JOURNAL OF BACTERIOLOGY,0021-9193/99/$04.0010

Sept. 1999, p. 5718–5724 Vol. 181, No. 18

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Identification of sB-Dependent Genes in Bacillus subtilisUsing a Promoter Consensus-Directed Search and

Oligonucleotide HybridizationANJA PETERSOHN,1 JORG BERNHARDT,1 ULF GERTH,1

DIRK HOPER,1 TORSTEN KOBURGER,1 UWE VOLKER,2

AND MICHAEL HECKER1*

Institut fur Mikrobiologie und Molekularbiologie, Ernst-Moritz-Arndt-Universitat,17487 Greifswald,1 and Laboratorium fur Mikrobiologie und Max-Planck-Institut

fur terrestrische Mikrobiologie, Philipps-Universitat Marburg,35043 Marburg,2 Germany

Received 23 February 1999/Accepted 23 June 1999

A consensus-directed search for sB promoters was used to locate potential candidates for new sB-dependentgenes in Bacillus subtilis. Screening of those candidates by oligonucleotide hybridizations with total RNA fromexponentially growing or ethanol-stressed cells of the wild type as well as a sigB mutant revealed 22 genesthat required sB for induction by ethanol. Although almost 50% of the proteins encoded by the newlydiscovered sB-dependent stress genes seem to be membrane localized, biochemical functions have so farnot been defined for any of the gene products. Allocation of the genes to the sB-dependent stress regulonmay indicate a potential function in the establishment of a multiple stress resistance. AldY and YhdF showsimilarities to NAD(P)-dependent dehydrogenases and YdbP to thioredoxins, supporting our suggestionthat sB-dependent proteins may be involved in the maintenance of the intracellular redox balance afterstress.

In Bacillus subtilis, the alternative sigma factor sB tightlycontrols a large stationary-phase and stress regulon (7–10, 16,17, 31). The functional characterization of members of the sB

regulon led to the assumption that the proteins encoded areinvolved in the protection of DNA, membranes, and proteinsagainst oxidative damage, which might represent an importantcomponent within the complex stress response (6, 14).Moreover, general stress proteins appear to contribute tosurvival of extreme environmental conditions such as severeheat or osmotic stress, repeated freezing and thawing, aswell as acid or alkaline shock of starving B. subtilis (15, 32).In summary, the expression of the sB-dependent generalstress regulon is expected to provide an unspecific, multipleand prospective stress resistance to nongrowing B. subtiliscells in anticipation of future stress (for a review, see ref-erence 17).

The discovery and characterization of new sB-dependentgenes will certainly improve our understanding of the physio-logical role of the entire sB regulon in B. subtilis. So far,members of the sB regulon have been defined mainly on thebasis of transposon mutagenesis or identification of proteinspots from two-dimensional protein gels (5, 10, 11, 31). In thisstudy, we used the combination of a consensus promoter-basedsearch for new sB targets and an oligonucleotide hybridizationto detect new members of the general stress regulon of B.subtilis.

MATERIALS AND METHODS

Bacterial strains and growth conditions. The B. subtilis wild-type strain 168 (3)and its isogenic sigB mutant ML6 (22) were cultivated in a shaking water bath at37°C in a minimal medium with glucose as the carbon source previously de-scribed (29). Ethanol stress was imposed by adding ethanol to exponentiallygrowing cells to a final concentration of 4% (vol/vol).

Analysis of transcription. Total RNA of both B. subtilis strains was isolated bythe acid phenol method of Majumdar et al. (25), with modifications as previouslydescribed (31). Decreasing amounts of total RNA were transferred onto apositively charged nylon membrane by slot blotting. Hybridizations at 50°C withdigoxigenin-end-labeled oligonucleotides specific for the various genes (Table 1)and detections were performed as instructed by the manufacturer (BoehringerMannheim). For 39-end labeling, 100 pmol of each oligonucleotide was incu-bated for 1 h in a 20-ml reaction mixture containing 12.5 nmol of digoxigenin-11-ddUTP, 2.5 mM cobalt chloride, 13 reaction buffer and 12.5 U of terminaltransferase. The whole volume was used for the hybridization. Primer extensionswere performed with radiolabeled primers as previously described (33) (Table 1).A DNA-sequencing ladder was generated with the same primers, using PCRproducts as a template (Table 1).

General methods. The BSOrf homology search tool of the database of the B.subtilis genome sequencing project was used for deriving of the primers. Data-base searches were performed with the Blast program (2).

