University of Groningen

Nonribosomal peptide synthesis in Bacillus subtilisDuitman, Erwin Hans

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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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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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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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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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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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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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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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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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