The Bacillus subtilis genome comprises two paralogous single-stranded DNA binding protein [SSB] genes, ssb and ywpH, which show distinctexpression patterns . The main ssb gene is strongly expressedduring exponential growth and is coregulated with genes encodingthe ribosomal proteins S6 and S18 . The gene organization rpsF-ssb-rpsRas observed in B . subtilis is found in many gram-positive aswell as some gram-negative bacteria, but not in Escherichiacoli . The ssb gene is essential for cell viability, and likeother SSBs its expression is elevated during SOS response . Incontrast, the paralogous ywpH gene is transcribed from its ownpromoter at the onset of stationary phase in minimal mediumonly . Its expression is ComK dependent and its gene product is required for optimal natural transformation.

 
Single-stranded DNA binding proteins [SSBs] in bacteria playcrucial roles in DNA replication, repair, and recombinationprocesses . The function of SSB in these processes, its biochemicalproperties, and its interaction with other proteins in the cellhave been studied extensively in Escherichia coli and severalbacteriophages [17, 26] . Relatively little is known about the regulation of SSB expression.

In Escherichia coli the ssb gene is preceded by three promoters,one of them being inducible by DNA damage [3, 4] . The DNA damageinducibility is due to the presence of a LexA binding site inthe upstreammost promoter . Interestingly, this SOS-box is sharedwith the divergently transcribed uvrA gene, coding for the Asubunit of the exonuclease ABC, which is involved in DNA repair[3] . The same organization was found for the uvrA and ssb genes in Sinorhizobium meliloti [24] . Although this gene organizationis also identical in Proteus mirabilis and Serratia marcescens,the ssb genes of these bacteria are not inducible by DNA damage[7] . It has been suggested that E . coli SSB negatively autoregulatesits own translation, because it is capable of binding to itsown mRNA, in this way inhibiting translation [21].

Two paralogous genes coding for SSB were found in the Bacillus subtilis genome, ssb and ywpH . The deduced amino acid sequencesof SSB and YwpH show 80% similarity and 63% identity . Notably,YwpH is lacking 66 amino acid residues of the C terminus of SSB . Although the amino acid sequences of bacterial SSBs are highly conserved within the first two thirds of the protein containing the DNA-binding domains, they diverge substantiallyin the C-terminal third region [7] . The C-terminal region of E . coli SSB is not required for DNA binding in vitro, but is essential for its in vivo function [6, 29] . In contrast to E.coli, neither of the B . subtilis ssb genes is organized adjacentto uvrA as the ssb gene is in E . coli . The first one, ssb, mapsat 358.6^o of the B . subtilis genome and is flanked by the rpsF and rpsR genes, coding for the ribosomal proteins S6 and S18, respectively [Fig . 1A] . A rho-independent transcriptional terminatoris situated downstream of the rpsR gene, and possibly rpsF,ssb, and rpsR belong to one operon . The second ssb-like gene,ywpH, maps at 319.4^o and is flanked by a gene of unknown function[ywpG] and the glcR gene, coding for a regulator involved incarbon catabolite repression [23] [Fig . 1B] . Between these genes,no obvious terminator structure could be identified.

 
In this paper we studied the transcriptional regulation of thetwo ssb-like genes in B . subtilis and address the question of why there are two SSBs in this organism.

 
Bacterial strains, medium, and growth conditions. Table 1 lists the bacterial strains and plasmids used in this study . Bacteria were grown at 37°C under vigorous agitationin rich medium [TY [1% tryptone, 0.5% yeast extract, 1.0% NaCl]or BFA [16] when appropriate] or minimal medium [22] . For the selection of transformants, appropriate antibiotics were addedto the growth media at the following concentrations: for B.subtilis, 5 µg of chloramphenicol per ml, 10 µgof kanamycin per ml, and 100 µg of spectinomycin per ml;for E . coli, 100 µg of ampicillin per ml and 150 µgof spectinomycin per ml . To visualize -amylase activity, TYplates were supplemented with 1% starch, and to visualize LacZactivity plates were supplemented with 0.004% 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside [X-Gal].