RESULTS AND DISCUSSION

Knowledge of the sequence of the entire B. subtilis genome(24) provides an excellent basis for a comprehensive analysis ofgene expression by using global approaches such as two-dimen-sional protein electrophoresis (proteome analysis) (5), chiptechnology (transcriptome analysis) (13), and consensus se-quence-based searches for members of individual regulons(20). In this report, we used the genome sequence for theidentification of new sB-dependent genes.

After an initial manual evaluation, the whole genome wassearched with the sequence pattern DGWKTNDN12–15GGRWAW (D 5 A, G, T; W 5 A, T; K 5 G, T; R 5 A, G; N 5A, C, G, T). Only targets deviating not more than three nu-cleotides from the consensus of sB-dependent promoters

* Corresponding author. Mailing address: Institut fur Mikrobiologieund Molekularbiologie, Ernst-Moritz-Arndt-Universitat, Friedrich-Ludwig-Jahn-Straße 15, 17487 Greifswald, Germany. Phone: 49-3834-864200. Fax: 49-3834-864202. E-mail: hecker@microbio7.biologie.uni-greifswald.de.

5718

on March 15, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

GTTTWWN12–15GGGWAW and lying within 400 bp up-stream of predicted open reading frames were considered forfurther analyses. The rather large deviation from the consensuswas intentionally permitted so that we could identify even weaksB-dependent promoters.

To demonstrate that the presence of a putative sB promoterindeed conferred sB-dependent stress induction, total RNAfrom isogenic wild-type and sigB mutant cells was isolatedbefore (control) and after ethanol stress (4% [vol/vol]) andhybridized with a digoxigenin-labeled oligonucleotide designedagainst the open reading frame downstream of the potential sB

target.Prior to the screening, oligonucleotide probes against known

sB-dependent genes were used to prove the specificity of thehybridization assay. Rigorously sB-dependent genes such asgspA displayed a signal only after treatment of the wild typewith ethanol; no signal was observed with RNA from growingcells or from the sigB mutant as the template (Fig. 1A; Table2, group A) (4). ctc illustrates the pattern for genes the tran-scription of which is driven by the vegetative sigma factor sA inaddition to sB (18). Basal-level expression was easily detectedfor such genes in the wild type and the sigB mutant prior to

stress, whereas induction by ethanol stress absolutely requiredsB (Fig. 1A; Table 2, group A). Genes such as trxA, which aresubject to a double or multiple control, may retain inductioneven in the sigB mutant albeit at a reduced level (Fig. 1A;Table 2, group A) (28).

Of the 28 genes induced by ethanol stress, 22 were inducedonly in the wild type and required sB for induction (Fig. 1B).The number of new sB-dependent genes may even exceed 22since some of the promoters were located in front of operonscontaining probably one or two additional genes (e.g., ykgA/ykgB, ypuB/ypuC, yqhQ/yqhP, and yoxC/yoxB/yoaA). Althoughthese oligonucleotide hybridizations clearly establish the sB

dependency of the induction, they do not provide informationabout the precise location of the sB-dependent promoter.Therefore, primer extension experiments were performed forselected genes in order to map the 59 ends of the correspond-ing transcripts and to ascertain that transcription initiateddownstream of the potential sB-dependent promoters after theimposition of ethanol stress. Figure 2A displays the resultsobtained for yhdF as a representative example. The 59 ends ofyhdF (Fig. 2A) were barely detectable with RNA from expo-nentially growing bacteria, but the intensity of the signal dra-

TABLE 1. Oligonucleotides used in this study

Gene-specific oligonucleotide or primer Sequence (59339)

Positive control oligonucleotidesctc.................................................................................................................................................................................CATGTCCTGAAGTACGGATATTCCgspA .............................................................................................................................................................................TTACCTCTCTCTCCTGATCCtrxA ..............................................................................................................................................................................CAAGGTCCGCACCAAGGAGC

