●/○ withpGSP12 and ◆/◇ without pGSP12). Closed symbols refer to
growth in minimal medium (MM) and opensymbols refer to growth in
rich medium (TY). The time scale indicates hours before and after
transition fromthe exponential to the stationary growth phase and
arrows indicate the time of transformation. B) Number
oftransformants obtained after transformation with 1 µg chromosomal
DNA.
encoding the surfactin -and mycosubtilinsynthetases, we had to
introducetranscriptional lacZ fusions and specificmutations in
these strains. However, the lowlevels of competence development in
theseB. subtilis strains precluded this approach.The low copy
plasmid pGSP12, containingthe comK gene under the control of its
ownpromoter, stimulates competencedevelopment in B. subtilis 168
derivatives,and bypasses the requirements for mediumcomposition
essential for optimalcompetence development (van Sinderenand
Venema, 1994). To examine whetherpGSP12 also would increase
competencedevelopment, and hence the transformationefficiencies, of
B. subtilis ATCC6633 andATCC21332, this plasmid was introducedinto
these strains by means of conventionalprotoplast transformation.
Indeed, thenumber of transformants obtained inminimal medium after
introduction of
pGSP12 increased dramatically for both B.subtilis ATCC6633 and
ATCC21332 (Fig.3.1). This figure also shows that similarresults
were obtained when both strainswere cultured in rich medium,
although thetotal number of transformants was lower.These results
clearly demonstrate thatpGSP12 is a very useful tool to
enhancetransformation in B. subtilis strains which donot normally
develop sufficient levels ofcompetence for genetic studies.
Two other methods have beendescribed, which could be used to
stimulatecompetence development in B. subtilis:knockout mutations
in mecA or clpC, andoverexpression of ComS by means of a highcopy
plasmid harbouring comS (Hahn, etal., 1995; Kong et al., 1993; Liu
et al., 1996).MecA, in consort with the protein chaperonClpC, binds
ComK and presents the latterfor degradation by the protease
ClpP.During the development of competence this
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Chapter 3
50
Figure 3.2. Stimulation of Campbell-type integration by
PEG-ligation. A) Ethidiumbromide stained agarose gelloaded with PCR
product containing the dacC region (lane1), linerized pLGW300
(lane2), DNA ligationproducts after ligation in the absence of
PEG8000 (lane 3), and in the presence of PEG8000 (lane 4). B)
Numberof transformants after transformation with 1 µg DNA ligation
products obtained in the absence and thepresence of PEG8000.
process is inhibited due to the accumulationof ComS in the cell.
ComS binds to MecAand prevents binding of MecA to ComK and,as such,
the degradation of ComK. Bothmutations in mecA or clpC, as well
asoverproduction of ComS result in anoverproduction of ComK and
increasedlevels of competence. However, theintroduction of mecA or
clpC mutations toraise competence will be very difficult if
notimpossible, in such poorly transformablestrains. In addition,
mutations in mecA orclpC are accompanied by a number ofundesirable
effects resulting in decreasedviability, and knockout mutations of
thesegenes do not bypass a possible mutation inthe comK gene. The
advantage of pGSP12over a comS bearing plasmid is thatpGSP12 is
able to bypass possible adversemutations in the original comK
resulting inpoor competence development.Producing Campbell-type
integration byligation products. Campbell-typeintegrations of
suitable cloning vectorsrequire transformation of competent B.
subtilis cells with multimeric DNA. Usually,plasmid multimers
are first isolated from E.coli. However, subcloning in E. coli may
beimpossible if the DNA fragments to becloned are deleterious to
the host. Theproduction of multimers by means of in vitroDNA
ligation could overcome this problem,were it not that standard
ligation reactionsare rather inefficient and result in asubstantial
amount of closed circularmonomeric DNA products. Ligation in
thepresence of high concentrations of PEG8000have been shown to
strongly stimulate theligation reaction, and in addition,
PEG8000inhibits the formation of closed circularmonomeric DNA, thus
resulting in a highpercentage of large linear multimeric
DNAmolecules (Pfeiffer and Zimmerman, 1983;Zimmerman and Pfeiffer,
1983). To examinewhether ligation products produced in thepresence
of PEG8000 might serve as moreefficient substrates for
Campbell-typeintegrations in competent B. subtilis, wetested the
integration efficiency of theintegration vector pLGW300 in the
dacC
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Transformation tools
51
region of B. subtilis. Linearized pLGW300and a PCR product of
the dacC region wereligated in the presence and absence of
15%PEG8000, and the DNA ligation productswere transformed to
competent B. subtiliscells. Clearly, addition of PEG8000
stronglystimulated the ligation efficiency, andresulted in the
formation of large multimericDNA complexes which did not enter the
gelmatrix (Fig. 3.2A). Most importantly, PEG8000increased the
transformation efficiency ofDNA ligation products 30 to 60 fold
(Fig.3.2B). In conclusion, PEG-ligation increasesthe suitability of
DNA ligation products assubstrate for Campbell-type integrations
incompetent B. subtilis. It should bementioned however, that
multimeric DNAobtained from E. coli is still a more
efficientsubstrate producing a 10-fold higher numberof
transformants compared to PEG-ligationproducts (data not
shown).