 
Strain constructions and transformation. Cloning and transformation were performed according to establishedtechniques [5], [20] and suppliers' manuals . The nucleotide sequences of the primers used for PCR are listed in Table 2. Enzymes were from Roche Molecular Biochemicals [Mannheim, Germany].

 
Upstream regions of the ssb and ywpH genes were amplified by PCR with Pwo DNA polymerase and chromosomal DNA of B . subtilis 168 as the template . The following fragments were amplified: fragment S1 [primers ssb-1 and ssb-2; from 295 bp upstream to11 bp downstream of the start codon of ssb], S2 [primers rps-1, ssb-2; from 602 bp upstream to 11 bp downstream of the startcodon of ssb], R1 [primers rps-1, rps-2; from 274 bp upstreamto 50 bp downstream of the start codon of rpsF], Y1 [primersywpH-1, ywpH-2; from 282 bp upstream to 10 bp downstream ofthe start codon of ywpH], and Y2 [primers ywpG-1, ywpG-2; from369 bp upstream to 50 bp downstream of the start codon of ywpG].These PCR fragments were cloned into the SmaI-digested promoter-screening vector pBTK2 [15] . The resulting plasmids carrying the insertin the correct orientation were linearized and transformed intoB . subtilis 168, selecting for kanamycin-resistant transformants.The transformants were screened for an amylase-deficient phenotypeto confirm that the construct had integrated in the amyE locus.

The ywpH deletion mutant was constructed as follows . A 1.493-bp DNA fragment containing the ywpH gene flanked by 486 bp of upstream sequence and 665 bp of downstream sequence was amplified byPCR with primers X-ywpH-3 and H-ywpH-4 and chromosomal DNA ofB . subtilis 168 as a template . The amplicon was cloned intoPvuII-digested plasmid pIC408, a pUC derivative carrying a spectinomycinresistance gene [M . Steinmetz, unpublished data] . Subsequently,a 150-bp internal fragment of ywpH was deleted in the resultingplasmid by PvuII and SmaI digestion and replaced with the 1.34-kb PvuII fragment of pUC7c [11] containing the chloramphenicolresistance marker . The resulting plasmid was transformed intoB . subtilis 168 and BIV12 . Transformants were selected for chloramphenicolresistance and subsequently screened for spectinomycin sensitivity,indicating the successful disruption of ywpH due to a double-crossoverevent.

The comK disruption mutant was constructed by transforming B. subtilis BIV12 carrying the Y1-lacZ fusion with chromosomal DNA of strain BV2004 carrying a spectinomycin cassette integrated into the comK gene [12] . The resulting spectinomycin- and chloramphenicol-resistantstrain was designated BIV24 and used for transcription studies.

ß-Galactosidase activity assay. To assay ß-galactosidase activities, overnight cultureswere diluted in fresh medium and samples were taken at differentintervals for optical density readings at 600 nm [OD₆₀₀] andß-galactosidase activity determinations . The ß-galactosidaseassay and the calculation of ß-galactosidase units[Miller units] were performed as described by Miller [18].

Northern blot analysis. Total RNA of B . subtilis was isolated from cultures grown inTY, BFA, or MM . Cells were harvested at hourly intervals, from3 h before until 3 h after transition point [T], and RNA wasextracted as previously described [12]; 1 µg of totalRNA was separated by formaldehyde-agarose gel electrophoresisand blotted onto a nylon membrane . This membrane was hybridizedwith digoxigenin-labeled probes detecting transcripts containingyyaF, rpsF, ssb, or ywpH, respectively . Probes were constructedby inserting internal fragments of the coding regions of yyaF,rpsF, ssb, or ywpH in the multiple cloning site of the pBluescriptSK[-] plasmid . Subsequently digoxigenin-labeled antisense RNA probeswere transcribed in vitro with the T7 or T3 RNA polymerase sitepresent in this plasmid . In vitro RNA labeling, hybridization,and signal detection were carried out according to the manufacturer's instructions [DIG Northern starter kit; Roche Diagnostics, Mannheim, Germany].