Oligonucleotides specific for tested genesaldY..............................................................................................................................................................................TTCCGTCGTGCTCTTGGCCCACTClctE...............................................................................................................................................................................CGCAAATGCATAACTGCTTCCyabJ ..............................................................................................................................................................................CCGCAAACTGTTCCATATCCGCGyacL .............................................................................................................................................................................CAGCTTCTTGTCTTGAACCTCATGycnH.............................................................................................................................................................................TGAGGAATCGTTTTGATCTCCTCGydaS .............................................................................................................................................................................GGCGAGGCTCGGTCCCCATGTGCCydaT .............................................................................................................................................................................CCCATTCCTTCGCTTTGCTTGTCGCydbP .............................................................................................................................................................................ATATTCATGCGTGTGCAGTCTGGGydhK.............................................................................................................................................................................AGAGATAACATCAGAATTCCCAGTGCyfhK..............................................................................................................................................................................ATTTGAGCCCAATCGGCATTTACGyflA...............................................................................................................................................................................TAGCGGCGGACCCCACAGCCAATCyhaR.............................................................................................................................................................................GCGTGATGGGCATCAGGCCGATTCydhF .............................................................................................................................................................................TGGAGATATCAGCCCCCTCTTTAGAyjbC..............................................................................................................................................................................CCCTCTTGCATCCTTAGACACATACyjgB ..............................................................................................................................................................................GGCATTTCCCCTTTATAGGCGGTGykgA .............................................................................................................................................................................GTGCTGGTCGTTAGCGGTTTTTACyotK..............................................................................................................................................................................CATCTTGTTTAACAGTGTTTGAATTGAATGyoxA .............................................................................................................................................................................GCCATACAATGTTGGTGTGTCGTGyoxC .............................................................................................................................................................................CTTGTTTTGGATCACGGTAACTCCypuB .............................................................................................................................................................................CTTAGCACCCAGTTTAACTTTTCTTGyqhA.............................................................................................................................................................................TACAAACTTCCAGCCATTGTTGCGyqhQ ............................................................................................................................................................................TCCGTTCTTCTGATGGCTGTAACGyqhZ.............................................................................................................................................................................GCTTCCAATTCACCAGATGCTTGGAGyqiS ..............................................................................................................................................................................CACTTCCTCATCCTCAGCATGAGCyqxL .............................................................................................................................................................................TCTTGCCTTACTTCTTTCTTGAGGyrvD .............................................................................................................................................................................CGGCCATTCTGATCTTCCAAACAGGysdB .............................................................................................................................................................................TTAGGATTCGCGACATATTTGACCyvrE ..............................................................................................................................................................................CTGTCATCCCGAAGATGATACAGGyxkO .............................................................................................................................................................................GGCAATGGCCCGGTACGTCTCTTCyycD .............................................................................................................................................................................TGTCGGCGCTACATTCTCCACCTC

Primers for primer extension and generation of PCR productsyacL-PE.......................................................................................................................................................................AATGAAGAACGCCTGAACTATTCGyhdF-PE ......................................................................................................................................................................TTCCTCGATAATCCTCGTCCTCTGyjbC-PE.......................................................................................................................................................................TCAATTGGAAAGTACTCGCTAAGCyacL-forward ..............................................................................................................................................................GGACAGAAGGGCATCTGATCyacL-reverse ...............................................................................................................................................................TCTTCTCCCTTTACAGCGCCyhdF-forward..............................................................................................................................................................GTGGTGCGCCCATGTACTGGyhdF-reverse ...............................................................................................................................................................CACCACACAAAAAACCAGCTCyjbC-forward...............................................................................................................................................................GCAGGAAGTTATTCCCGAGCyjbC-reverse................................................................................................................................................................CATCGTTGTTTTCCAAGACCTC

VOL. 181, 1999 sB-DEPENDENT GENES IN B. SUBTILIS 5719

on March 15, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

FIG. 1. Screening of potential targets for sB-dependent transcription by slot blot hybridization with digoxigenin-labeled oligonucleotides specific for the corre-sponding genes. The lanes contain decreasing amounts (3, 1.5, and 0.75 mg) of total RNA isolated from wild-type bacteria and sigB mutant cells before (controls 1 and2, exponential growth) and 10 min after the imposition of ethanol stress. (A) Hybridization pattern of known sB-dependent genes. Whereas the stress induction of ctcand gspA strictly requires sB, induction of trxA can occur at both sB-dependent and sB-independent promoters. (B) New sB-dependent genes requiring sB for inductionby ethanol stress. (C) Genes displaying induction by ethanol stress even in the absence of sB. (D) Examples of target genes that lack induction by ethanol stress despitedisplaying a putative sB promoter.

5720 PETERSOHN ET AL. J. BACTERIOL.

on March 15, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

matically increased after stress. In agreement with the sB de-pendency, the signal was not observed with RNA isolated froma sigB mutant.

