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Adaptor Protein MecA Is a Negative Regulator of the Expression of Late Competence Genes in Streptococcus thermophilus Céline Boutry, Astrid Wahl, Brigitte Delplace, André Clippe, Laetitia Fontaine, and Pascal Hols Biochimie et Génétique Moléculaire Bactérienne, Institut des Sciences de la Vie, Université catholique de Louvain, Louvain-la-Neuve, Belgium In Streptococcus thermophilus, the ComRS regulatory system governs the transcriptional level of comX expression and, hence, controls the early stage of competence development. The present work focuses on the posttranslational control of the activity of the sigma factor ComX and, therefore, on the late stage of competence regulation. In silico analysis performed on the S. thermo- philus genome revealed the presence of a homolog of mecA (mecA St ), which codes for the adaptor protein that is involved in ComK degradation by ClpCP in Bacillus subtilis. Using reporter strains and microarray experiments, we showed that MecA St represses late competence genes without affecting the early competence stage under conditions that are not permissive for com- petence development. In addition, this repression mechanism was found not only to act downstream of comX expression but also to be fully dependent on the presence of a functional comX gene. This negative control was similarly released in strains de- leted for clpC, mecA, and clpC-mecA. Under artificial conditions of comX expression, we next showed that the abundance of ComX is higher in the absence of MecA or ClpC. Finally, results of bacterial two-hybrid assays strongly suggested that MecA in- teracts with both ComX and ClpC. Based on these results, we proposed that ClpC and MecA act together in the same regulatory circuit to control the abundance of ComX in S. thermophilus. S treptococcus thermophilus is of major economical value due to its extensive use for the production of yogurt and hard cooked cheese by the dairy industry (3, 21). Recently, S. thermophilus was shown to be able to develop natural competence for transforma- tion, a transitory physiological state enabling the capture and sta- ble integration of naked DNA in the chromosome of this species (2, 14, 15, 18). It is well established that natural competence regulation usu- ally involves a set of regulators, which together form a cascade from the early stage to the late stage of competence development (8, 20). For S. thermophilus, competence was shown to be natu- rally induced when S. thermophilus cells were grown in a chemi- cally defined medium. This induction results in the transitory ex- pression of the comX gene, coding for the master regulator of late competence (com) genes, which are necessary for DNA uptake, protection, and chromosomal integration (14, 18). ComX is an alternative sigma factor ( X ) that binds to the RNA polymerase in order to transcribe specifically the late com genes by recognizing a small sequence (Com box) in their upstream regions (31, 33, 46). Recently, we have shown that the activation of comX expression in S. thermophilus relies on a novel quorum-sensing system, named ComRS (14). The proposed activation model starts with the intra- cellular production of a precursor peptide (pre-ComS), which is then secreted and matured (ComS*) (14, 34). When its concen- tration in the extracellular medium increases and reaches a thresh- old, ComS* binds to the oligopeptide-binding protein AmiA3 and is internalized by the multicomponent oligopeptide transporter AmiCDEF. Inside the cell, ComS* activates the transcriptional regulator ComR. The ComRS complex in turn activates comX and comS transcription, resulting in an autoamplification loop (14). Homologs of the ComRS system were also found in all sequenced genomes of streptococci belonging to the mutans, pyogenic, and bovis groups (34). Furthermore, this system was shown to be functional in Streptococcus mutans as the most proximal regula- tory system for comX activation and competence development (34). Besides the transcriptional control of comX, the ComX protein was shown to be the target of posttranslational control in strepto- cocci. In Streptococcus pneumoniae, the amount of ComX is posi- tively regulated by ComW and negatively controlled by ClpEP and ClpCP. ComW is produced in the early competence phase to sta- bilize and activate ComX. ClpEP and ClpCP are protease machin- eries that degrade ComX and ComW, respectively (32, 36, 42, 46). In Streptococcus pyogenes, the mechanism of ComX stabilization/ degradation is still unknown, except for the implication of the ClpP protease (38). For S. thermophilus, previous extensive in silico analyses did not allow the identification of a homolog of ComW, but during the course of that study, the ClpC ATPase subunit was shown to act as a negative regulator of competence development in this species (1). For Bacillus subtilis, the posttrans- lational control of the master regulator of competence develop- ment, ComK, has also been highlighted (25–27, 30, 36, 37, 40, 43, 45, 47–49). To prevent competence development under inappro- priate conditions, ComK is sequestered by MecA, an adaptor pro- tein which targets ComK to the ClpCP protease system (25, 40, 43, 47–49). ComK is released from the inhibitory complex by the small antiadaptor peptide ComS, which interacts directly with MecA and is produced when the cell density increases, leading to the activation of ComK transcription and of the late com genes (11, 12, 37, 40, 43). B. subtilis also contains a paralog of MecA, YpbH, which binds ClpC and affects both competence and spo- rulation by an unknown mechanism (39). Received 22 December 2011 Accepted 13 January 2012 Published ahead of print 27 January 2012 Address correspondence to Pascal Hols, [email protected]. L.F. and P.H. contributed equally to this work. Supplemental material for this article may be found at http://jb.asm.org/. Copyright © 2012, American Society for Microbiology. All Rights Reserved. doi:10.1128/JB.06800-11 0021-9193/12/$12.00 Journal of Bacteriology p. 1777–1788 jb.asm.org 1777 on May 5, 2018 by guest http://jb.asm.org/ Downloaded from

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Adaptor Protein MecA Is a Negative Regulator of the Expression ofLate Competence Genes in Streptococcus thermophilus

Céline Boutry, Astrid Wahl, Brigitte Delplace, André Clippe, Laetitia Fontaine, and Pascal Hols

Biochimie et Génétique Moléculaire Bactérienne, Institut des Sciences de la Vie, Université catholique de Louvain, Louvain-la-Neuve, Belgium

In Streptococcus thermophilus, the ComRS regulatory system governs the transcriptional level of comX expression and, hence,controls the early stage of competence development. The present work focuses on the posttranslational control of the activity ofthe sigma factor ComX and, therefore, on the late stage of competence regulation. In silico analysis performed on the S. thermo-philus genome revealed the presence of a homolog of mecA (mecASt), which codes for the adaptor protein that is involved inComK degradation by ClpCP in Bacillus subtilis. Using reporter strains and microarray experiments, we showed that MecASt

represses late competence genes without affecting the early competence stage under conditions that are not permissive for com-petence development. In addition, this repression mechanism was found not only to act downstream of comX expression butalso to be fully dependent on the presence of a functional comX gene. This negative control was similarly released in strains de-leted for clpC, mecA, and clpC-mecA. Under artificial conditions of comX expression, we next showed that the abundance ofComX is higher in the absence of MecA or ClpC. Finally, results of bacterial two-hybrid assays strongly suggested that MecA in-teracts with both ComX and ClpC. Based on these results, we proposed that ClpC and MecA act together in the same regulatorycircuit to control the abundance of ComX in S. thermophilus.

Streptococcus thermophilus is of major economical value due toits extensive use for the production of yogurt and hard cooked

cheese by the dairy industry (3, 21). Recently, S. thermophilus wasshown to be able to develop natural competence for transforma-tion, a transitory physiological state enabling the capture and sta-ble integration of naked DNA in the chromosome of this species(2, 14, 15, 18).

