Regulation and Physiological Significance of ClpC and ClpP in Streptococcus mutans

Tolerance of environmental stress, especially low pH, by Streptococcusmutans is central to the virulence of this organism. The ClpATPases are implicated in the tolerance of, and regulation ofthe response to, stresses by virtue of their protein reactivationand remodeling activities and their capacity to target misfoldedproteins for degradation by the ClpP peptidase. The purposeof this study was to dissect the role of selected clp genesin the stress responses of S. mutans, with a particular focuson acid tolerance and adaptation. Homologues of the clpB, clpC,clpE, clpL, clpX, and clpP genes were identified in the S. mutansgenome. The expression of clpC and clpP, which were chosen asthe focus of this study, was induced at low pH and at growthabove 40°C. Inactivation of ctsR, the first of two genesin the clpC operon, demonstrated that CtsR acts as a repressorof clp and groES-EL gene expression. Strains lacking ClpP, butnot strains lacking ClpC, were impaired in their ability togrow under stress-inducing conditions, formed long chains, aggregatedin culture, had reduced genetic transformation efficiencies,and had a reduced capacity to form biofilms. Comparison of two-dimensionalprotein gels from wild-type cells and the ctsR and clpP mutantsrevealed many changes in the protein expression patterns. Inparticular, in the clpP mutant, there was an increased productionof GroESL and DnaK, suggesting that cells were stressed, probablydue to the accumulation of denatured proteins.

INTRODUCTION

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Introduction

In order to survive adverse environmental conditions, bacteriaundergo a complex program of differential gene expression thatleads to the transient induction of a subset of proteins thatprotect the cell from damage caused by the accumulation of unfoldedand misfolded proteins (22). Most of these polypeptides, alsoknown as heat shock proteins (HSPs), are molecular chaperonesor proteases that play crucial roles in promoting protein folding,assembly, and degradation during normal growth and, in particular,under stress-inducing conditions (5, 13). The HSP100/Clp ATPasesconstitute a large family of closely related proteins that performimportant housekeeping functions, including protein reactivationand remodeling activities typical of molecular chaperones, andtarget specific proteins for degradation by the ClpP peptidase(35, 40). The number of identified members of the Clp familyhas significantly increased in the past few years (33). Thesehighly conserved and ubiquitously distributed proteins are classifiedon the basis of the presence of one or two ATP-binding domainsand the occurrence of specific signature sequences (33). Theclass 1 Clp proteins, such as ClpA, ClpB, and ClpC, have twoATP-binding domains and are distinguished by the length of thespacer regions separating the ATP-binding domains, whereas theclass 2 Clp proteins (such as ClpX and ClpY) display a singledomain. Although designated a Clp protein, for caseinolyticprotease, ClpP itself is unrelated to the Clp ATPase familyand has only peptidase activity. However, when ClpP associateswith members of the Clp ATPase family, it acts as a serine proteaseand prevents the accumulation of altered proteins that mightbe toxic for the bacteria under stress-inducing conditions (25).

Despite their ubiquity and high degree of conservation, mechanismsfor the regulation of heat shock genes in bacteria are fairlydiverse. In Escherichia coli, transcription of the heat shockregulon requires either the ${sigma}$ ³² subunit of RNA polymerase orthe minor sigma factor ${sigma}$ ^E (4). It was demonstrated that the DnaK,DnaJ, and GrpE chaperones play a central role in the controlof the general stress response by targeting ${sigma}$ ³² for degradationby the FtsH protease. In Bacillus subtilis, at least four classesof stress-regulated genes have been identified (10, 16). Genescoding for the ClpP protease and for the ClpC and ClpE ATPasesof B. subtilis are designated as part of the class 3 stressregulon (10, 11, 20). Class 3 stress genes are negatively controlledby CtsR, which is the product of the first gene of the clpCoperon and which binds to a directly repeated heptad that usuallyoverlaps with the transcriptional initiation site or the -35and -10 sequences of the ctsR, clpE, and clpP genes. This targetsequence and the CtsR repressor were also shown to be presentin the regulatory region of clp genes from several gram-positivebacteria, including some Streptococcus species (10).