The remaining six genes were induced by ethanol in the wildtype as well as in the sigB mutant, indicating a more complexcontrol (Fig. 1C). However, induction seemed to be morepronounced in the wild type than in the sigB mutant, implyingan involvement of sB (Fig. 1C). Evidence for a role of sB inethanol induction was obtained by primer extension experi-ments, two of which are displayed in Fig. 2B and C. One 59 endof the yacL mRNA was mapped to a site which is preceded bya putative sB-dependent promoter. The signal was exclusivelydetected after ethanol stress in the wild type and was missingwith RNA from the sigB mutant as template (Fig. 2B). Asecond sB-independent start site was mapped further down-stream (data not shown).

Using a yjbC-specific primer, we mapped two 59 ends of theyjbC mRNA. The intensity of both reverse transcripts in-creased after ethanol treatment (Fig. 2C). The sequence pre-ceding the downstream signal resembled a typical sB-depen-dent promoter, and this start site was not used in the sigBmutant. The promoter sequence located in front of the up-stream start site resembled sW-dependent promoters (19), andthe intensity of the signal increased even in a sigB mutant afterethanol stress (Fig. 2C). ysdB, a second gene displaying par-tially sB-dependent ethanol induction, also possesses a sW-dependent promoter (21) and moreover contains fairly wellconserved recognition sequences for sB (Table 2,C). sW is anew extracytoplasmic function sigma factor recently describedby Huang et al. (19–21). The authors suggest that sW activatesa large stationary-phase regulon that functions in detoxifica-

tion or production of antimicrobial compounds. Our data in-dicate that in addition to entry into stationary phase, ethanolstress induces transcription at sW-dependent promoters (Fig.1C and 2C) and that genes such as yjbC seem to be subject toa control by sB and sW.

The new presumably sB-dependent promoters as well as thedefined promoters of the control genes are summarized inTable 2. An alignment of all the sB-dependent promoterscurrently available yields consensus sequences GTTTaa andGGG(A/T)A(A/T) for the 235 and 210 regions, respectively,which are separated by 13 to 15 nucleotides. Capital lettersindicate bases which are conserved in more than 80% of the58 promoters analyzed. Residues 236 (G), 233 (T), 215(G), and 212 (A) seem to be particularly important sincethey are absolutely conserved in all 58 promoters. All ofthese residues exception the T at position 233 have previ-ously been recognized as being critical for sB promoteractivity (27, 30). Less than 10% of all known sB-dependentpromoters deviate in more than two residues from this con-sensus sequence.

For most of the proteins encoded by the new sB-dependentgenes, biochemical functions have not been determined al-though similarities to sequences found in databases suggestedputative functions for some of them. aldY and yhdF encodegene products with similarities to aldehyde and glucose dehy-drogenases, respectively. Among the gene products which areinduced by ethanol stress in the sigB mutant are two moredehydrogenases: LctE, an L-lactate dehydrogenase, and YcnH,similar to succinate-semialdehyde dehydrogenases. These dataappear to confirm our earlier suggestion that several generalstress proteins with similarities to NAD- or NAD(P)-depen-

FIG. 1—Continued.

VOL. 181, 1999 sB-DEPENDENT GENES IN B. SUBTILIS 5721

on March 15, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

TABLE 2. Defined sB promoters of the control genes and promoters of the presumable new sB-dependent genes found in this study

Group and promoter sequencea Gene

Protein

Presumed functionand/or similarity

Probablycotranscribed

gene(s)Molecular

mass(kDa)

pI

A (sB-dependent genes [positive controls])CGAGGTTTAAATCCTTATCGTTAT-GGGTATTGTTTGTAATAG-N33-ATG ctc 22.0 4.24 Belongs to the L25P family of ribosomal

proteinsACGTGTTTATTTTTTTGAAAAA---GGGTATGTAACTTG-TAC-N23-ATG gspA 33.5 5.19 Similar to lipopolysaccharide-1,3-galacto-

syltransferases and to lipopolysaccha-ride-1,2-glucosyltransferases

TCAGGTTTTAAAACAGCTCCGGCA-GGGCATGGTAAAGTACAT-N241-ATG trxA 11.39 4.34 Thioredoxin

B (genes controlled by sB)ATTCGTTTACATATTGGCTTCAG--CGGAAATAGAAGAAGACA-N25-ATG aldY 52.8 5.07 Probable aldehyde dehydrogenase, con-

tains an aldehyde dehydrogenase sig-nature (glutamic acid active site)