It is well established that natural competence regulation usu-ally involves a set of regulators, which together form a cascadefrom the early stage to the late stage of competence development(8, 20). For S. thermophilus, competence was shown to be natu-rally induced when S. thermophilus cells were grown in a chemi-cally defined medium. This induction results in the transitory ex-pression of the comX gene, coding for the master regulator of latecompetence (com) genes, which are necessary for DNA uptake,protection, and chromosomal integration (14, 18). ComX is analternative sigma factor (�X) that binds to the RNA polymerase inorder to transcribe specifically the late com genes by recognizing asmall sequence (Com box) in their upstream regions (31, 33, 46).Recently, we have shown that the activation of comX expression inS. thermophilus relies on a novel quorum-sensing system, namedComRS (14). The proposed activation model starts with the intra-cellular production of a precursor peptide (pre-ComS), which isthen secreted and matured (ComS*) (14, 34). When its concen-tration in the extracellular medium increases and reaches a thresh-old, ComS* binds to the oligopeptide-binding protein AmiA3 andis internalized by the multicomponent oligopeptide transporterAmiCDEF. Inside the cell, ComS* activates the transcriptionalregulator ComR. The ComRS complex in turn activates comX andcomS transcription, resulting in an autoamplification loop (14).Homologs of the ComRS system were also found in all sequencedgenomes of streptococci belonging to the mutans, pyogenic, andbovis groups (34). Furthermore, this system was shown to befunctional in Streptococcus mutans as the most proximal regula-tory system for comX activation and competence development(34).

Besides the transcriptional control of comX, the ComX proteinwas shown to be the target of posttranslational control in strepto-cocci. In Streptococcus pneumoniae, the amount of ComX is posi-tively regulated by ComW and negatively controlled by ClpEP andClpCP. ComW is produced in the early competence phase to sta-bilize and activate ComX. ClpEP and ClpCP are protease machin-eries that degrade ComX and ComW, respectively (32, 36, 42, 46).In Streptococcus pyogenes, the mechanism of ComX stabilization/degradation is still unknown, except for the implication of theClpP protease (38). For S. thermophilus, previous extensive insilico analyses did not allow the identification of a homolog ofComW, but during the course of that study, the ClpC ATPasesubunit was shown to act as a negative regulator of competencedevelopment in this species (1). For Bacillus subtilis, the posttrans-lational control of the master regulator of competence develop-ment, ComK, has also been highlighted (25–27, 30, 36, 37, 40, 43,45, 47–49). To prevent competence development under inappro-priate conditions, ComK is sequestered by MecA, an adaptor pro-tein which targets ComK to the ClpCP protease system (25, 40, 43,47–49). ComK is released from the inhibitory complex by thesmall antiadaptor peptide ComS, which interacts directly withMecA and is produced when the cell density increases, leading tothe activation of ComK transcription and of the late com genes(11, 12, 37, 40, 43). B. subtilis also contains a paralog of MecA,YpbH, which binds ClpC and affects both competence and spo-rulation by an unknown mechanism (39).

Received 22 December 2011 Accepted 13 January 2012

Published ahead of print 27 January 2012

Address correspondence to Pascal Hols, [email protected].

L.F. and P.H. contributed equally to this work.

Supplemental material for this article may be found at http://jb.asm.org/.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JB.06800-11

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In silico analyses revealed the existence of a unique MecA-likeprotein in S. thermophilus. The aim of this study was to investigateits role in the control of competence development. We showedusing reporter strains and microarray analyses that MecA re-presses the expression of a large set of late com genes under non-permissive competence conditions. Importantly, the expressionof early com genes, including comX, was not affected. In addition,we found evidence that MecA, together with ClpC, but not ClpE,exerts posttranslational control on the abundance of ComX. Wepropose that MecA may act as an anti-sigma factor for targetingComX to the ClpCP degradation machinery, similarly to the post-translational control of ComK in B. subtilis.

MATERIALS AND METHODSBacterial strains, plasmids, and growth conditions. The bacterial strainsand plasmids used in this study are listed in Table 1. Escherichia coli wasgrown with shaking at 37°C in Luria-Bertani (LB) broth (44). S. thermo-philus strains were grown anaerobically (BBL GasPak systems; BectonDickinson, Franklin Lakes, NJ) at 29°C, 37°C, or 42°C in M17 broth,Todd-Hewitt broth (THB) (Difco Laboratories Inc., Detroit, MI) orCDML (29). These media were supplemented with either 1% (wt/vol)glucose (M17 broth with glucose [M17G] and THB with glucose[THBG]) or 1% (wt/vol) lactose (CDML). Solid-agar plates were pre-pared by adding 2% (wt/vol) agar to the medium. When necessary, anti-biotics were added to the media at the following concentrations: ampicil-lin at 250 �g ml�1 for E. coli, kanamycin at 50 �g ml�1 for E. coli,erythromycin at 250 �g ml�1 for E. coli and at 2.5 or 5 �g ml�1 for S.thermophilus, and chloramphenicol at 5 �g ml�1 and spectinomycin at 75�g ml�1 for S. thermophilus.

Detection of absorbance and luminescence. Growth (optical densityat 600 nm [OD600] and luciferase (Lux) activity (expressed in relative lightunits [RLU]) were monitored at 10-min intervals with a Varioskan Flashmultimode reader (ThermoFisher Scientific, Zellic, Belgium) as previ-ously described (14).

DNA techniques and electrotransformation. For general molecularbiology techniques, we followed methods reported previously by Sam-brook et al. (44). The preparation of S. thermophilus chromosomal DNAand the electrotransformation of E. coli and S. thermophilus were per-formed as previously described (10, 13, 15). The primers used in this studywere purchased from Eurogentec (Seraing, Belgium) and are listed inTable S1 in the supplemental material. PCRs were performed with Phu-sion high-fidelity DNA polymerase (Finnzymes, Espoo, Finland) with aGeneAmp 2400 PCR system (Applied Biosystems, Foster City, CA).

Construction of the PcomGA-luxAB reporter strains. Reporter strainsCB007 and CB006 were constructed by replacing part of the blp locus ofstrains LMD-9 and LMG18311, respectively, with the transcriptional fu-sion PcomGA-luxAB carried by plasmid pGICB007, as previously described(14). Strains CB007 and CB006 were confirmed by PCR with primerslocated upstream and downstream of the recombination regions (primersare listed in Table S1 in the supplemental material). Plasmid pGICB007 isa derivative of pGICB001 (14), where the comX expression signals (PcomX)were replaced with the comGA expression signals (PcomGA) amplified withprimer pair DPGA1-DPGA2.

Construction of mecA deletion mutants. LMD-9 derivative strainsCB0011, CB0071, and CB0051 and LMG18311 derivative strain CB0061were constructed by replacing the sequence between the start and stopcodons of mecA with the chloramphenicol resistance cassette lox66-P32-cat-lox71 (mecA::lox66-P32-cat-lox71) according to procedures describedpreviously by Fontaine et al. (16). The primers used to construct thesestrains are listed in Table S1 in the supplemental material. Strains CB0012,CB0072, and CB0052 were constructed by excising the chloramphenicolmarker from strains CB0011, CB0071, and CB0051, respectively, usingthe Cre-loxP system. This procedure was performed by using plasmidpGhostcre as described previously (16).

