Clp-HSP100 ATPases are a widespread family of ubiquitous proteins that occur in both prokaryotes and eukaryotes and play important roles in the folding of newly synthesized proteins and refoldingof aggregated proteins . They have also been shown to participatein the virulence of several pathogens, including Listeria monocytogenes. Here, we describe a member of the Clp-HSP100 family of L . monocytogenes that harbors all the characteristics of the ClpB subclass, which is absent in the closely related gram-positive model organism, Bacillus subtilis. Transcriptional analysis of clpB revealed a heat shock-inducible ^A-type promoter . Potential binding sitesfor the CtsR regulator of stress response were identified inthe promoter region . In vivo and in vitro approaches were usedto show that expression of clpB is repressed by CtsR, a findingindicating that clpB is a novel member of the L . monocytogenesCtsR regulon . We showed that ClpB is involved in the pathogenicityof L . monocytogenes since the clpB mutant is significantly affectedby virulence in a murine model of infection; we also demonstratethat this effect is apparently not due to a defect in generalstress resistance . Indeed, ClpB is not involved in toleranceto heat, salt, detergent, puromycin, or cold stress, even thoughits synthesis is inducible by heat shock . However, ClpB wasshown to play a role in induced thermotolerance, allowing increasedresistance of L . monocytogenes to lethal temperatures . Thiswork gives the first example of a clpB gene directly controlledby CtsR and describes the first role for a ClpB protein in inducedthermotolerance and virulence in a gram-positive organism.

 
Listeria monocytogenes is a gram-positive pathogen implicated in food-borne infections and is responsible for meningitis, septicemia, and gastroenteritis—diseases with a high degreeof mortality for immunocompromised hosts . During the past fewyears, this bacterium has been extensively studied, and it hasbecome a model for intracellular growth [51] because of its abilities to escape from the phagosome, grow in the cytosol,and efficiently invade neighboring cells.

Several virulence proteins that are required for the key stepsof the infectious process have been identified to date . InlAand InlB are required for entrance of L . monocytogenes intoepithelial cells; listeriolysin O is required for escape fromthe phagosome; ActA is required for actin polymerization, cell-cellmobility, and invasion; and PlcB is required for lysis of thetwo-membrane vacuole [7] . All virulence genes identified sofar are under the positive control of a single regulator, PrfA.Due to the secondary structure of its mRNA, this activator,which acts as a thermosensor [28], is present only at the host temperature.

In addition to these major virulence factors are many proteins that are involved in pathogenicity of Listeria . These proteins, known as stress proteins, are important because they allow persistence and rapid adaptation during the infectious process . The accumulating body of data regarding several pathogens indicates that acidor oxidative stress proteins and [more recently] heat shockproteins [HSPs] and chaperones play an important role in virulence.Indeed, synthesis of the two major Staphylococcus aureus chaperones, DnaK and GroESL, was shown to be induced during infection ofhuman epithelial cells [52]; in L . monocytogenes, expression of the groESL operon is induced during intracellular infection, while DnaK is required for efficient phagocytosis with macrophages [15, 22] . Another class of stress proteins, the Clp family,has also been shown to play a major role in the virulence ofseveral pathogens: ClpP was shown to control expression of theattachment invasion locus [ail] of Yersinia enterocolitica,whereas in Salmonella enterica serovar Typhimurium, inactivationof clpP prevents growth and survival within macrophages [24,69] . Systematic genome-wide approaches such as signature-tagged mutagenesis revealed the roles of several clp genes, including clpE, clpC, and clpL of Streptococcus pneumoniae [23, 33, 48],as well as clpX of S . aureus [38].

Clp proteins are ubiquitous among prokaryotes and eukaryotes,and they function both as proteases and chaperones [19] . Bacterialgenomes are endowed with different sets of clp paralogs encodingClp-HSP100 ATPase subunits, belonging to groups A, B, C, D,E, or L, and that are distinguished by their N-terminal domainsand the central spacer regions between the two ATP-binding sites.clpP, which encodes the proteolytic subunit of the Clp ATP-dependentprotease, is usually present as a single copy, but up to fivecopies per genome can coexist, as shown in Streptomyces lividansand Streptomyces coelicolor [8, 65, 66] . The ClpP proteolytic subunit requires association with an ATPase subunit in orderto be active, giving rise to a multimeric complex presentingstructural and functional analogies with the eukaryotic proteasome[50] . The ATPase subunits can also act in the absence of ClpP,forming a smaller complex with chaperone activity . It is interestingthat ClpB of Escherichia coli does not interact with the proteolytic subunit and is exclusively considered a chaperone [68] . However,although some ClpB proteins have been characterized for both eukaryotes and bacteria, no phenotypes have been described as yet for low-G+C gram-positive bacteria.