AAGTGTTTCGAAATGATCAGGAGC-GGATATTGATTGGTAAAT-N21-ATG ydaS 8.64 12.22 Membrane protein, unknown functionATGCGTTTTTATTTTTCACCTGC--GGGTACCATTTTTATAAA-N19-ATG ydaT 16.5 6.38 Unknown ydaSGTTTGATTACAGCCAGAGCTGTCTGCAGGGAAACATTATGTTCTG-N193-ATG ydbP 12.4 4.42 Similar to thioredoxins of A. fulgidus and

S. cerevisiaeTTATGTTTGGCTTTGCAAACAAAG-GGGAATAGGACAAACTGC-N76-TTG ydhK 22.5 7.81 Protein with 1 transmembrane domain,

unknown functionACATGTTTCACCAGCCTGTCAATCAGGGAATACCACTTATATC-N26-ATG yfhK 18.7 10.37 Protein probably with signal sequence,

similar to proteins with unknown func-tion

AAAGGTTTATGTTTTTCCATCTAT-GGGAAATGATTCATAAAC-N17-ATG yflA 50.6 10.4 Integral membrane protein, similar toamino acid carrier protein

TGGCGTTTATTCATTTCTGTCGTG-TGGTAACGTTCAGTATCA-N24-GTG yhdF 31.4 5.9 Similar to glucose-1-dehydrogenase, be-longs to the short-chain dehydroge-nases/reductases family

ACATGTTTCGTTGCAAAAACACG--GGGAAACGGAATGGTAGA-N19-ATG yjgB 20.8 6.68 Membrane lipoprotein, unknown func-tion

AATGGTTTAATGATTTTCATGATGAGGGAATAATAAATGTATT-N25-ATG ykgA 29.6 4.88 Function unknown, similar to streptococ-cal acid lipoprotein

ykgB

TCTAGTTTCTCTTTTTAAAAGAGTAGGGTATTGCAAACAACAG-N58-ATT yotK 7.2 7.69 UnknownAACGGTGTTTTTTTATTTGATAG--GGGAAAATATAAAATGGA-N64-ATG yoxA 37.2 6.00 Similar to hypothetical proteins of B.

subtilispbp

TTCTGATTAAAAAAACGGATACA--GGGTAATGACATAAGAAA-N12-ATG yoxC 11.39 10.37 Protein probably with signal or trans-membrane sequence, similar to thegeneral stress protein YtxG

yoxB, yoaA

TGCTGATTATACAAAAAGTGGATT-GGGAATGATAAAAGAACA-N21-ATG ypuB 7.23 4.24 Protein with 1 transmembrane domain,unknown function

ypuC

TTCGGTTTTTCACCTGTCCAGAAACTGGGCTAGCTGGATTCGAAC-N136-ATG yqhA 31.8 5.17 Membrane protein with 1 transmem-brane domain, similar to RsbR of B.subtilis

CTGCGAGTAAAATTTGAAAATAAC-GGGTATAATGCATGTAGG-N118-ATG yqhQ 36.0 9.71 Integral membrane protein with un-known function

yqhP

ACTGGTTTAGTGACGCGGTTATT--GGGCAATTAAAGAATAAA-N25-ATG yqxL 37.7 10.00 Membrane protein, similar to divalentcation transport proteins

CCATGTTTAAAACCAGTCTTGATT-GGGAAAGTTACCTCAATA-N21-ATG yrvD 12.2 9.16 Membrane protein with unknown func-tion

yrvE, apt

ATAAGTTTTTCAGCTTTTTAAAAA-GGGAAAATAAAAAAAACA-N24-ATG ytkL 10.9 6.41 Similar to proteins with unknown func-tion

AGTGGTTTGGACACCTCTTTGCC--GGGAATAACAATATATAG-N24-ATG yvrE 33.2 4.65 Similar to the eukaryotic SMP-30/CGR1protein family

TTTTGTTTGAAAAAGAAAAGGGAC-AGGAAAAATAGGAAAAGA-N19-ATG yxkO 29.9 5.8 Membrane protein with unknown func-tion, similar to hypothetical family ofuncharacterized proteins (UPF0031)

GATCGTTTCGGACAGTAACAAGGC-GGGAAAAATGCAATAAAA-N19-ATG yycD 7.4 4.81 Unknown

C (complex-regulated genes, sB could be involved)AATTGAATTGACCGGATCTTGGCC-TGGAAAACCAATGACTAA-N363-ATG lctE 34.9 5.64 L-Lactate dehydrogenase lctPTTTCGGTTAAAACCTTATGAATAC-GGGTATATTAATGTTGGT-N74-ATG yacL 40.8 5.01 Membrane protein, similar to hypotheti-

cal proteinsyacM, yacN,

gltXAACGGATTACTTTTGCTGACAGC--GGGAATTAACGGTAATAT-N153-ATG ycnH 50.2 4.8 Similar to the succinate-semialdehyde

dehydrogenase GabD of E. coli, con-tains an aldehyde dehydrogenase sig-nature (cysteine active site)