Construction of comX deletion mutants. Strain CB0078 (�comX)was obtained by the double homologous recombination of plasmidpGICB003 in strain CB007, as previously described (14). Double mutantstrain AW007 (mecA::lox72 comX::P32-cat) was obtained by the naturaltransformation of strain CB0072 (mecA::lox72) using plasmid pGIBD001as the donor DNA. pGIBD001 is a derivative of pGICB002 (14), in whichthe luxAB genes have been replaced by a chloramphenicol resistance cas-sette (P32-cat). To obtain plasmid pGIBD001, plasmid pGICB002 wasreverse amplified by PCR with primers pJUDdelcomX1 and pJUDdel-comX2, which hybridize upstream and downstream of luxAB, respec-tively, and then ligated to the P32-cat cassette obtained as a PvuII restric-tion fragment from pUC18Cm (19).

Construction of clpC and clpE deletion mutants. LMD-9 derivativestrains AW001 (ClpC�) and AW003 (ClpE�), CB007 derivative reporterstrains AW002 (ClpC�) and AW004 (ClpE�), and CB0072 derivativereporter strains AW005 (MecA� ClpC�) and AW006 (MecA� ClpE�)were constructed by replacing the coding sequence of clpC or clpE with aspectinomycin resistance cassette (spc). This was achieved by naturalcompetence (16) using PCR fragments amplified from chromosomalDNA extracted from the corresponding mutant strains of S. thermophilusLMG 18311 (a generous gift from L. S. Håvarstein) (1). The primers usedare listed in Table S1 in the supplemental material.

Construction of comX::StrepTagII replacement strains. LMD-9 andCB0072 derivative strains CB0053 and CB0054 (MecA�), respectively,were constructed by the chromosomal exchange of comX with a comX::StrepTagII fusion (purification affinity tag StrepTagII [IBA, Germany]fused at the C terminus of ComX) carried by plasmid pGIBD002, as pre-viously described (14). Plasmid pGIBD002 is a derivative of pGICB002(14), where the luxAB genes have been replaced by comX::StrepTagII.Plasmid pGIBD002 was obtained as follows. Plasmid pGICB002 was re-verse amplified with primers pJUDdelcomX1 and pJUDdelcomX2. Theopen reading frame (ORF) of comX was amplified by PCR from theLMD-9 chromosome with primers IntcomXSTREP1 and Intcom-XSTREP2 to generate the comX::StrepTagII fusion. Both PCR productswere restricted by NcoI and HindIII, ligated, and then transformed into E.coli EC1000 cells.

Construction of the mecA complementation vector. mecA expres-sion plasmid pGICB005 contains a transcriptional fusion between thepromoter PblpU and mecA. This plasmid was constructed as follows. Plas-mid pXL was reverse amplified with primers pXLdeltacomX1 andpXLdeltacomX2, which hybridize the upstream and downstream regionsof comX, respectively. The promoterless mecA gene was amplified by PCRfrom the LMD-9 chromosome with primers SURTRADMECA1 andSURTRADMECA2. Both PCR products were restricted by ApaI andXmaI, ligated, and then transformed into E. coli EC1000. The controlplasmid (pXL�1) used in complementation experiments is a pXL deriv-ative containing a comX deletion. This plasmid was constructed by thereverse amplification of pXL with primers DelcomX1 and DelcomX2. Theresulting PCR fragment was restricted by NcoI, ligated, and transformedinto E. coli EC1000. The induction of the promoter PblpU was achieved bythe addition of 250 ng ml�1 D9C-30 synthetic peptide to the cultures atthe beginning of growth, as reported previously (15).

Construction of the pMGXstrep expression vector. In plasmidpMGXstrep, the comX::StrepTagII fusion is cloned under the control ofthe constitutive P32 promoter. The ORF of comX::StrepTagII was ampli-fied by PCR from the CB0053 chromosome with primers MX1 and MX2.The PCR product was restricted by SalI and SmaI and then cloned intopMG36eT (16) digested by the same restriction enzymes.

Detection of ComX-StrepTagII by Western analysis. PlasmidpMGXstrep was electroporated into wild-type (WT) strain LMD-9 and itsderivative strains deficient in MecA, ClpC, and ClpE (CB0051, AW001,and AW003, respectively). Lactococcus lactis overproducing ComX-StrepTagII (A. Wahl, unpublished data) and WT LMD-9 carrying plasmidpMGX (producing ComX without StrepTagII) (16) were used as positiveand negative controls, respectively. These strains were grown overnight in

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THBG containing 5 �g ml�1 erythromycin at 37°C. The OD600 was thenadjusted to 0.05 in THBG, and cells were grown until they reached anOD600 of 0.4 at 37°C or 42°C. Cells from 50-ml cultures were resuspendedin 1 ml lysis buffer (100 mM Tris [pH 8.0], 150 NaCl, 1 mM EDTA, 1 mMphenylmethylsulfonyl fluoride [PMSF]) containing 1/5 (vol/vol) glassbeads (G8893, �106 �m; Sigma). Bacterial cells were then lysed by using

a Precellys24 homogenizer (three times for 30 s at 5,000 rpm). Eightymicroliters of total cell extract was mixed with 20 �l of 5� Laemmli bufferand heated at 98°C for 10 min. Samples (25 �g of total protein) wereloaded onto gradient 4 to 20% SDS-PAGE precast gels (Thermo-Scientific), separated by electrophoresis, and transferred onto a nitrocel-lulose membrane. ComX-StrepTagII was detected by using a mouse

TABLE 1 Bacterial strains and plasmids used in this study

Strain or plasmid Characteristic(s)a Reference or source

StrainsE. coli

EC1000 Kmr RepA�; MC1000 containing a copy of the repA gene of pWV01 in its chromosome 28BTH101 F= cya-99 araD139 galE15 galK16 rpsL1 hsdR2 mcrA1 mcrB1 23

S. thermophilusLMD-9 Wild type ATCCCB003 LMD-9 �comX 14CB001 LMD-9 blpD-blpX::PcomX-luxAB 14CB0011 CB001 mecA::lox66-P32-cat-lox71 This studyCB0012 CB001 mecA::lox72 This studyCB007 LMD-9 blpD-blpX::PcomGA-luxAB This studyCB0071 CB007 mecA::lox66-P32-cat-lox71 This studyCB0072 CB007 mecA::lox72 This studyCB0078 CB007 �comX This studyCB0051 LMD-9 mecA::lox66-P32-cat-lox71 This studyCB0052 LMD-9 mecA::lox72 This studyCB0053 LMD-9 comX::StrepTagII This studyCB0054 CB0011 comX::StrepTagII This studyAW001 LMD-9 clpC::spc This studyAW002 CB007 clpC::spc This studyAW003 LMD-9 clpE::spc This studyAW004 CB007 clpE::spc This studyAW005 CB0072 clpC::spc This studyAW006 CB0072 clpE::spc This studyAW007 CB0072 comX::P32-cat This studyCB006 LMG18311 blpD-blpX::PcomGA-luxAB This studyCB0061 CB006 mecA::lox66-P32-cat-lox71 This study

PlasmidspGICB001 Emr TS; pJIM4900 derivative containing the PcomX-luxAB transcriptional fusion flanked by the upstream and

downstream sequences of blpD and blpX, respectively14

pGICB007 Emr TS; pGICB001 derivative in which the PcomX-luxAB fusion was replaced by a PcomGA-luxAB fusion This studypGICB002 Emr TS; pJIM4900 derivative containing the luxAB genes flanked by the upstream and downstream

sequences of comX14

pGICB003 Emr TS; pJIM4900 derivative containing the upstream and downstream sequences of comX; this plasmid wasused to delete comX