In L . monocytogenes, three clp genes have been shown to play a role in virulence . ClpC is required for intracellular growth and in vivo survival in host tissues by promoting early escapefrom the phagosomal compartment [54, 55] and is also necessaryfor cell adhesion and invasion [44] . The ClpE ATPase plays arole in L . monocytogenes virulence also [43], and an L . monocytogenesclpP mutant presents a defect in intracellular replication [16].

A clpB mutant of Y . enterocolitica, a major gastrointestinal pathogen, presents a decrease in invasin and flagellin expression, characteristics that are encoded by the two virulence genesinv and fleB [2] . For S . enterica serovar Typhimurium, the clpBmutant was discovered during a systematic search for mutantsdeficient in colonization of the chicken alimentary tract andwas shown to be attenuated for virulence in 1-day-old chicks[64] . Finally, a clpB mutant of Francisella novicida was isolatedduring a screen for genes required for in vitro growth in thioglycolate-elicited mouse peritoneal macrophages [21].

Analysis of the complete genome of L . monocytogenes EGDe [18] reveals several uncharacterized genes encoding proteins belonging to the Clp family, two of which are preceded by potential binding sites for the CtsR regulator of stress response [10] . Here wehave characterized the clpB gene of L . monocytogenes . Usingboth in vivo and in vitro approaches, regulation of clpB wasstudied, showing a direct control by CtsR . This repression wasdemonstrated to be thermosensitive . A deletion mutant was constructed,and functional analysis revealed a role for ClpB in terms ofthe virulence of L . monocytogenes . We also show that, althoughClpB has no obvious role in terms of stress tolerance, it isrequired for induced thermotolerance of L . monocytogenes.

 
Bacterial strains, growth conditions, and transformation. Bacterial strains used in this work are listed in Table 1 . E.coli K12 strain TG1 [[lac proAB] supE thi hsd5 [F' traD36 proABlacI^q lacZ M15]] [17] was used for cloning experiments.

 
E . coli was grown in Luria-Bertani [LB] medium . Electroporation procedures were used for transformation with selection on LB plates supplemented with ampicillin [100 µg/ml], erythromycin[200 µg/ml], or kanamycin [25 µg/ml] . L . monocytogenesLO28 was routinely grown in brain heart infusion [BHI] complexmedium . Constructs were introduced into LO28 strains by electroporation.The following antibiotics were used at the indicated concentrations: erythromycin [8 µg/ml], kanamycin [50 µg/ml], andspectinomycin [60 µg/ml] . Bacillus subtilis was grownin LB medium and transformed as previously described by usingplasmid DNA [40] . Transformants were selected on SP plates supplementedwith chloramphenicol [5 µg/ml] or spectinomycin [100 µg/ml].

ß-Galactosidase activity was estimated on plates by 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside [X-Gal] hydrolysis . ß-Galactosidase-specific activities weredetermined as previously described [39-41] and were expressedas Miller units per milligram of protein.

Basal stress resistance experiments were performed as follows. Overnight cultures were diluted 100-fold in BHI medium and grownat 37°C with vigorous shaking until the optical densityat 600 nm [OD₆₀₀] reached 0.3 . Exponentially growing cultureswere then divided into two parts, and one of which was subjectedto one of the following stress conditions: 2% NaCl [wt/vol];0.01% sodium dodecyl sulfate [SDS] [wt/vol]; 15, 30, or 60 µgof puromycin per ml [values indicate final concentrations];or growth at 42, 44, 48, or 55°C . Growth was then monitoredfor an additional 3 h . For induced thermoresistance, overnightcultures were diluted 100-fold in BHI medium and placed at 37°Cwith vigorous shaking until the OD₆₀₀ reached 0.3 . Prior toheat treatment at 60°C, cultures were divided into two parts,one of which was maintained at 37°C, while the other half was preincubated at the nonlethal temperature of 48°C for20 min . Both cultures were then incubated at 60°C, and aliquotswere quickly transferred to ice, diluted in ice-cold BHI, andimmediately plated on BHI; CFU were then counted . The inducedthermoresistance experiment was repeated four times, yieldingthe same results.