Streptococcus mutans is recognized as one of the major etiologicalagents of human dental caries (23). In the oral cavity, S. mutanscolonizes the teeth and is able to withstand substantial environmentalinsults, including pH fluctuations, severe carbohydrate restriction,and oxidative stresses. Among the environmental stresses encounteredin dental biofilms, rapid and significant acidification maybe the most extreme challenge. S. mutans is intrinsically moreacid resistant than many oral bacteria and can mount an acidtolerance response (ATR), which allows the organisms to adaptto growth at a low pH. The ATR of S. mutans is characterizedthus far by enhanced glycolytic capacities, increased activityof the proton-translocating F-ATPases, enhanced DNA repair capacity,and acquisition of cross-protection against other stresses (2,14, 15, 31). The scope of the ATR appears significant as assessedby two-dimensional (2-D) gel electrophoresis (36), but few ofthe gene products that appear to be necessary for acid tolerancein S. mutans have been identified and essentially nothing isknown about genetic control of the expression of the ATR.

Our laboratory is primarily interested in dissecting the roleof stress genes in acid tolerance and the regulation of adaptationto acidic conditions. Previously, it was demonstrated that themajor molecular chaperones, DnaK and GroEL, are controlled bythe HrcA repressor and are intimately involved in responsesto environmental acidification (18, 21). In addition, we haveshown that DnaK is essential for cell survival and that reductionin the levels of this chaperone leads to an acid-sensitive phenotype(21). Here, we identified and analyzed the transcriptional regulationof the genes coding for the Clp protease machinery in S. mutans.The expression and functional significance of the S. mutansClpC and ClpP proteins in stress tolerance, competence development,and biofilm formation were also investigated.

MATERIALS AND METHODS

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Materials and Methods

Bacterial strains and growth conditions. E. coli strain DH10B was grown in Luria broth, and S. mutansstrain UA159 was grown in brain heart infusion (BHI) broth.When needed, kanamycin (40 µg ml^-1 for E. coli or 500µg ml^-1 for S. mutans) or ampicillin (100 µg ml^-1)was added to the media. For heat shock of S. mutans, cells weregrown in BHI at 37°C to mid-exponential phase (optical densityat 600 nm [OD₆₀₀] ${cong}$ 0.5) and aliquots of the cultures were thentransferred to a water bath at 42°C for various periodsof time. For studies involving acid shock and acid adaptation,S. mutans was grown in continuous chemostat culture at pH 7.0or 5.0 as previously described (18). To evaluate the impactof the loss of the clpP, clpC, or ctsR gene on stress resistance,the wild-type and mutant strains were grown overnight in BHI,diluted 50-fold into BHI, and incubated at 37°C (control)or at high temperatures (40, 42, or 43°C), at a low pH (pH5.0, 37°C), or at 37°C in the presence of streptomycin(25 µg ml^-1). The ability to form stable biofilms wasassessed by growing the cells in a 96-well (flat-bottom) cellculture cluster (Costar 3595; Corning, Inc., Corning, N.Y.)containing biofilm medium as originally described by Loo etal. (24), with minor modifications for S. mutans developed byBhagwat et al. (3).

DNA methods. Chromosomal DNA was prepared from S. mutans as previously described(6). Plasmid DNA was isolated from E. coli by using QIAgen columns(Qiagen, Chatsworth, Calif.), and restriction and DNA-modifyingenzymes were obtained from Life Technologies, Inc. (Gaithersburg,Md.), New England Biolabs (Beverly, Mass.), or MBI Fermentas(Amherst, N.Y.). PCRs were carried out with 100 ng of S. mutanschromosomal DNA by using Taq DNA polymerase, and PCR productswere purified using the QIAquick kit (Qiagen). DNA was introducedinto S. mutans by natural transformation (30) and into E. coliby electroporation (32). Southern blot analyses were carriedout under high-stringency conditions as detailed by Sambrooket al. (32).

Strain construction. The strains used in this study are listed in Table 1. To constructinsertion mutations in ctsR, clpC, and clpP, DNA fragments,each approximately 1.7 kb in length, containing the genes ofinterest were amplified by PCR and cloned onto pGEM5 (Promega,Madison, Wis.) as SphI-SpeI fragments. The resulting plasmidswere used to insert a kanamycin resistance gene (Kan^r) thatis flanked by strong transcription and translation terminators( ${Omega}$ Kan) (29) into the ctsR, clpC, and clpP genes. A second clpPmutant was generated by the insertion of a nonpolar Kan^r marker(19) to exclude the possibility that the phenotype observedfor the clpP polar insertion was due to polar effects on downstreamgenes. To inactivate ctsR in a way that would allow for theexpression of clpC, which is located immediately downstreamof ctsR, a nonpolar construct with the ${Omega}$ Kan element followedby the Streptococcus salivarius urease promoter (PureI) (8)was used. This approach was successfully used to generate anonpolar insertion in the S. mutans hrcA gene that allowed forexpression of the genes downstream of hrcA in the dnaK operon(21). Plasmids containing the Kan^r gene marker were then isolatedand used to transform S. mutans. Transformants were selectedon BHI agar with kanamycin, and Southern blot analysis was usedto confirm that the gene inactivation desired had occurred asplanned.