14

pUC18Cm Apr Cmr; pUC18 derivative containing the P32-cat cassette from pGIZ850 19pGIBD001 Emr Cmr TS; pGICB002 derivative for chromosomal exchange of comX by P32-cat This studypGIBD002 Emr TS; pGICB002 derivative for chromosomal exchange of comX by comX::StrepTagII This studypXL Emr; pTRKH2 derivative containing a PblpU-comXLMG18311 fusion 2pGICB005 Emr; pXL derivative in which comX has been replaced by mecA and which contains a PblpU-mecALMD-9 fusion This studypXL�1 Emr; pXL derivative containing a deletion of comX This studypMG36eT Emr; pMG36e derivative for constitutive expression under the control the P32 promoter 16pMGXstrep Emr; pMG36eT derivative containing a P32-comXLMD9::StrepTagII fusion This studypGhostcre Emr TS; pG�host9 derivative containing a P1144-cre fusion 16pUT18 Apr; pUC19 derivative containing the T18 fragment of CyaA under the control of the Plac promoter for in-

frame X-T18 fusions24

pUT18C Apr; pUC19 derivative containing the T18 fragment under the control of the Plac promoter for in-frame T18-X fusions

24

pKNT25 Kmr; pSU40 derivative encoding the T25 fragment of CyaA under the control of the Plac promoter for in-frame X-T25 fusions

22

pKT25 Kmr; pSU40 derivative encoding the T25 fragment of CyaA under the control of the Plac promoter for in-frame T25-X fusions

24

a Kmr, kanamycin resistance; Emr, erythromycin resistance; Apr, ampicillin resistance; Cmr chloramphenicol resistance; TS, thermosensitive RepA protein.

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monoclonal antibody against StrepTagII (StrepMAB-Classic) as the pri-mary antibody and a horseradish peroxidase-conjugated rabbit anti-mouse polyclonal antibody (pAb) as a secondary antibody, according tothe manufacturer’s instructions (IBA, Germany).

B2H plasmid construction and bacterial two-hybrid assay. Methodsused for bacterial two-hybrid (B2H) plasmid construction and the bacte-rial two-hybrid assay were described previously by Karimova et al. (22–24). The coding sequences of comX (ster_0189), mecA (ster_0216), clpC(ster_0109), and clpE (ster_0648) were amplified by PCR from S. thermo-philus LMD-9 genomic DNA by using primers reported in Table S1 in thesupplemental material and inserted into plasmids pUT18, pUT18C,pKT25, and pKNT25. The full list of constructed plasmids for B2H assaysis shown in Table S1 in the supplemental material. To perform a two-hybrid assay, a combination of two recombinant plasmids was electro-transformed into E. coli BTH101 (cya-99). Six clones from each combina-tion were tested on indicator MacConkey (Difco) agar plates containingkanamycin and ampicillin and supplemented with 1% (wt/vol) maltoseand 0.5 mM IPTG (isopropyl-�-D-thiogalactopyranoside). Plates wereincubated at 30°C for 36 h.

RNA extraction. For transcriptome comparisons between the LMD-9�mecA:: lox72 mutant (CB052) and WT LMD-9, strains were grown dur-ing 16 h at 37°C in THBG. For the comparison between WT LMD-9 andthe LMD-9 �comX mutant (CB003), strains were grown at 37°C during 16h in M17G, washed twice (5,000 � g for 9 min at room temperature) in 1volume of CDML, and resuspended in 1 volume of CDML. Cultures werethen diluted 30-fold in their respective media (either THBG or CDML).When cells reached the mid-log phase (OD600 of 0.4), 25-ml aliquots werecollected. Cells were harvested by centrifugation (7,000 � g for 4 min) andmechanically broken with 0.18-mm-diameter glass beads in a Braun ho-mogenizer (three 1-min periods of homogenization with 1-min intervalson ice). Total RNA was extracted by using the High Pure RNA isolation kit(Roche, Basel, Switzerland).

Microarray experiments and data analysis. The slide for S. thermo-philus LMD-9 was a custom-designed Agilent Technologies oligonucleo-tide microarray containing eight 15K arrays (Gene Expression Omnibus[GEO] accession number GPL13365), and each coding sequence was rep-resented by one to five 60-mer probes. Microarray experiments were per-formed as previously described (14). Gene expression analysis was carriedout by using GeneSpring GX v11.0 software (Agilent Technologies). Datawere filtered for outliers, negative and positive controls, and flagged sig-nals as previously described (14). Probes from triplicates were filtered by at test for significance at a threshold of a P value of �0.05. Significantlyregulated probes were then defined based on a fold change (FC) higherthan 1.5. Significantly regulated genes were defined as genes for which atleast 50% of the probes were significantly regulated and with a meanabsolute FC of total probes of at least 1.5. Significantly induced probesadjacent to an induced coding DNA sequence (CDS) were also retained ifthe FC of the total probes of the adjacent CDS was at least of 1.5.

Microarray data accession numbers. The normalized transcriptomedata have been deposited in the GEO database under accession numbersGSE28371 and GSE28372.

RESULTSThe genome of S. thermophilus contains a unique homolog ofMecA from B. subtilis. Five S. thermophilus genomes (strainsLMD-9, LMG18311, CNRZ1066, ND03, and JIM8232) have beensequenced so far. In silico analyses of the 5 genomes identified aunique gene encoding a putative homolog of MecA from B. sub-tilis (MecABs) in each of them. The MecA-like protein of S. ther-mophilus (MecASt [Ster_0216]; 249 amino acids [aa]) is moreclosely related to MecABs (60.6% and 26.0% similarity and iden-tity, respectively) than YpbH (53.5% and 20.4%) of B. subtilis (seeFig. S1 and S2 in the supplemental material). Among streptococci,MecA-like proteins were identified in all streptococcal groups,

where the closest orthologs of MecASt were found in the genomesof Streptococcus vestibularis (94.8% identity), S. salivarius (94.0%),and S. gallolyticus (60.7%) (see Fig. S1 in the supplemental mate-rial). These in silico data are consistent with work reported previ-ously by Persuh et al. (40), which showed that MecA was wide-spread among Gram-positive bacteria. Both MecA and YpbH areorganized into two domains but display different lengths of theinterdomain linker region (39, 40). In the ClpC-MecA-ComKcomplex, the N-terminal domain (NTD) was shown to interactwith ComK, while the C-terminal domain (CTD) is the site ofrecognition of the ClpC ATPase subunit of the Clp machinery (35,40, 43, 50). An alignment between a set of streptococcal MecAproteins including MecASt, MecABs, and YpbH revealed a highlevel of conservation in both the NTD and CTD, with the NTDbeing the most conserved, while the interdomain linker regionexhibited a very low level of conservation and is longer in strepto-coccal proteins (see Fig. S2 in the supplemental material). SinceMecASt shows a significant level of identity with MecABs and thesame organization in two domains, we hypothesized that it couldplay a role in S. thermophilus similar to that found for B. subtilis,i.e., a repressor of competence development.

MecA is a repressor of the late comGA operon under condi-tions that are not permissive for competence development. Inthe first set of experiments, the functional role of MecA from S.thermophilus was evaluated by growing cells in THBG medium.These growth conditions were shown previously to be nonpermis-sive for spontaneous competence development, since the level ofcomX expression is very low (1, 2, 16).