GGCTGTTTAAACAAGAAGAAAATG-GGGTATATCTAAAAGTAT-N30-ATG yjbC 23.1 5.22 Unknown yjbDTTCGGAATTTATATTACAAAAT---GGATAAATATGGCCTTGC-N143-ATG yqiS 31.7 8.18 Protein with 1 transmembrane domain,

similar to phosphate butyryltrans-ferases, belongs to the phosphateacetyltransferase and butyryltrans-ferase family

yqiT, yqiV,yqiW,bfmBAA,bfmBAB,bfmBB

ATACGACTATTTCACTTGAAAATC-GGGTATATGTTTTTACAG-N83-ATG ysdB 15.6 9.85 Membrane protein with unknown func-tion

D (noninducible genes preceded by a putative sB promoter sequence)ATGAGTTTAACGCAAATGTGGC---GGGAATCGGCGTCTTAGT-N157-ATG yabJ 13.6 5.24 Unknown, similar to proteins with un-

known functionAAAGGTTTATTTGGGGATGGCG---GGGAATGTATAGGAGAAA-N34-ATG yhaR 29.5 6.27 Protein with 1 transmembrane domain,

similar to 3-hydroxybutyryl-coenzymeA-dehydratase

yhaQ, yhaP

AGGTGTGTGAAAATATTCGGTAATAGGGTAAAAAAACCTTGAT-N86-ATG yqhZ 14.8 4.8 Unknown, similar to NusB (transcriptiontermination)

folD

a Boldface sequences represent, from left to right, 235 region, 210 region, and transcriptional start site (11). Start codons are indicated on the far right.

5722

on March 15, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

dent dehydrogenases might be involved in the maintenance ofthe redox balance during stress (17). YdbP, which needed sB

for induction, is similar to thioredoxins of Archaeoglobus fulgi-dus and Saccharomyces cerevisiae (44% identity in a 85-amino-acid overlap and 42% identity in a 75-amino-acid overlap,respectively) and could therefore also be involved in the pro-tection against oxidative stress.

YqhA is highly similar to modulators of sigma factor activitysuch as RsbR and IspU of B. subtilis (1, 26). RsbR functions asa positive regulator of sB activity by interaction with theantagonist protein RsbS (1). YqhA and RsbR share a highdegree of identity in their C-terminal portion, which alsocomprises a conserved phosphorylation site (1). Conse-quently, it would be attractive to analyze whether YqhAbelongs to the family of antagonist proteins in the partner-switching regulatory mechanism and might be involved inconveying environmental signals in the sB signal transduc-tion network.

Gaidenko and Price pointed out that sB-dependent stressproteins may be involved in the maintenance of the cell enve-lope integrity during stress (15). Supporting their hypothesis,many new sB-dependent genes described in this study seem tocode for integral membrane proteins. The high proportion ofmembrane proteins found can partially be attributed to the factthat two-dimensional protein electrophoresis as one of the twoapproaches of identification of stress genes failed to detect thisclass of proteins due to their alkaline isoelectric point andsolubilization problems (5). Induction of all of those genes bythe stress sigma factor sB provides only a first hint for theirinvolvement in stress protection. Knockout mutations in thegenes need to be analyzed for resistance to oxidative, acid,

FIG. 2. Mapping of the 59 ends of yhdF (A), yacL (B), and yjbC (C) duringexponential growth (co), after exposure to ethanol stress (e) and salt stress (s),by primer extension analyses with RNA from wild-type (168) and sigB mutant(ML6) cells. The transcriptional start sites are marked with asterisks. The 235and 210 regions (underlined), the 59 ends of the messages, and the translationalstart sites are in boldface; putative sW-recognition sequences in front of yjbC areunderlined.

5723

on March 15, 2020 by guest

http://jb.asm.org/

Dow

nloaded from

alkaline, heat or salt stress in order to gather more informationon their function.