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TABLE 1. S. mutans strains used in this study

Assay of genetic competence. Transformation efficiencies of S. mutans UA159 and its derivativeswere determined by preparing competent cells as previously describedand transforming them with plasmid-borne copies of the luxSor brpA (for biofilm regulatory protein A) genes of S. mutansinto which erythromycin antibiotic resistance (Erm^r) markershad been inserted (41). Transformants were enumerated by platingcells on BHI agar containing erythromycin (5 µg ml^-1).

2-D polyacrylamide gel electrophoresis. 2-D electrophoresis was performed at Kendrick Labs, Inc. (Madison,Wis.) by following the protocol described by O'Farrell (28)but using conditions described elsewhere (21). The protein concentrationof the samples was determined by a bicinchoninic acid assay(Sigma, St. Louis, Mo.). Gels containing equal amounts of proteinwere silver stained and dried between sheets of cellophane.

RNA methods. Total RNA was extracted from chemostat-grown cultures of S.mutans or from mid-exponential phase batch cultures by followinga protocol described by Chen et al. (8). Total RNA was separatedon a 1.0% formaldehyde gel as described elsewhere (1). RNAswere UV cross-linked to the membranes, and filter membraneswere probed with internal fragments of the S. mutans clpC, clpE,clpP, ctsR, or groEL gene labeled with [ ${alpha}$ -³²P]dATP (NEN LifeScience, Boston, Mass.). All hybridizations and washes werecarried out under high-stringency conditions. Signals obtainedon autoradiographs were quantified using an IS1000 digital imagingsystem (Alpha Innotech Co., San Leandro, Calif.). Primer extensionswere carried out with the oligonucleotide designated EXTCTSR(5'-CTGACTGGGAACAACAC-3') by a protocol described previously(1), with primer annealing and reverse transcription performedat 42°C.

RESULTS

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Results

Identification of clp genes in S. mutans. The highly conserved amino acid sequence of the Clp proteinsof E. coli, B. subtilis, Streptococcus pneumoniae, and Lactococcuslactis were used to identify putative clp genes in the S. mutansgenome by using resources available at the University of Oklahoma'sAdvanced Center for Genome Technology. A total of five openreading frames (ORFs) were identified within the S. mutans genome.BLAST search analysis of the deduced amino acid sequences ofthe five ORFs indicated high degrees of similarity to ClpB,ClpC, ClpE, ClpL, and ClpX from other organisms and, therefore,the genes were designated clpB, clpC, clpE, clpL, and clpX.The structural organization and location of the clpB, clpE,clpL, and clpX genes in the chromosome are shown in Fig. 1,and the clpC operon organization is shown in Fig. 2. We alsotook advantage of the complete sequence of the S. mutans genometo identify the clpP gene and the gene coding for the putativerepressor of clp gene expression in gram-positive bacteria,ctsR. In order to begin investigating the CtsR regulon in S.mutans, we began a more detailed analysis of the ctsR, clpC,and clpP genes. The clpC gene (2,373 bp in length), which islocated at nucleotides 1899196 to 1901634 in the S. mutans genome,encoded a polypeptide with a predicted molecular mass of 87.8kDa that shared the highest levels of similarity with a putativeS. pyogenes ClpC (73% identity). Immediately upstream, and overlappingby 1 nucleotide with the clpC start codon, was another ORF of463 bp. The 17.5-kDa product encoded by this ORF showed 47%identity (65% similarity) with the class 3 heat shock gene repressorCtsR from B. subtilis. Finally, elsewhere in the chromosome(positions 1587548 to 1588136), a clpP homologue coding fora predicted 21.6-kDa protein that shared 86% identity with aputative S. pyogenes ClpP and high levels of homology to otherknown ClpP proteins was identified.