To assess the role of MecA in the regulation of early and latecompetence genes, the mecA open reading frame was replaced bya chloramphenicol resistance cassette (lox66-P32-cat-lox71) inLMD-9 reporter strains harboring either PcomX-luxAB or a PcomGA-luxAB inserted into the blp locus (strains CB0011 and CB0071,respectively). To avoid polar effects on adjacent genes, the chlor-amphenicol resistance marker was removed by using the Cre-loxPsystem as previously reported (16) (yielding strains CB0012 andCB0072, respectively). The promoter of the comX gene was chosensince it integrates all the signals originating from the early stage ofcompetence, while the promoter of comGA controls one of themost important late com operons, which codes for a part of theDNA uptake machinery (7). All the constructed reporter strainswere then grown in THBG medium in order to monitor the lucif-erase (Lux) activity. The specific Lux activities (RLU/OD600) of thecontrol reporter strains (CB001 and CB007) were compared tothose of their isogenic mecA::lox66-P32-cat-lox71 (CB0011 andCB0071) and mecA::lox72 (CB0012 and CB0072) mutant strains(Fig. 1 and Table 2). The inactivation of MecA had no impact onthe expression of the PcomX-luxAB fusion (Fig. 1A). In contrast, themaximum Lux activities of the two MecA-deficient strains(CB0071 and CB0072) harboring the PcomGA-luxAB fusion weresimilarly increased more than 100-fold compared to that of thecontrol strain (CB007) (Fig. 1B). To enlarge our investigation ofthe functional role of MecA, the corresponding gene was deletedin LMG18311 derivative strains harboring a PcomGA-luxAB fusion(CB006 and CB0061). Similarly to LMD-9, specific PcomGA activitywas highly induced (200-fold) in the MecA-deficient strain(CB0061) (Table 2). To further support that the observed pheno-type results from the mecA deletion solely, a mecA complementa-tion plasmid (the pXL derivative pGICB005) was constructed andelectrotransformed into MecA-deficient reporter strain CB0071.

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Plasmid pGICB005 fully restored PcomGA activity to a level similarto that of WT reporter strain CB007 (Table 2). In contrast, thecontrol strain harboring the empty vector [CB0071(pXL�1)]showed a maximum Lux activity similar to that of the parentalMecA-deficient strain (CB0071) (Table 2).

We next studied whether the repression activity exerted byMecA on PcomGA expression was dependent on the presence of afunctional ComX protein. To this end, we compared the PcomGA-luxAB activities of a comX mutant strain (CB0078) to those of acomX mecA double mutant strain (AW007). The deletion of comXsolely or in combination with mecA similarly decreased the level ofactivity of PcomGA compared to that of the WT reporter strain(Table 2).

Altogether, these results show that MecA is a negative regulatorof a major late com operon in S. thermophilus. Notably, MecA

exerts its control without affecting the transcriptional activity ofcomX. Moreover, the role of MecA as a repressor of late compe-tence gene expression absolutely requires the presence of an intactComX protein, strongly suggesting that MecA acts on the ComXprotein to achieve its regulatory function.

MecA is not a repressor of the comGA operon under permis-sive competence conditions. In a second set of experiments, thefunctional role of MecA from LMD-9 and LMG18311 strains wasevaluated by growing cells in CDML medium. These growth con-ditions were shown previously to be permissive for spontaneouscompetence development at high and low levels for LMD-9 andLMG18311, respectively (16).

LMD-9 and LMG18311 PcomGA-luxAB reporter strains weregrown in CDML, and PcomGA activity was measured (Table 2).For LMD-9, the maximum Lux activities were similar betweenthe two MecA-deficient strains (CB0071 and CB0072) and thecontrol (CB007). In contrast, the MecA-deficient strain ofLMG18311 (CB0061) displayed a 27-fold-higher level of Luxactivity (Table 2).

These data strongly suggest that when the level of competenceinduction reached a certain threshold, such as that which wasobserved for strain LMD-9 under CDML conditions, the deple-tion of MecA has no impact. In contrast, MecA could still act as arepressor in LMG18311, which is poorly competent under theseconditions. These results suggest that when the transcription ofcomX is strongly activated by the ComRS system, the latter repre-sents the dominant mechanism for the control of competencedevelopment.

The inactivation of MecA partially activates the late comregulon. To further investigate the functional role of MecA, wedecided to evaluate the overlap between the late com regulon con-trolled by ComX and the MecA regulon in S. thermophilus LMD-9using microarrays.

In order to determine the late com regulon, we compared thetranscriptomes of WT LMD-9 and its isogenic comX mutant strain(ComX�) (CB003) under natural comX-inducing conditions(CDML). Microarray experiments were performed as described inMaterials and Methods. RNAs were extracted when culturesreached the mid-log phase, when the maximum PcomGA activity ofreporter strain CB007 was measured (data not shown). We iden-tified 135 genes that were upregulated (FC � 1.5; P � 0.05) in thewild type compared to the ComX-deficient strain (see Table S2 inthe supplemental material). From this gene list, 93 genes weredistributed into 13 clusters that include at least one predictedComX box (therefore defined here as the ComX-regulated loci).All late com genes that were identified previously as being essentialfor competence development in S. pneumoniae (41) were har-bored in the ComX-regulated loci, except pilD, giving confidencein the extent of the regulon that was established (Fig. 2, and seeTable S3 in the supplemental material).

In order to identify the set of MecA-regulated genes, the tran-scriptomes of the LMD-9 mecA::lox72 mutant (CB0052) and WTLMD-9 were compared under THBG growth conditions (mid-logphase). As expected, mecA was included in the list of downregu-lated genes (FC � 67.7) (see Table S4 in the supplemental mate-rial). We also noticed that comX expression was not altered, con-firming the results obtained with PcomX-luxAB reporter strainCB001. Moreover, none of the early com genes previously identi-fied were up- or downregulated (14) (Table 3, and see Table S4 inthe supplemental material). The set of upregulated genes with an

FIG 1 Growth (OD600) and luciferase activities (RLU/OD600) of LMD-9 re-porter strains and their isogenic MecA-deficient strains grown in THBG at37°C. (A) Growth (white symbols) and specific luciferase activities (black sym-bols) of PcomX-luxAB fusions of strains CB001 (MecA�) (triangles) andCB0012 (MecA�) (circles). (B) Growth (white symbols) and specific luciferaseactivities (black symbols) of PcomGA-luxAB fusions of strains CB007 (MecA�)(triangles) and CB0072 (MecA�) (circles).

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FC of �1.5 (P � 0.05) consists of 47 genes, including 27 late comgenes and 25 genes distributed in ComX-regulated loci (Table 3and Fig. 2, and see Table S2 in the supplemental material). AmongComX-regulated loci, the gene cluster (ster_1834 to ster_1842)

that includes comGA (FC � 46.7) is the cluster most stronglyinduced by the inactivation of MecA. All late com genes that wereidentified as being essential for competence development in S.pneumoniae, except pilD and recA, are members of the MecA regu-

TABLE 2 Maximum luciferase activities of S. thermophilus strains

Strain Promoter Relevant feature(s)b

Mean max luciferase activity � SEMa

THBG conditions(RLU/OD600 � 104)

CDML conditions(RLU/OD600 � 106)

LMD-9 derivativesCB007 PcomGA Wild type 3.5 � 1.0 14.6 � 0.5CB0071 PcomGA MecA� (Cmr) 353.6 � 32.1 14.5 � 0.3CB0072 PcomGA MecA� 389.0 � 77.3 16.7 � 0.5CB0078 PcomGA ComX� 1.6 � 0.5 NTAW007 PcomGA MecA� ComX� 1.7 � 0.4 NTAW002 PcomGA ClpC� 280.6 � 26.1 NTAW004 PcomGA ClpE� 4.2 � 1.3 NTAW005 PcomGA MecA� ClpC� 300.0 � 33.5 NTAW006 PcomGA MecA� ClpE� 318.0 � 49.7 NTCB0071(pXL�1) PcomGA MecA�, empty vector 337.1 � 8.5 NTCB0071(pGICB005) PcomGA MecA�, complementation vector 4.1 � 0.6 NTCB001 PcomX Wild type 6.3 � 0.6 33.1 � 2.0CB0011 PcomX MecA� (Cmr) 6.7 � 0.7 28.6 � 5.9CB0012 PcomX MecA� 8.0 � 0.5 16.2 � 2.7

LMG18311 derivativesCB006 PcomGA Wild type 0.2 � 0.02 0.2 � 0.04CB0061 PcomGA MecA� (Cmr) 39.2 � 1.6 5.3 � 0.4

a Mean values from triplicate experiments � SEM. NT, not tested.b Cmr, chloramphenicol resistance resulting from the replacement of mecA with cat.