Induction of DNA damage. Cells of the strains carrying either the S1- or the R1-lacZfusion were grown in TY and cells carrying the Y1-lacZ fusionwere grown in MM . After they reached an OD₆₀₀ of about 0.1,the cultures were divided and mitomycin C was added to one portionof the culture at a final concentration of 100 ng/ml and theexpression of ssb and ywpH was monitored.

Assessment of competence. To test the involvement of YwpH in natural competence a deletionwas introduced into the ywpH gene of B . subtilis and transformabilityassays were performed as described previously [5].

 
Transcription analysis of the ssb and ywpH genes. To study the expression of the ssb and ywpH genes, transcriptional fusions of the potential promoter-containing fragments witha promoterless lacZ were constructed and integrated into the B . subtilis chromosome at the site of amyE.

Strains BIV7 [S1], BIV17 [S2], BIV8 [R1], BIV12 [Y1], and BIV13 [Y2] containing the different lacZ fusions, schematically depicted in Fig . 1, were screened on rich [TY] and minimal medium [MM]agar plates containing X-Gal for blue or white phenotypes . This revealed promoter activity only for the constructs in strains BIV17, BIV8, BIV12, and BIV13, but not in BIV7 . In BIV12 andBIV13, promoter activity was detected only in MM, indicatinga medium-dependent expression of the genes ywpG and ywpH [Fig. 1].

No promoter activity could be detected when the 295 bp immediately upstream of the ssb start codon [S1] were used to drive lacZ expression [Fig . 1] . However, strong promoter activity was detectedwith the S2 fragment containing the complete rpsF gene and 274bp upstream of the rpsF start codon . Apparently, ssb and rpsFare part of one operon, which presumably also includes rpsR.This is confirmed by the fact that promoter activity was alsofound from the smaller R1 fragment comprising the 274 bp upstreamof the rpsF start codon only . In contrast, ywpH was found tobe transcribed from a promoter directly upstream of the gene.

In order to study the expression pattern of ssb and ywpH in more detail, strains containing the S1-, R1-, and Y1-lacZ fusions [BIV7, BIV8, and BIV12, respectively] were grown in TY and MM and ß-galactosidase activity was examined . No expressioncould be detected from the S1 fragment under the conditionsemployed [data not shown], but the ssb operon appeared to bestrongly transcribed from the rpsF promoter in both rich andminimal media . The highest values [between 200 and 300 Millerunits per OD] were reached during exponential growth [Fig . 2A and B].After the transition point [T] between logarithmic growthand stationary growth, transcription from the rpsF promoterdecreased . There is still transcription in the stationary phaseat a two- to fourfold lower level . These results are in agreementwith the higher need for SSB protein for DNA replication infast-growing and thus frequently dividing cells . In contrast,no expression of the ywpH gene could be detected in cells ofB . subtilis BIV12 grown in TY and only low expression was observedin exponentially growing cells in MM . However, expression ofywpH was strongly induced when cells entered the stationaryphase and reached its highest level after the transition point[Fig . 3].

 
Northern blot analysis. For a more detailed picture of the transcription of ssb andywpH, Northern blots with probes for yyaF [upstream of rpsF],rpsF, ssb, and ywpH were performed . Upstream of ssb two promoters are present, a promoter upstream of rpsF and a promoter upstream of yyaF . Downstream of rpsR a terminator structure is present.Transcription from these two promoters would result in two mRNAfragments . A transcript containing only rpsF-ssb-rpsR of 1.2kb and a transcript also containing yyaF of 2.4 kb [Fig. 1A].Figure 4A shows fragment sizes of 2.4 and 1.3 kb with the rpsFprobe, which correspond to the expected transcripts . With thessb probe, a smaller fragment of about 0.9 kb is also visible.Since no promoter activity was detected directly upstream ofssb, this fragment is likely to be caused by selective cleavageor degradation of the mRNA leading to the removal of rpsF fromthe ssb-rpsR fragment . The size of such a fragment would be0.85 kb, which corresponds to the size observed.