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FIG. 1. Schematic diagram of the clpB, clpE, clpL, and clpX loci and flanking regions. ORF1 and ORF3 in the clpB locus encode putative phosphosugar mutase and dihydrolipoamide dehydrogenase proteins, respectively. ORF1 in the clpE locus encodes a putative transposase, and mutT codes for a MutT/nudix family protein. The rplJ and pdxK genes in the clpL locus encode putative 50S ribosomal L10 and pyridoxal kinase proteins, respectively. The dfr gene in the clpX locus is predicted to encode a dihydrofolate reductase protein, and ORF4 encodes a putative GTP-binding protein. ORF2 and ORF4 in the clpE locus, ORF3 in the clpL locus, and ORF2 in the clpX locus code for hypothetical proteins. See the text for more details. Arrows represent the orientation of the genes. The molecular mass, isoelectric points, and nucleotide positions (nt.) in the chromosome of the putative Clp proteins are shown underneath the genes.

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FIG. 2. (A) Genetic organization of the S. mutans ctsR/clpC operon and flanking regions. The tsf gene is predicted to encode an elongation factor, and ftf encodes a levansucrase precursor. Arrows indicate the direction of transcription. The estimated molecular masses from deduced amino acid sequences are shown below the genes, and the numbers above the schematic indicate the size of the intergenic regions in base pairs. (B) Northern blot analysis of S. mutans UA159 clpC. RNA was isolated from batch- or chemostat-grown cells that were subjected to heat shock (42°C) or acid shock (pH 5.0) (ss [steady state]). Total RNA (10 µg per lane) was separated in a 1.0% gel, transferred to a nylon membrane, and hybridized to a clpC-specific probe.

Transcriptional analysis of the clpC operon. The clpC and ctsR genes were found to be part of a bicistronicoperon. An ORF coding for a protein highly similar to a putativeelongation factor (Tsf) from S. pyogenes was found 5' to ctsR,and a levansucrase precursor from S. mutans GS-5 (ftf [34])was identified 3' to clpC (Fig. 2A). A promoter was mapped byprimer extension (data not shown) 42 bases upstream of the ctsRstart codon with a -10 sequence identical to ${sigma}$ ^A-type promotersand a partially conserved -35 sequence (TTCCAA versus TTGACAof ${sigma}$ ^A-type promoters) located 16 bp upstream of the -10 sequence.

Northern analyses with RNA isolated from the wild-type strainby using ctsR or clpC internal fragments as probes revealeda transcript of approximately 2.9 kb, indicating that ctsR andclpC are cotranscribed from the ${sigma}$ ^A-type promoter located upstreamof ctsR (Fig. 2B). Curiously, a smaller transcript of approximately2 kb in size was detected with the ctsR and clpC probes, butthe size of this transcript does not correlate well with theknown sizes of ctsR and clpC. However, both transcripts wereequally induced after heat and acid stresses (twofold when cellswere subjected to heat shock at 42°C for 15 min and fourfoldafter 30 min following acid shock at pH 5.0). No significantdifferences in the clpC mRNA levels in steady-state cells grownat a pH of either 7.0 or 5.0 were observed (Fig. 2B).

Transcriptional analysis and stress induction profile of clpP. Recently, a ${sigma}$ ^A-type promoter was identified upstream of the clpPgene in S. pneumoniae (7). Analysis of the sequence of the regionupstream of the S. mutans clpP gene revealed a putative ${sigma}$ ^A-typepromoter identical in sequence to the one that was mapped inS. pneumoniae (TTGACC-N₁₇-TATAAT). Upstream from clpP and separatedby 535 bp, an ORF that coded for a protein that shares 93% identitywith an S. salivarius uracil phosphoribosyltransferase (upp)was identified (Fig. 3A). Analysis of the region 3' to the clpPstop codon revealed two ORFs with the highest degree of similarityto those coding for the B. subtilis ylbF and ylbG gene products(35 and 43% identities, respectively). Recently, it was demonstratedthat, in B. subtilis, YlbF and perhaps YlbG are involved insporulation and in competence development by increasing thestability or synthesis of the competence factor ComK at theposttranscriptional level (37).

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FIG. 3. (A) Schematic representation of the S. mutans clpP gene and flanking regions. The upp gene is predicted to encode a uracil phosphoribosyltransferase, and ylbF and ylbG have been implicated in competence development and sporulation in B. subtilis. Arrows indicate the direction of transcription. The estimated molecular masses from deduced amino acid sequences are shown below the genes, and the numbers above the schematic indicate the size of the intergenic regions in base pairs. (B) Northern blot analysis of S. mutans UA159 clpP. RNA was isolated from batch- or chemostat-grown cells that were subjected to heat shock (42°C) or acid shock (pH 5.0) (ss [steady state]). Total RNA (10 µg per lane) was separated in a 1.0% gel, transferred to a nylon membrane, and hybridized to a clpP-specific probe.