FIG 2 Organization of the ComX-regulated loci in S. thermophilus LMD9. The selected set of upregulated genes (red and red-hatched pentagons) is based on the microarrayexperiment performed on WT LMD-9 versus the LMD-9 comX mutant (loci I to XIII) (see Table S2 in the supplemental material). These genes are organized into 13 loci on thebasis of the presence of predicted ComX boxes (arrows with white rectangles) (see Table S3 in the supplemental material). Shared upregulated genes resulting from mecAinactivationandobtainedfromthemicroarrayexperimentwithCB0052(mecA::lox72)versusWTLMD-9(Table3)areshownasred-hatchedpentagons.Pentagonssurroundedby a dotted line and a black line correspond to late and essential late com genes in S. pneumoniae TIGR4, respectively (41). The ORF orientation in each locus is indicated by thedirection of the pentagons. The number below each pentagon corresponds to the STER locus tag of LMD-9. �, pseudogene.

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TABLE 3 Genes upregulated in the LMD-9 MecA� strain compared to WT strain LMD-9 grown under THBG conditions

Category and locus tag inLMD-9 Gene Descriptione Strand

FCa

Late comgene inS. pneumoniaeb

Presence of:

MecA�

strainComX�

strain

Essentialcom inS. pneumoniaec

ComXboxd

MecA-regulated genes sharedwith the late com regulon

STER_0406 comFA Late competence protein required forDNA uptake

� 1.6 7.2 SP_2208 Y Y

STER_0407 comFC Late competence protein � 1.9 7.6 SP_2207 YSTER_0488 coiA Competence protein, transcription

factor� 27.0 42.0 SP_0978 Y Y

STER_0489 pepB Oligopeptidase � 2.4 18.9 SP_0979STER_0922 dprA DNA-processing protein, Smf family � 31.3 72.2 SP_1266 Y YSTER_0923 topA DNA topoisomerase I � 2.6 29.9STER_1029 Pseudo 1.8 11.3STER_1030 Hypothetical protein � 1.8 13.4STER_1031 Pseudo 1.6 8.7STER_1519 murZ UDP-N-Acetylglucosamine

1-carboxyvinyltransferase� 2.0 6.8

STER_1520 comEC Late competence protein required forDNA uptake

� 22.1 23.2 SP_0955 Y

STER_1521 comEA Late competence protein required forDNA binding and uptake

� 15.1 10.7 SP_0954 Y Y

STER_1818 Hypothetical protein � 1.5 7.1STER_1819 Cytidine/deoxycytidylate deaminase

family protein, putative� 1.6 16.3

STER_1820 Hypothetical protein � 2.3 21.1STER_1821 ssbA Single-strand DNA-binding protein � 1.7 23.3 SP_1908 Y YSTER_1834 ackA Acetate kinase � 3.4 25.9STER_1835 Hypothetical protein � 8.4 72.6 SP_2045STER_1836 Hypothetical protein � 62.0 181.4 SP_2047 YSTER_1837 Competence protein, putative � 67.2 163.2 SP_2048STER_1838 Pseudo 77.0 190.4STER_1839 comGD Competence protein � 72.8 151.3 SP_2050 YSTER_1840 comGC Late competence protein, exogenous

DNA-binding protein� 83.5 203.3 SP_2051 Y

STER_1841 comGB Late competence protein, ABCtransporter subunit

� 77.4 147.3 SP_2052 Y

STER_1842 comGA Late competence protein, ABCtransporter subunit

� 46.7 50.0 SP_2053 Y Y

STER_1677 ABC transporter, permease protein � 1.8 2.8STER_1678 ABC-type multidrug transport system,

ATPase component� 1.8 2.7

Unique MecA-regulated genesSTER_0596 Pyridine nucleotide-disulfide

oxidoreductase� 1.9 �1.5

STER_0588 Hypothetical protein � 2.3 �1.5STER_0870 ptpS 6-Pyruvoyl tetrahydrobiopterin

synthase� 1.5 �1.5

STER_0871 Putative coenzyme PQQ synthesisprotein

� 1.6 �1.5

STER_0872 7-Cyano-7-deazaguanine reductase � 1.6 �1.5STER_1022 High-affinity Fe2�/Pb2� permease � 4.4 �1.5STER_1023 Hypothetical protein � 4.4 �1.5STER_1024 Hypothetical protein � 5.3 �1.5STER_1025 ABC-type Fe3�-hydroxamate transport

system, periplasmic component� 7.3 �1.5

STER_1026 fatA ABC-type spermidine/putrescinetransport system, ATPasecomponent

� 7.7 �1.5

STER_1027 fatC ABC-type Fe3�-siderophore transportsystem, permease component

� 7.4 �1.5

(Continued on following page)

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lon. In addition, 22 upregulated genes are unique to MecA inac-tivation.

Taken together, these microarray results show that the absenceof MecA activates to some extent the late com regulon withoutaffecting the early stage of competence.

ClpC and MecA display similar repressing effects on the ex-pression of the late comGA operon. For S. thermophilusLMG18311, both ClpC and ClpE were recently shown to repressthe activity of a late com promoter (1). Furthermore, the depletionof ClpC in this strain had no impact on the activity of PcomX, butthe abundance of ComX was shown previously to be increased inthe clpC mutant (1). In order to compare the effects of the depri-vation of MecA, ClpC, and ClpE on the activity of PcomGA inLMD-9, a range of reporter strains (CB007 derivatives) were con-structed, and their Lux activities were compared under conditionsnot permissive for competence (THBG). Both MecA- and ClpC-deficient strains (strains CB0072 and AW002, respectively)showed similar maximum specific Lux activities and similar kinet-ics of activation of the PcomGA-luxAB fusion (Fig. 3A and Table 2).In contrast, the inactivation of ClpE in strain LMD-9 (AW004)had nearly no impact on the expression of the PcomGA-luxAB fu-sion (Fig. 3A and Table 2). In order to evaluate the synergetic orcumulative effects of the inactivation of MecA and Clp, doubleMecA-ClpC- and MecA-ClpE-deficient reporter strains were con-structed (AW005 and AW006, respectively). Notably, both doublemutants showed behaviors similar to those of the single clpC andmecA mutants (Fig. 3B and Table 2).

These genetic results strongly suggest that MecA and ClpC, butnot ClpE, are involved in the same regulatory circuit for the tran-scriptional control of the comGA operon in strain LMD-9.