 
In MM ssb and rpsF are transcribed highly during logarithmic growth from their own promoter, and after the transition point they are also transcribed from the yyaF promoter . The results from both the ß-galactosidase assay and the Northernblot analyses suggest that ssb is cotranscribed and coregulatedwith genes coding for ribosomal proteins, thereby coupling theregulation of protein synthesis to DNA metabolism.

In rich medium yyaF is transcribed during log phase and its transcription stops at the onset of stationary phase [Fig . 4A]. However, in minimal medium yyaF is highly transcribed after the transition point [Fig . 4B] . The RNA fragment that hybridizeswith the yyaF probe corresponds to the upper band visible inboth the rpsF and ssb blots . This indicates that transcriptionfrom the yyaF promoter continues into the rpsF-ssb-rpsR operon,causing additional transcription of this operon from the yyaFpromoter . In MM the transcription of rpsF, ssb, and rpsR istherefore boosted from the yyaF promoter, which itself is shownto be ComK dependent by means of DNA array analysis [2, 12, 19] . Two of these studies also showed the transcription of rpsFand ssb to be ComK dependent . Our results show that it is likelythat this observation is caused by readthrough from the yyaFpromoter rather than direct regulation of the rpsF promoterby ComK . The apparent transcription of yyaF in rich medium duringlate logarithmic phase indicates multiple modes of transcriptionregulation of this gene.

The transcription of ywpH in TY is virtually absent, but two clear mRNA fragments are present when the cells were grown in MM [Fig . 4C] . The fragments observed have sizes of 2.4 and 1.4 kb . The 1.4-kb band corresponds to a transcript containing ywpH and glcR . The longer fragment observed at time points 2 and 4 of 2.4 kb is likely to be a result of readtrough into the downstream gene ywpJ due to an enhanced transcription rate. During logarithmic growth a small amount of ywpH containing mRNA is detected, and when the cells reach stationary growththe transcription of ywpH increases.

In conclusion, the two ssb-like genes obviously show opposite expression patterns, one being expressed to the highest level during exponential growth and the other one being expressedduring the stationary phase in MM, indicating a distinct functionof their gene products . These results should be interpretedwith care, since no direct protein concentration measurementswere carried out.

ssb gene is controlled by the SOS response. It has been reported that the ssb gene of E . coli is induced by DNA damage [3] . However, its DNA damage inducibility is stilla matter of discussion [17] . E . coli SSB is supposed to be involvedin the induction of the SOS response by promoting RecA dependentcleavage of LexA [17] . Likely SSB serves a similar role in B.subtilis . Since B . subtilis has two paralogues of SSB that areexpressed under different growth conditions, we wondered whetherone or both of these SSBs could be induced by DNA-damaging agentstoo.

When DNA damage was induced by the addition of mitomycin C tothe growth medium, an increase of expression of the ssb genefrom the rpsF promoter was observed, starting about 1.5 h afterthe addition of mitomycin C . A maximum of about threefold elevated expression was reached at 2.5 to 3 h after addition of mitomycinC [Fig . 5] . The observed increase of expression is similar to the induction level observed in E . coli, which is very slow compared to that of other genes in the recA-lexA SOS regulon.Even in the presence of mitomycin C there was no promoter activitydetectable from the fragment directly upstream of ssb [strainBIV7], confirming the absence of an additional ssb promoter[data not shown] . These results suggest the involvement of theribosomal proteins S6 and perhaps also S18 in the SOS response of B . subtilis, because their expression is subject to the same control, which mediates the SOS response . Several genes under the control of DinR, the B . subtilis LexA homologue, have been identified, and a consensus sequence for its binding to DNAwas proposed [30] . However, no such target sequence could be found in the regulatory region of the rpsF gene . Therefore, its SOS-dependent induction might be indirect . From the promoter of ywpH no significant mitomycin C-dependent induction could be detected [data not shown].