Northern blot analysis with RNA isolated from the wild-typestrain was used to demonstrate a stress-inducible transcriptof 0.58 kb, which corresponds to the length of clpP, as wellas two larger transcripts that were not stress inducible butnevertheless corresponded in size to the 16S and 23S ribosomalRNAs (Fig. 3B). It has been demonstrated in other organismsthat ClpP is an HSP whose synthesis can be induced by a varietyof stresses. Densitometric analysis of Northern blots of RNAsfrom batch- or chemostat-grown cells revealed that the levelsof clpP mRNA were increased threefold when cells were subjectedto heat shock at 42°C for 30 min or fourfold after 30 minfollowing acid shock at pH 5.0 (Fig. 3B). Comparisons of theclpP mRNA levels from steady-state cultures at pH 7.0 or 5.0showed a 1.5-fold induction at low pH, similar to what has beenobserved for the dnaK gene (18).

Effects of the inactivation of ctsR, clpC, and clpP on growth under stress-inducing conditions. The clpC, clpP, and ctsR genes were inactivated by insertingthe ${Omega}$ Kan cassette in the 5' portion of the genes to give thestrains listed in Table 1. Although there was strong evidencefrom Northern analyses that clpP is a monocistronic transcript,a second clpP mutant strain (JL-NPclpP) was constructed usinga nonpolar kanamycin cassette to eliminate the possibility thatthe phenotype observed in the clpP mutant strain could be dueto polar effects on the downstream genes. Similarly, a secondCtsR-deficient strain (JL-NPctsR) was constructed with a nonpolarelement that allowed for clpC transcription. Cell morphology,competence development, and stress sensitivities of the polarand nonpolar ctsR and clpP mutants were examined, but no differenceswere observed. Therefore, because we had already conducted adetailed characterization of the nonpolar ctsR mutant (JL-NPctsR)and the polar clpP mutant strain (JL-clpP), these two strainswere used throughout the studies.

Under standard growth conditions, i.e., 37°C in a 5% CO₂atmosphere, the JL-NPctsR and JL-clpC mutant strains grew aswell as the wild-type strain (Fig. 4A). At temperatures above37°C, the wild-type, JL-NPctsR, and JL-clpC strains wereable to grow at temperatures of up to 43°C. However, attemperatures above 40°C, growth rates of the strains werereduced and the cells tended to clump and adhere to the sidesof the glass culture tubes. We also tested the ability of thestrains to grow at pH 5.0 or in the presence of streptomycin(25 µg ml^-1), an antibiotic known to increase mistranslation.Under all those conditions, the JL-NPctsR and JL-clpC strainsdisplayed growth rates comparable to those for the wild type(Fig. 4B, C, and D).

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FIG. 4. Growth curves of S. mutans wild-type ( ${square}$ ), JL-clpC ( ${lozenge}$ ), JL-NPctsR ( ${circ}$ ), and JL-clpP ( ${triangleup}$ ) strains grown at 37°C (A), at 42°C (B), in the presence of streptomycin (25 µg ml^-1) (C), and at pH 5.0 (D). A typical result representative of three independent experiments is shown.

Under standard growth conditions, the clpP mutant grew moreslowly than the wild-type strain but was able to reach a similarOD₆₀₀ before entering stationary phase (Fig. 4A). Although theClpP-deficient strain was able to grow at temperatures of upto 40°C, growth of JL-clpP was impaired at 42°C (Fig.4B) and completely inhibited at 43°C (data not shown). Whengrown at 42°C at pH 5.0 or in the presence of streptomycin(25 µg ml^-1), the lag phase and early log phase of theclpP mutant were comparable to those of the wild-type strain.However, under every stress-inducing condition tested, the cellyields achieved by the clpP mutant strain were significantlylower than those achieved by the wild-type strain (Fig. 4B,C, and D). These results clearly indicated a stress-sensitivephenotype for the clpP mutant strain.