Another interesting problem raised in this study is the ques-tion of why genes with presumable sB promoters, which areconserved in all the positions known to be critical for pro-moter recognition by sB (27, 30), still lacked stress induction(Fig. 1D). A potential reason for such a failure of inductioncan be the presence of operator elements blocked by repres-sor molecules. Recently, we obtained evidence that CtsR, aglobal repressor of class III heat stress genes (12, 23), pre-vents the sB-dependent induction of the clpC operon byglucose starvation (23). A detailed transcriptional analysis isneeded to determine whether the genes in question arecontrolled by sB only under special circumstances or not atall.

Although the number of sB-dependent genes is now morethan 80, the use of sophisticated approaches such as DNAchip technology and detailed analysis of genes subject tocomplex transcriptional regulation by several networks willhelp to define still undiscovered members of this importantregulon.

ACKNOWLEDGMENTS

We thank M. Messenger (University of Western Ontario, London,Ontario, Canada) for help at the beginning of this study and A. Harangfor technical assistance.

This work was supported by grants from the Deutsche Forschungs-gemeinschaft, the Fonds der Chemischen Industrie, and the EU Bio-technology Programme (BIO 4-CT95-0278) to M.H.

REFERENCES

1. Akbar, S., C. M. Kang, T. A. Gaidenko, and C. W. Price. 1997. Modulatorprotein RsbR regulates environmental signalling in the general stress path-way of Bacillus subtilis. Mol. Microbiol. 24:567–578.

2. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990.Basic local alignment search tool. J. Mol. Biol. 215:403–410.

3. Anagnostopoulos, C., and J. Spizizen. 1961. Requirements for transforma-tion in Bacillus subtilis. J. Bacteriol. 81:741–746.

4. Antelmann, H., J. Bernhardt, R. Schmid, and M. Hecker. 1995. A gene at333° on the Bacillus subtilis chromosome encodes the newly identified sB-dependent general stress protein GspA. J. Bacteriol. 177:3540–3545.

5. Antelmann, H., J. Bernhardt, R. Schmid, H. Mach, U. Volker, and M.Hecker. 1997. First steps from a two-dimensional protein index towards aresponse-regulation map for Bacillus subtilis. Electrophoresis 18:1451–1463.

6. Antelmann, H., S. Engelmann, R. Schmid, A. Sorokin, A. Lapidus, and M.Hecker. 1997. Expression of a stress- and starvation-induced dps/pexB ho-mologous gene is controlled by the alternative sigma factor sB in Bacillussubtilis. J. Bacteriol. 179:7251–7256.

7. Benson, A. K., and W. G. Haldenwang. 1992. Characterization of a regula-tory network that controls sB expression in Bacillus subtilis. J. Bacteriol.174:749–757.

8. Benson, A. K., and W. G. Haldenwang. 1993. The sB-dependent promoter ofthe Bacillus subtilis sigB operon is induced by heat shock. J. Bacteriol.175:1929–1935.

9. Boylan, S. A., A. R. Redfield, M. S. Brody, and C. W. Price. 1993. Stress-induced activation of the sB transcription factor of Bacillus subtilis. J. Bac-teriol. 175:7931–7937.

10. Boylan, S. A., A. R. Redfield, and C. W. Price. 1993. Transcription factor sB

of Bacillus subtilis controls a large stationary-phase regulon. J. Bacteriol.175:3957–3963.

11. Boylan, S. A., M. D. Thomas, and C. W. Price. 1991. Genetic method to

identify regulons controlled by nonessential elements: isolation of a genedependent on alternate transcription factor sB of Bacillus subtilis. J. Bacte-riol. 173:7856–7866.

12. Derre, I., G. Rapoport, and T. Msadek. 1999. CtsR, a novel regulator ofstress and heat shock response, controls clp and molecular chaperone geneexpression in Gram-positive bacteria. Mol. Microbiol. 31:117–132.

13. Desaizieu, A., U. Certa, J. Warrington, C. Gray, W. Keck, and J. Mous. 1998.Bacterial transcript imaging by hybridization of total RNA to oligonucleotidearrays. Nat. Biotechnol. 16:45–48.

14. Engelmann, S., and M. Hecker. 1996. Impaired oxidative stress resistance ofBacillus subtilis sigB mutants and the role of katA and katE. FEMS Microbiol.Lett. 145:63–69.

15. Gaidenko, T. A., and C. W. Price. 1998. General stress transcription factor sB

and sporulation transcription factor sH each contribute to survival of Bacil-lus subtilis under extreme growth conditions. J. Bacteriol. 180:3730–3733.