MecA and ClpC negatively affect ComX accumulation invivo. Analogously to the role played by MecA and ClpC in B.subtilis, the results collected so far were in favor of a direct controlof the abundance of ComX by the MecA and ClpC proteins. In thefirst approach to support this hypothesis, we tagged the chromo-somal copy of ComX with StrepTagII fused at its C terminus inorder to monitor its abundance using anti-StrepTagII antibodiesin Western blotting experiments. Although ComX-StrepTagII

was not altered in its ability to develop competence, we were un-able to detect the fusion protein in the wild type and its isogenicMecA-deficient strain (CB0053 and CB0054) under conditionsnot permissive for competence (THBG) (data not shown). Thissuggests that the abundance of ComX under these conditions re-mains very low, even when MecA is inactivated. To circumventthis problem, the comX::StrepTagII fusion was constitutively ex-pressed under the control of the P32 promoter on a multicopyplasmid (pMGXstrep). pMGXstrep was then transferred toPcomGA reporter strain CB007 and its derivatives deficient forMecA (CB0072), ClpC (AW002), and ClpE (AW004). At 37°C,the maximum specific Lux activities were similar between the WTand mutant strains (data not shown). Similarly to the situationwith LMD-9 growing in CDML, it is probable that the level ofComX-StrepTagII production is too high under these conditionsto observe the negative effect of MecA and ClpC on PcomGA expres-sion. As expected, Western blotting experiments showed that theabundance of ComX-StrepTagII remained unchanged between allthese strains (data not shown). For Gram-positive bacteria, thelevel of the ClpCP machinery was shown previously to be in-creased in response to high temperatures (6, 9). Therefore, thesame experiments were reproduced at 42°C instead of at 37°C. Inthis experimental setup, the levels of specific Lux activities of theMecA- and ClpC-deficient strains were 4-fold increased com-pared to those of their isogenic parental strain (CB007), while theinactivation of ClpE had no effect (Fig. 4A). Consistently, Westernblotting experiments showed that the abundance of the ComX-StrepTagII protein was similarly increased when MecA or ClpCwas inactivated, compared to the wild-type and ClpE-defectivestrains (Fig. 4B).

These data show that MecA and ClpC, but not ClpE, negativelyaffect the abundance of ComX in vivo in S. thermophilus LMD-9,confirming the above-described expression results obtained withthe reporter strains.

MecA interacts with both ComX and ClpC in bacterial two-hybrid assays. To obtain the first evidence of direct physical in-teractions between the MecA, ComX, and Clp machineries, arange of bacterial two-hybrid (B2H) system experiments was set

TABLE 3 (Continued)

Category and locus tag inLMD-9 Gene Descriptione Strand

FCa

Late comgene inS. pneumoniaeb

Presence of:

MecA�

strainComX�

strain

Essentialcom inS. pneumoniaec

ComXboxd

STER_1028 fatD ABC-type Fe3�-siderophore transportsystem, permease component

� 6.8 �1.5

STER_1257 ldh L-Lactate dehydrogenase � 1.8 �1.5STER_1601 Hypothetical protein � 1.9 �1.5STER_1602 Pseudo 1.8 �1.5STER_1639 Hypothetical protein � 1.5 �1.5STER_1683 Hypothetical protein � 3.4 �1.5STER_1684 Response regulator � 3.6 �1.5STER_1709 scrK Fructokinase � 1.6 �1.5STER_1710 scrA Sucrose PTS, enzyme IIBCA � 2.0 �1.5

a Mean absolute fold change (FC) with a cutoff value of �1.5 and a P value of �0.05 for the LMD-9 MecA� strain (WT versus CB0052) and the LMD-9 ComX� strain (WT versusCB003).b Late com genes identified in S. pneumoniae TGIR4 (41). Orthologous genes with an identity of 40% were identified by BlastP.c Late com genes identified in S. pneumoniae TGIR4 as being essential for competence (41). Y, presence of an orthologous gene in LMD-9.d Presence (Y) of a conserved ComX box ([T/A]NCGAAT[A/G]) in the upstream intergenic region.e Pseudo, pseudogene; PQQ, pyrroloquimoline quinone; PTS, phosphotransferase system.

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up. In the B2H system, the T25 and T18 fragments of the Borde-tella pertussis adenylate cyclase are used as fusion partners. Whenthe fusion proteins interact with each other, a functional comple-mentation between the T25 and T18 fragments takes place, result-ing in adenylate cyclase activity and cyclic AMP (cAMP) produc-tion, which turns on the transcription of the lac operon (23).

Plasmids allowing the production of all possible combinationsof N and C fusion proteins between T18 and T25 and full-lengthMecA and ComX were constructed. The production of adenylatecyclase from transformants of the cya mutant of E. coli (BTH101)was detected on MacConkey agar plates. As shown in Fig. 5, strainBTH101, producing the following pairs of fusion proteins, formedred colonies on MacConkey indicator plates: T18-MecA and T25-ComX, T18-MecA and ComX-T25, MecA-T18 and T25-ComX,and MecA-T18 and ComX-T25 (Fig. 5A, red rectangle). In con-

trast, the negative-control strains (T18 and T25, T18-MecA andT25, MecA-T18 and T25, T18 and T25-ComX, and T18 andComX-T25) remained white on the indicator plates (Fig. 5A,black rectangles). The T18 fusions with ComX carried by high-copy-number plasmid pUT18 were excluded due either to theiractivation in the negative-control strains, possibly by aggregationwith the partnerless T25, or to not-reproducible results (data notshown). Concerning MecA self-interactions, positive signals wereobserved for the following two pairs of fusions proteins: MecA-T18 and T25-MecA, and T18-MecA and MecA-T25 (Fig. 5B andC, dark blue squares). Therefore, only two out of four possiblecombinations of MecA fusion partners gave positive signals. Thisfinding may be explained by the fact that both the NTD and CTDof MecA are involved in the oligomerization process in B. subtilis,and therefore, fusion partners could affect the self-interaction(40). Next, all combinations of fusions proteins between MecAand ClpC or MecA and ClpE were tested in the B2H system asdescribed above (Fig. 5B and C). All MecA and ClpC fusion pro-teins gave positive interaction signals with each other (Fig. 5B,green squares), while all MecA and ClpE fusions proteins werenegative with each other (Fig. 5C, dotted black squares). BothClpC and ClpE showed strong self-interactions, as expected, andall the control strains remained negative (Fig. 5B, light bluesquare, and C, magenta square, respectively).

Altogether, these B2H results were the first indication that

FIG 3 Growth (OD600) and luciferase activities (RLU/OD600) of LMD-9 re-porter strains deficient for MecA, ClpC, ClpE, and MecA-ClpC grown inTHBG at 37°C. (A) Growth (white symbols) and specific luciferase activities(black symbols) of PcomGA-luxAB fusions of strains CB0072 (MecA�) (circles),AW002 (ClpC�) (triangles), and AW004 (ClpE�) (squares). (B) Growth(white symbols) and specific luciferase activities (black symbols) of PcomGA-luxAB fusions of strains CB0072 (MecA�) (circles) and AW005 (MecA�-ClpC�) (triangles).