Inactivation of CtsR leads to high-level expression of clpC, clpE, clpP, and groEL. Northern blot analysis with RNA isolated from mid-exponential-phasecultures of CtsR-deficient (JL-NPctsR) and wild-type strainsof S. mutans demonstrated that groEL, clpE, and clpP mRNAs werepresent in greater amounts in the mutant strain (two-, six-,and sixfold increases, respectively) (Fig. 5). It was not possibleto determine the effect of ctsR inactivation on clpC expression,since the transcription from the cognate promoter of clpC inthe ctsR mutant was disrupted by a polar Kan^r marker and clpCwas driven by the S. salivarius urease promoter. The levelsof expression of groEL, clpC, clpE, and clpP mRNA were alsocompared in the wild-type strain and the clpC and clpP mutants.In the JL-clpC strain, the levels of clpE and clpP mRNA werecomparable to what was found for the wild-type strain whereasthe level of groEL mRNA was increased about fourfold (Fig. 5).This finding suggests that ClpC may have a regulatory effecton groEL expression or that the levels of groEL mRNA were elevateddue to the accumulation of damaged proteins that were once targetedby ClpC to the protease machinery. Northern analyses with RNAisolated from the clpP mutant strain grown to mid-log phaseunder normal conditions revealed that levels of groEL, clpC,and clpE mRNA were induced nine-, three-, and sixfold, respectively(Fig. 5). Thus, lack of ClpP also causes an effective inductionof these stress genes.

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FIG. 5. Northern blot analysis of UA159 (WT) and its derivatives. Cells were grown in BHI at 37°C to mid-exponential phase. RNA (10 µg per lane) was isolated from cells and separated in a 1.0% gel, transferred to a nylon membrane, and hybridized to groEL-, clpC-, clpE-, and clpP-specific probes.

Protein expression patterns of clp and ctsR mutants. A 2-D gel approach was used to determine whether there weredetectable changes in the synthesis of the proteome of S. mutansdue to the loss of ClpP (JL-clpP) or CtsR (JL-NPctsR). In theclpP mutant strain, the levels of the molecular chaperones DnaKand GroEL-ES were elevated at least twofold, indicating thatcells were perceiving a stress-inducing condition, probablydue to the accumulation of denatured proteins that are normallydegraded by ClpP. Although the twofold increase of GroEL levelsin the clpP mutant strain does not correlate very well withthe mRNA levels (ninefold increase), it has been observed thatlarge increases in the amount of groEL mRNA are needed to effectsignificant changes in the absolute quantity of this proteinin the cells (21). Additionally, the levels of at least 25 otherproteins were altered (10 up-regulated, 15 down-regulated) inthe clpP mutant compared to those in the wild-type strain growingunder the same conditions (Fig. 6B). When compared to the wild-type2-D pattern, at least 13 proteins were up-regulated in the JL-NPctsRstrain whereas 17 proteins showed reduced synthesis (Fig. 6C).Two protein spots, approximately 22 kDa each, were stronglyexpressed in the JL-NPctsR strain and absent in the JL-clpPstrain. The predicted molecular mass of the S. mutans ClpP proteinis 21 kDa, so perhaps one or both of the two spots is the ClpPprotein.

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FIG. 6. 2-D protein gels of S. mutans strains UA159 (A), JL-clpP (B), and JL-NPctsR (C). Cells were grown in BHI to an OD₆₀₀ of 0.5. The silver-stained proteins that exhibited more obvious differences in the clpP and ctsR mutants in comparison to the wild type are indicated. Proteins with enhanced expression are indicated with arrows and a plus symbol, and proteins with reduced synthesis are indicated by arrows only. The DnaK, GroEL, GroES, and putative ClpP proteins are indicated as such. The filled triangle indicates tropomyosin protein (33 kDa, pI 5.2) loaded as an internal control.

Role of ClpC and ClpP in competence development. To assess the role of the S. mutans ClpC and ClpP proteins incompetence, the efficiency of transformation of the ctsR, clpC,and clpP mutants was evaluated by transforming the strains withconstructs containing an Erm^r marker as detailed in Materialsand Methods. Similar results were obtained regardless of thetransforming DNA. As shown in Table 2, the JL-clpC strain showeda modest although not statistically significant increase intransformation efficiency whereas the inactivation of ctsR hadno effect. When compared to the parental strain, the clpP mutantshowed a fourfold reduction in the number of transformants (Table2).

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TABLE 2. Transformation frequency of S. mutans strains

Role of clp genes in biofilm formation. The capacity of the JL-NPctsR, JL-clpC, and JL-clpP strainsto form biofilms in the wells of 96-well microtiter plates wasevaluated. The data obtained after 24 h of growth indicatedthat the inactivation of clpC and ctsR led to a slight, albeitconsistent, increase in biofilm formation (>9 and >19%,respectively) compared to that in the wild-type strain. Inactivationof clpP resulted in a fivefold reduction in biofilm formation(Fig. 7). To exclude the possibility that the differences observedwere due to variations in the growth capacity of the strains,the final OD₆₀₀ of the cultures in microtiter plates was assessed.All the strains grew equally well over a 24-h period (data notshown), but the clpP strain showed a consistent four- to fivefoldreduction in stable biofilm formation after 24, 48, and 72 h.