16. Hecker, M., W. Schumann, and U. Volker. 1996. Heat-shock and generalstress response in Bacillus subtilis. Mol. Microbiol. 19:417–428.

17. Hecker, M., and U. Volker. 1998. Non-specific, general and multiple stressresistance of growth-restricted Bacillus subtilis cells by the expression of thesB regulon. Mol. Microbiol. 29:1129–1136.

18. Hilden, I., B. N. Krath, and B. Hove-Jensen. 1995. Tricistronic operonexpression of the genes gcaD (tms), which encodes N-acetylglucosamine1-phosphate uridyltransferase, prs, which encodes phosphoribosyl diphos-phate synthetase, and ctc in vegetative cells of Bacillus subtilis. J. Bacteriol.177:7280–7284.

19. Huang, X., K. L. Fredrick, and J. D. Helmann. 1998. Promoter recognitionby Bacillus subtilis sW: autoregulation and partial overlap with the sX regu-lon. J. Bacteriol. 180:3765–3770.

20. Huang, X., and J. D. Helmann. 1998. Identification of target promoters forthe Bacillus subtilis sX factor using a consensus-directed search. J. Mol. Biol.279:165–173.

21. Huang, X., A. Gaballa, M. Cao, and J. D. Helmann. 1999. Identification oftarget promoters for the Bacillus subtilis extracytoplasmic function s factor,sW. Mol. Microbiol. 31:361–371.

22. Igo, M., M. Lampe, C. Ray, W. Schafer, C. P. Moran, Jr., and R. Losick.1987. Genetic studies of a secondary RNA polymerase sigma factor in Ba-cillus subtilis. J. Bacteriol. 169:3464–3469.

23. Kruger, E., and M. Hecker. 1998. The first gene of the Bacillus subtilis clpCoperon, ctsR, encodes a negative regulator of its own operon and other classIII heat shock genes. J. Bacteriol. 180:6681–6688.

24. Kunst, F., N. Ogasawara, I. Moszer, A. M. Albertini, and A. Danchin. 1997.The complete genome sequence of the Gram-positive bacterium Bacillussubtilis. Nature 390:249–256.

25. Majumdar, D., Y. J. Avissar, and J. H. Wyche. 1991. Simultaneous and rapidisolation of bacterial and eukaryotic DNA and RNA: a new approach forisolating DNA. BioTechniques 11:94–101.

26. Mizuno, M., S. Masuda, K. Takemaru, S. Hosono, T. Sato, M. Takeuchi, andY. Kobayashi. 1996. Systematic sequencing of the 283 kb 210°–232° region ofthe Bacillus subtilis genome containing the skin element and many sporula-tion genes. Microbiology 142:3103–3111.

27. Ray, C., R. E. Hay, H. L. Carter, and C. P. Moran, Jr. 1985. Mutations thataffect utilization of a promoter in stationary-phase Bacillus subtilis. J. Bac-teriol. 163:610–614.

28. Scharf, C., S. Riethdorf, H. Ernst, S. Engelmann, U. Volker, and M. Hecker.1998. Thioredoxin is an essential protein induced by multiple stresses inBacillus subtilis. J. Bacteriol. 180:1869–1877.

29. Stulke, J., R. Hanschke, and M. Hecker. 1993. Temporal activation of beta-glucanase synthesis in Bacillus subtilis is mediated by the GTP pool. J. Gen.Microbiol. 139:2041–2045.

30. Tatti, K. M., and C. P. Moran, Jr. 1984. Promoter recognition by sigma-37RNA polymerase from Bacillus subtilis. J. Mol. Biol. 175:285–297.

31. Volker, U., S. Engelmann, B. Maul, S. Riethdorf, A. Volker, R. Schmid, H.Mach, and M. Hecker. 1994. Analysis of the induction of general stressproteins of Bacillus subtilis. Microbiology 140:741–752.

32. Volker, U., B. Maul, and M. Hecker. 1999. Expression of the sB-dependentgeneral stress regulon confers multiple stress resistance in Bacillus subtilis. J.Bacteriol. 181:3942–3948.

33. Wetzstein, M., U. Volker, J. Dedio, S. Lobau, U. Zuber, M. Schiesswohl, C.Herget, M. Hecker, and W. Schumann. 1992. Cloning, sequencing, andmolecular analysis of the dnaK locus from Bacillus subtilis. J. Bacteriol.174:3300–3310.

5724 PETERSOHN ET AL. J. BACTERIOL.

on March 15, 2020 by guest

http://jb.asm.org/

Dow

nloaded from