The use of ligation products forCampbell-type integrations in B.
subtilisrenders the use of shuttle vectorsredundant. This was
demonstrated byconverting B. subtilis 168 into a surfactinproducing
strain, just by using a singlekanamycin resistance gene to the
3'-end ofthe wild-type sfp gene (Fig. 3.3). B. subtilis168 does not
produce surfactin due to apoint mutation in the 3'-end of the sfp
gene.This gene encodes a phosphopanteteinetransferase, which
couples the essentialcofactor phosphopanteteine to the
surfactinsynthetase. The 3'-end of a wild-type sfpgene from the
surfactin producing strain B.subtilis ATCC21332, obtained by PCR,
wasligated in the presence of PEG8000 to akanamycin resistance
marker obtained byPCR as well. To ensure the desiredorientation of
both genes in the multimericligation products both PCR products
weredigested with BamHI and EcoRI prior toligation. The obtained
ligation products weredirectly transformed to competent B.
subtilis168 cells. Most of the obtained kanamycin
Figure 3.3. Schematic representation of the strategyto convert
B. subtilis 168 into a surfactin producingstrain. The 3'-end of
wild-type sfp gene ('sfp') and thekanamycin resistance marker (KmR)
were obtainedby PCR, and digested with BamHI and EcoRI toensure
proper orientation. After ligation in thepresence of PEG8000, the
ligation products weretransformed into B. subtilis 168. A
Campbell-typeintegration will result in 2 possible
phenotypes;kanamycin resistance colonies which do not
producesurfactin (sfp-mut), and kanamycin resistant colonieswhich
do produce surfactin (sfp-wt); a and b in figure3,
respectively.
resistant colonies produced surfactin andthe majority of
transformants contained asingle integrated kanamycin resistance
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Chapter 3
52
marker adjacent to sfp (data not shown).Another effect of
macro-molecular crowdingis that the activity of
T4-polynucleotdekinase is strongly stimulated as well(Harrison and
Zimmerman, 1986). We haverepeated the integration of the
kanamycinresistance marker at the sfp locus, yetwithout digestion
of the PCR fragments priorto ligation. In addition to PEG8000, we
addedT4-polynucleotide kinase to the ligationmixture.
Transformation of the ligationproducts to competent B. subtilis 168
gavealmost the same results to that described infigure 2. A
substantial number oftransformants produced surfactin andcontained
a single kanamycin resistancemarker at the sfp locus (data not
shown).These experiments demonstrate that asingle antibiotic
resistance marker can bespecifically integrated into the genome of
B.subtilis when using PEG-ligation.
Both methods are now routineouslyused in our laboratory and
together thesemethods enabled us to study the expressionand
transcriptional regulation of themycosubtilin- and surfactin
synthetaseoperons in B. subtilis ATCC6633 and B.subtilis 168
(Cosmina et al., 1993; Duitmanet al., 1999). In addition, both
methodscould increase the genetic accessibility ofother poorly
transformable B. subtilisstrains, many of which are producers
ofimportant industrial products.
Acknowledgements
This work was supported by the EuropeanUnion (EU Grant
PL950176).
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acts as an autoregulatory controlswitch in the signal transduction
route tocompetence in Bacillus subtilis. J. Bacteriol.176:
5762-70.van Sinderen, D., Luttinger, A., Kong, .,Dubnau, D.,
Venema, G. and Hamoen, L.W. 1995. ComK encodes the
competencetranscription factor, the key regulatoryprotein for
competence development inBacillus subtilis. Mol. Microbiol. 15:
455-462.
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Chapter 3
54
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