 
ywpH gene is regulated by ComK and required for optimal competence. In the wild-type strain induction of ywpH expression in MM wasobserved when exponential growth ceased [Fig . 3] . ywpH expressionreached its maximum 2 h after the onset of stationary phase.This pattern for the regulation of ywpH is similar to that ofthe late competence genes in B . subtilis . The development ofnatural genetic competence is a typical postexponential featureof B . subtilis and occurs in response to certain growth conditionssuch as amino acid limitation in the presence of high glucoseconcentrations and at high cell density [for reviews see references8 and 10] . The expression of late competence genes is activatedby ComK [28] . An important step in transformation is recombination,and stable single-stranded DNA is required in this process.

To test the possibility that ywpH is regulated by ComK, we introduced a comK disruption in B . subtilis BIV12, carrying the Y1-lacZ fusion . In this comK null mutant expression of ywpH was totallyabolished [Fig . 3], which indicates that ywpH belongs to thelate competence genes controlled by ComK . In the course of thisstudy we and others also showed by means of transcription analysisof several B . subtilis comK deletion strains that the transcriptionof ywpH is indeed ComK dependent [2, 12, 19].

We tested the involvement of YwpH in competence . When ywpH was disrupted the transformation efficiency dropped approximately 50-fold, although not to zero . In the course of this study Oguraet al . and Berka et al . observed the effect of a ywpH disruption on competence within a similar order of magnitude . Competenceis not completely annulled in a ywpH mutant . This might be dueto the presence of the ssb gene product which might be ableto substitute for YwpH, although only partially.

This result demonstrates an important role of the ywpH gene product in competence . In vitro experiments with mutant proteinsof the E . coli SSB revealed that the C terminus is not required for DNA binding [29] . Moreover, the truncated SSB is functionalin promoting RecA protein-dependent homologous pairing and strandexchange in vitro [9] . Although YwpH is lacking the C terminuspresent in SSB, it is likely to be able to bind single-strandedDNA and to allow DNA recombination . However, YwpH is probablynot able to replace SSB in DNA replication and/or DNA repair,since the C terminus is required for in vivo function [6] . Inagreement with that, the ssb gene was found to be essential for viability in B . subtilis [13], whereas the ywpH gene couldbe knocked out without consequences for bacterial growth underthe employed conditions [data not shown] . Likely YwpH is involvedin homologous DNA recombination processes, which are necessaryfor acquiring foreign DNA in competent cells of B . subtilis.Presumably SSB also can participate in competence-related recombination,since although transformability is greatly reduced, an ywpHknockout strain can still be transformed.

Organization of ssb genes in other bacteria. The ssb gene organization in B . subtilis differs from the organization observed in E . coli . We therefore addressed the question of how common this gene organization is in other bacterial species. For that purpose, genomes of bacteria were screened for SSB homologues and their gene organization with the NCBI database[http://www.ncbi.nlm.nih.gov] . At this moment 87 complete sequencesof bacterial genomes are available, including 69 different species.In all genomes, one or more genes coding for an SSB homologuewere found . While only 15 species show the same gene order asin E . coli with an ssb gene divergently situated to the uvrAgene, 35 species show the ssb gene flanked by the rpsF and rpsRgenes as it is observed for B . subtilis . These 35 species include all gram-positive bacteria sequenced until now, as well as representatives of the Thermotogales and Thermus/Deinococcus group, and gram-negativespecies from the phyla Spirochaetales, Aquificales, Thermotogales,and Chlorobi and the epsilon subdivision of the proteobacteria.All other proteobacteria and representatives of the Chlamydiales,Cyanobacteria, and Fusobacteria do not possess this gene organization.On the basis of the ssb gene and number of ssb paralogues per species organization, the sequenced bacteria can be classifiedin four different groups . An overview is given in Table 3.