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FIG. 7. Biofilm formation by S. mutans UA159 (WT) and its derivatives. Cultures were grown in a 96-well microtiter plate containing biofilm medium at 37°C in 5% CO₂ for 24 h. Absorbance at 575 nm of the crystal violet extracted from the 24-h biofilms with ethanol is indicated. The graph shows the averages and standard deviations from three independent experiments.

DISCUSSION

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Discussion

It is recognized that protein degradation is a critical processthat ensures the optimal functioning of the cell by preventingthe accumulation of denatured proteins or by regulating thestability of regulatory proteins. The Clp protein complex, whichpossesses both proteolytic and chaperone activities, has beenimplicated in stress survival in many organisms. In B. subtilis,mutations in the clpC, clpP, and clpX genes were shown to affectcompetence development, motility, growth at high temperatures,degradative enzyme synthesis, and sporulation (26, 38, 39).In L. lactis, clpE mutants formed smaller colonies on agar mediacontaining puromycin although growth of this mutant as wellas of the ${Delta}$ clpB and ${Delta}$ clpC strains did not seem to be affectedby heat or high concentrations of salt (17). It was also shownthat inactivation of the L. lactis clpP gene results in a stress-sensitivephenotype (12). Finally, in S. pneumoniae, it was shown thatClpP and ClpE are required for thermotolerance but that ClpCis dispensable for growth at a high temperature (7).

In this study, we have identified and characterized the clpCand clpP genes of S. mutans. Additional members of the Clp ATPasefamily, namely, clpB, clpE, clpL, and clpX, were also identifiedin the S. mutans genome, but the roles of these genes in homeostasisand stress tolerance remain to be elucidated. Similar to whathas been observed in other gram-positive organisms, loss ofClpP in S. mutans resulted in a stress-sensitive phenotype.The clpP mutant grew more slowly than the wild-type strain,formed long chains, aggregated, and had an 80% reduction inbiofilm formation. Moreover, the levels of the chaperones DnaK,GroEL, ClpC, and ClpE were elevated in the ClpP-deficient strain,indicating that ClpP deficiency induced a stress response inthe cells, probably as a result of the accumulation of denaturedproteins that are normally targeted to the ClpP protease. Ithas been observed that clp genes play a role in cell division.In B. subtilis, ClpC- and ClpP-deficient strains produce elongatedcells and form long chains, especially under stress-inducingconditions. The longer chains observed in the S. mutans clpPmutant clearly suggest an impairment in cell septation and/orautolysis. However, the precise role of ClpP in cell divisionand autolysis remains to be investigated. In contrast to theClpP-deficient strains, the ClpC-deficient strain was able togrow as well as the wild-type strain and, under certain stress-inducingconditions, the clpC mutant grew slightly faster and reacheda higher yield than the parental strain. Additionally, the clpCmutant did not show alterations in cell morphology, chain length,or biofilm-forming capabilities. Therefore, unlike what wasobserved in B. subtilis, it appears that ClpC does not playa major role in stress tolerance in S. mutans and possibly inother lactic acid bacteria.

In B. subtilis, the cellular concentration of the competencefactor comK has been shown to be controlled by the ClpP andClpC proteolytic machinery, along with the MecA protein, whichacts as an adapter, connecting ComK to ClpC (38, 39). Therehas been a report of a MecA-like gene in S. mutans, but it isnot known whether this gene product has a function similar tothat of B. subtilis MecA (9). By searching the S. mutans databases,we found no evidence of a comK homologue. In S. pneumoniae,it appears that ClpP is responsible for controlling the levelsof the competence genes comC, comD, and comE whereas ClpC doesnot appear to be involved in competence (7). The efficiencyof transformation of the S. mutans clpP, clpC, and ctsR mutantswas determined by the acquisition of an Erm^r marker via homologousrecombination. Compared to the parental strain, the clpP mutantdisplayed a fourfold reduction in the number of transformants.In contrast, there was a relatively small increase in the frequencyof transformation for the JL-clpC and JL-NPctsR strains. Thereduced competence in the S. mutans clpP mutant strain differsfrom the complete absence of transformation in a B. subtilisclpP mutant (26). Notably, the clpP gene is located upstreamof two ORFs, with homologies to the products encoded by ylbFand ylbG of B. subtilis. In B. subtilis, disruption of ylbFleads to a dramatic decrease in the expression of comK (37).There was no evidence of a comK homologue in the S. mutans databases,and a nonpolar clpP mutant strain also showed reduced competence(data not shown). However, we did not analyze the expressionof ylbF and ylbG in the clpP mutant strains and it is possiblethat the observed effects on competence in the clpP mutant aredue, at least in part, to the altered expression of downstreamgenes. In S. pneumoniae, it was shown that ClpP acts as a negativeregulator of comCDE expression under conditions that do notsupport competence development (7). In order to definitivelyaddress whether ClpP has a similar regulatory function in competencedevelopment by S. mutans, further studies to investigate expressionof the S. mutans com genes in the clpP mutant strain are underway.