FIG 4 Luciferase activity (RLU/OD600) and detection of ComX-StrepTagII byWestern blotting of WT strain LMD-9 and strains deficient for MecA, ClpC,and ClpE expressing comX::streptagII constitutively from pMGXstrep andgrown in THBG at 42°C (OD600 � 0.4). (A) Maximum specific luciferaseactivities of PcomGA-luxAB fusions of strains CB007 (WT), CB0072 (MecA�),AW002 (ClpC�), and AW004 (ClpE�). Shown are mean values from triplicateexperiments � standard errors of the means (SEM). Significance between WTand mutant strains is based on a t test. ��, P value of �0.01; �, P value of �0.05.(B) Detection of ComX-StrepTagII in strains LMD-9 (WT), CB0052(MecA�), AW001 (ClpC�), and AW003 (ClpE�). Lactococcus lactis overpro-ducing ComX-StrepTagII and WT LMD-9 producing ComX withoutStrepTagII were used as a positive control (Ctl�) and a negative control (Ctl�),respectively.

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MecA could interact in vivo with ComX and ClpC to form a ter-nary complex (Fig. 5D).

DISCUSSION

Although previous studies unraveled the regulatory cascade lead-ing to the transcriptional activation of comX, encoding the masterregulator of competence development in S. thermophilus, theposttranslational control of ComX activity in this species re-mained nearly unexplored. In this work, we show by a geneticapproach that the adaptor protein MecA acts as a negative regu-lator of competence development by a mechanism that requiresthe presence of ComX. Using luciferase reporter strains and tran-scriptome comparisons, we demonstrate that the inactivation ofMecA has no effect on the expression of early com genes such ascomX but largely affects the expression of late com genes. However,the inactivation of mecA is insufficient to activate natural trans-formation under these conditions (data not shown), while theoverexpression of comX led to a low but detectable transformationrate (16). Similarly, no improvement in the transformation rate ofa ClpC-deficient strain of LMG18311 grown under nonpermissiveTHBG conditions was reported (1). Various hypotheses could ex-plain this apparent discrepancy, such as the absence of an induc-tion of one or more key late com genes that could be required fornatural transformation or an indirect effect of the inactivation ofMecA that could negatively alter the cell status for natural trans-formation.

Similarly to the role played by MecA in B. subtilis, our resultsstrongly suggest that MecA of S. thermophilus interacts withComX to target ComX to the ClpCP machinery for its degrada-tion. This hypothesis is supported by the following results: (i)MecA, ClpC, and combined MecA-ClpC deprivations release thetranscriptional control of the late comGA operon with a similar

response and without cumulative effects; (ii) the inactivation ofMecA or ClpC showed a similar in vivo accumulation of the ComXprotein; and (iii) B2H experiments showed that MecA interactsseparately with ComX and ClpC, suggesting the formation of aternary complex, ClpC-MecA-�X, analogous to the ClpC-MecA-ComK complex of B. subtilis.

In a recent study, Biornstad and Havarstein reported that bothClpC and ClpE act as negative regulators of the activity of a latecom promoter in S. thermophilus LMG18311 (1). The implicationof ClpC in the posttranslational control of ComX was clearly dem-onstrated, while the role of ClpE in competence development wasnot further investigated (1). Concerning the role of ClpE, ourresults based on reporter strains, Western blotting experiments,and B2H assays show that ClpE does not play a significant role inthe control of competence development in strain LMD-9 of S.thermophilus. In S. pneumoniae, the posttranslational control ofComX involves mainly ComW and the ClpEP degradation ma-chinery. The role of ComW as a possible adaptor protein forComX stabilization or as an antiadaptor protein has yet to beclarified (42). Concerning ClpC, it seems to play an indirect role inthe stabilization of ComX by degrading ComW (42). Interest-ingly, a MecA homolog is present in S. pneumoniae, but its impli-cation in the posttranslational control of ComX has never beenreported. Altogether, these results support the hypothesis thatClpC and ClpE independently interact with different actors tocontrol the abundance of ComX in streptococci.

Remarkably, a similar mechanism for the posttranslationalcontrol of the master regulator of competence development in-volving both MecA and ClpC seems to be conserved between rel-atively distant species, such as B. subtilis and S. thermophilus. Thisreinforces the importance of a strict control of competence devel-opment that has been positively selected through evolution. In the

FIG 5 Interactions between MecA and ComX, ClpC, or ClpE evaluated by B2H assays. (A to C) Matrices of bacterial two-hybrid interactions between MecA andComX (A), MecA and ClpC (B), and MecA and ClpE (C) on MacConkey indicator plates supplemented with 1% maltose. Plates were incubated at 30°C for 36h. Red, green, and dotted black rectangles indicate MecA-ComX, MecA-ClpC, and MecA-ClpE interaction assays, respectively. Dark blue, light blue, andmagenta rectangles indicate MecA, ClpC, and ClpE self-interaction assays, respectively. Controls are surrounded by a black line; 25 and 18 correspond to theempty vectors pKT25 and pUT18, respectively. (D) Summary of interactions detected by B2H assays suggesting a ternary complex, ClpC-MecA-ComX. Thearrow color code refers to the interaction assays described above for panels A, B, and C.

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case of S. thermophilus, the MecA adaptor protein seems to act asan anti-sigma factor to control competence development. Theimplication of an adaptor protein targeting a sigma factor to theClp machinery was reported previously in the case of the RssBadaptor protein, which specifically interacts with �S for its target-ing to the ClpXP machinery, thereby controlling the master regu-lator of the stationary phase and the general stress response in E.coli (4, 5). Other adaptor proteins were also shown previously toplay the role of anti-sigma factors, such as RsiW in the control of�W during alkaline stress or SpoIIAA/SpoIIAB in the control of �H

during the sporulation of B. subtilis (17).Based on the results presented in this study and current knowl-

edge of the functional role of MecA in B. subtilis (48), we proposethe following model for the posttranslational control of ComX inS. thermophilus (Fig. 6). Under inappropriate growth conditions,ComX is produced at a low level but sequestered by the anti-sigmafactor MecA in a ternary complex with the ATPase subunit ClpC,which itself binds to the serine protease subunit ClpP, resulting inComX degradation. In contrast, the negative control exerted byMecA would be bypassed when conditions are suitable for com-petence development, possibly via MecA saturation, due to a highlevel of accumulation of ComX. Consequently, ComX (�X) wouldbe free to associate with the core RNA polymerase and specificallyrecognize the Com box in front of the late com genes to activatetheir transcription. In order to strengthen this model, future workwill be dedicated to confirming the interactions between the dif-ferent partners of the ternary complex and to investigating ComXdegradation by ClpCP using in vitro studies.

ACKNOWLEDGMENTS

This research was carried out with financial support from FNRS.We are grateful to E. Bouveret for helpful discussions on the use of the

B2H system. We warmly thank L. S. Håvarstein for providing plasmidpXL and chromosomal DNA from Clp mutants. We are grateful to M.Wels for his help in the design of custom Agilent microarrays of S. ther-mophilus LMD-9.

C.B. held a doctoral fellowship from FRIA. L.F. and A.W. are postdoc-toral researchers at FNRS. P.H. is research associate at FNRS.

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FIG 6 Model of the role of MecA in competence regulation of S. thermophilus. The alternative sigma factor �X, encoded by comX, is recognized and sequestratedby MecA, which also binds the ClpC ATPase subunit of the Clp machinery. This ternary complex interacts with the serine protease ClpP, leading to thedegradation of �X by ClpCP, resulting in a small amount of free �X, which is insufficient to induce the transcription of late com genes. However, when conditionsare appropriate for competence development, �X accumulates above a critical threshold, allowing its association with the core RNA polymerase for the specifictranscription of late com genes.

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