 
Group I contains bacteria with the same ssb gene organization as B . subtilis, rpsF-ssb-rpsR . Most bacteria within this group are Gram positive . All bacteria in this group contain multiple SSB paralogues . Group II contains bacteria with the same organization as B . subtilis, rpsF-ssb-rpsR, but they do not possess multiple ssb paralogues . In addition to some gram-positive bacteria, this group also contains bacteria from the epsilon subdivision of the proteobacteria [Helicobacter pylori and Campylobacter jejuni], Ureaplasma urealyticum, and Borrelia burgdorferi . GroupIII contains bacteria with the same gene organizations as E. coli; ssb is divergently located to uvrA . Ralstonia solanacearum,Salmonella enterica serovar Typhimurium, Shewanella oneidensis,Pseudomonas aeruginosa, and Pseudomonas putida are also classifiedwithin this group . They have one gene located between uvrA andssb . All bacteria in this group contain only one ssb gene intheir chromosome . Furthermore, all these bacteria belong tothe alpha or gamma subdivision of the proteobacteria [gram negative].Group IV contains bacteria with ssb neither placed between rpsF and rpsR nor divergently located to uvrA . Most of these bacteria contain one ssb gene, but Nostoc sp . strain PCC 7120 and Xylellafastidiosa contain multiple ssb genes.

Since we propose a functional division of the gene productsfrom both SSB paralogues, one being the main SSB and the otherbeing competence related, we checked whether the occurrenceof multiple SSB homologues in a bacterial genome could be relatedto natural competence . Most bacteria known to be naturally transformable[14] are classified in group I, which contains species withmultiple ssb homologues . In group IV only Nostoc sp . strainPCC 7120 is known to be naturally transformable, and this speciesalso contains multiple SSBs . The only bacteria that are knownto be naturally competent but do not contain multiple ssb genesare H . pylori and C . jenuni [14] . These bacteria are both membersof the epsilon subdivision of the proteobacteria.

To further investigate if the identified groups can also be divided on the basis of their evolutionary descent, we usedall 69 SSB protein sequences to calculate a phylogenetic tree[Fig. 6] . The tree shows a clear grouping of groups I and II compared to group III . All the proteobacteria in group IV cluster with the other proteobacteria [group III], but the cyanobacteria, Chlamydiales, and Fusobacterium nucleatum cluster with the gram-positivebacteria from groups I and II . Xanthomonas citri [proteobacteria,gamma subdivision] also clusters outside of the proteobacteriagroup . Likely this is caused by horizontal gene transfer . Inthis tree the species from groups I and II are not separatedbased on their SSB homologies . These results were expected becauseit is unlikely that the development of the extra SSBs is directlycorrelated to the sequence of a bacterium's main SSB.

 
Next to the well-studied ssb gene organization observed in E. coli and some other gram-negative bacteria, the gene organization rpsF-ssb-rpsR observed in B . subtilis is a suitable model forthe study of ssb, since it is found in all gram-positive speciessequenced until now and several gram-negative bacteria . Moreover,additional ssb genes are found associated with this configuration,frequently in correlation with natural competence.

 
We thank Wiep Klaas Smits for the gift of strain BV2004 andSacha van Hijum for excellent technical assistance in the constructionof the phylogenetic tree . Piet Nuijten and Johan van den Boschare acknowledged for valuable discussions.

This work was supported by Intervet International B.V . [Boxmeer, The Netherlands].

 
* Corresponding author . Mailing address: Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, Kerklaan 30, NL-9751 NN Haren, The Netherlands . Phone: 31 50 3632093 . Fax: 31 50 3632348 . E-mail: o.p.kuipers@biol.rug.nl.

C.L . and R.N . contributed equally to this article.

Present address: FCDF Corporate Research, 7418 BA Deventer,The Netherlands.

Present address: PURAC Biochem, 4200 AA Gorinchem, The Netherlands.

^¶ Present address: Sir William Dunn School of Pathology, Universityof Oxford, Oxford OX1 3RE, United Kingdom.

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