In gram-positive bacteria, there are at least two differenttranscriptional repressors, HrcA and CtsR, that regulate expressionof heat shock genes (27). In many gram-positive organisms, thelevels of ClpC, ClpE, and ClpP are controlled by the CtsR repressorthat binds to a consensus heptad direct-repeat sequence (A/GGTCAAA-NAN-A/GGTCAAA)located in the regulatory regions of these genes (10). Morerecently, it was demonstrated in S. pneumoniae that the CtsRrepressor binds to DNA elements in the clpC, clpE, clpP, andgroESL promoter regions that differ in sequence from the consensussequence originally suggested for CtsR binding (7). Comparisonsof the regulatory regions of the clpC, clpE, clpP, and groESLgenes from S. pneumoniae and S. mutans disclosed almost identicaltarget sequences for CtsR binding, strongly supporting the ideathat CtsR negatively controls the expression of those genesin S. mutans. The role of CtsR in the regulation of clpP andgroES-EL was confirmed in the ctsR mutant in this study by Northernblotting and 2-D gel electrophoresis. Previously, it was shownthat HrcA is a major regulator of groESL expression (21), andthus this is the second report (7) to demonstrate that the groESLoperon is under dual heat shock control by the HrcA and CtsRrepressors. In addition to GroES-EL and the Clp proteins, whichhave been shown to be repressed by CtsR, proteomic analysisrevealed that levels of an additional 13 proteins were elevatedin the ctsR mutant strain. It is reasonable to speculate thatthe up-regulated proteins in the strain bearing the ctsR mutationmay constitute new members of the CtsR regulon.

It is also interesting to note that the clpP mutant strain showeda reduced capacity to form biofilms. Although the basis forthis phenotype is unknown, we can speculate that ClpP may actby controlling the stability or activity of transcriptionalregulators of biofilm maturation. Another possibility, whichis not mutually exclusive, may be that tolerance of nutrientlimitation or accumulation of growth-inhibitory metabolitesresulting from mass transport limitations in biofilms may requirea functional ClpP for protein turnover. The linkage of stresstolerance with biofilm formation is currently under investigationin our laboratory.

In conclusion, this study describes the genetic and transcriptionalorganization, and begins to shed light on the physiologicalsignificance, of the clp genes of S. mutans. The data obtaineddemonstrate that ClpP plays a critical role in the survivalof common environmental stresses, most likely by preventingthe accumulation of denatured proteins. In contrast, ClpC doesnot appear to be essential, although proteomic analysis revealsthat there is a significant impact of ClpC deficiency on theproteome of S. mutans (data not shown). The disparity betweenthe changes in expression profiles and the lack of apparentstress sensitivity may be explained by compensation for ClpCdeficiency by other chaperones. In addition, CtsR and ClpP maybe part of complex regulatory networks, either through the directrepression of expression or by the modulation of the stabilityof other proteins. Work is in progress to identify the proteinswith altered expression in the ctsR and clpP mutants, with thegoals of revealing members of the CtsR regulon and substratesof the Clp protease and of shedding light on how these geneproducts interact to achieve physiologic homeostasis and stressresistance.

ACKNOWLEDGMENTS

We thank Jacqueline Abranches and Margaret Chen for criticalreading of the manuscript.

This study was supported by grants DE12236 and DE13239 fromthe National Institute for Dental and Craniofacial Research.

FOOTNOTES

* Corresponding author. Mailing address: Department of Oral Biology, University of Florida, College of Dentistry, P.O. Box 100424, Gainesville, FL 32610. Phone: (352) 392-4370. Fax: (352) 392-7357. E-mail: rburne{at}dental.ufl.edu.

REFERENCES

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