DNA manipulations. Chromosomal DNA preparation, plasmid isolation, restrictionenzyme analysis, and amplification by PCR were performed accordingto standard protocols [57] . DNA sequences were determined bythe dideoxy chain termination method [59] using modified T7DNA polymerase [63] [Pharmacia] . DNA concentrations were calculatedby measuring UV spectroscopy at 260 nm.

Mutant and plasmid constructions. All oligonucleotide positions are given relative to the translationinitiation codon . The plasmids used in this study are listedin Table 1 . Plasmid pDL [71] was used for constructing transcriptional fusions with the Bacillus stearothermophilus bgaB gene, which encodes a thermostable ß-galactosidase [25], with subsequent integration at the B . subtilis amyE locus . A clpB'-bgaB transcriptional fusion was constructed using a 189-bp EcoRI/BamHI DNA fragment corresponding to the clpB upstream region, which was generated by PCR by using oligonucleotides ID73 [-193] [5'-GAAGAATTCATGTTCTTACTCCGCC-3']and ID74 [-5] [5'-GGAGGATCCTTATAAAAGATAAGTC-3'] . This fragmentwas cloned between the EcoRI and BamHI sites of plasmid pDLto give plasmid pDL73/74 . Linearization of this plasmid at theunique PstI site and transformation of the B . subtilis QB4991strain with selection for chloramphenicol resistance yieldedstrain QB8059, in which the clpB'-bgaB fusion was integratedas a single copy at the amyE locus . The linearized pxyl59/60plasmid [42] was then introduced in these strains by transformationand selection for spectinomycin to give strain QB8060, in whichthe L . monocytogenes ctsR gene is placed under the control ofthe PxylA xylose-inducible promoter and integrated as a singlecopy at the thrC locus.

A markerless clpB deletion mutant of L . monocytogenes was constructedusing plasmid pMADclpB . The mutant was constructed by firstusing PCR to generate two DNA fragments of 829 and 761 bp, usingoligonucleotide pairs AC189 [5'-AATGGATCCCACATCCGAGCGAGTAAACAC-3']and AC190 [5'-TAAGTCGACTCATTCGTCCTCCTTATAAAA-3'] and AC192 [5'-CACGTCGACTGAAAGGGAAAACTTTGGTTG-3']and AC193 [5'-TATCCATGGAATATTTATTTACTGGTTTTA-3'], corresponding,respectively, to the chromosomal DNA regions that are directlyupstream and downstream from the clpB gene . These fragmentswere cloned in pMAD, a pRN5101 derivative carrying a thermosensitiveorigin of replication [M . Arnaud and M . Débarbouillé,unpublished data], and the resulting pMADclpB plasmid was electroporatedin the LO28 strain with selection for erythromycin . Integrationand excision of pMADclpB was performed as previously described[4] but with a nonpermissive temperature growth of 40°C,thus yielding strain LM2000 [clpB], in which the entire clpBcoding sequence was removed . PCR amplifications were performedin order to confirm the gene deletion.

The ctsR deletion mutant was obtained by transforming the LO28 strain by plasmid pMADctsR . For this purpose, DNA fragmentsof 1,044 and 1,031 base pairs corresponding to the upstreamand downstream chromosomal DNA regions from ctsR were amplifiedby using oligonucleotide pairs AC212 [5'-GGCGGATCCCTCCTAAAGAGTAACGGAGGC-3']and AC213 [5'-ACTGAATTCCAATACTTGTTTCAAATAAGC-3'] and AC214 [5'-TGAGAATTCGGATTTTAGAGGCGATGTTAG-3']and AC215 [5'-TATCCATGGTCTTTATCAAAAGCATAAC-3'], respectively.The aphA3 kanamycin resistance gene, deprived of its transcriptioninitiation and termination signals, was then cloned at the EcoRIsite between the two fragments just described . The resultingpMADctsR plasmid was introduced into strain L028, and the integration-excision procedure was performed as was described for the clpB deletion, thus yielding strain LM2001.

Mouse virulence assay. Six- to 8-week-old pathogen-free Swiss female mice [Janvier,Le Genset St . Isle, France], were used in this study . Groupsof five mice were injected intravenously with doses of L . monocytogenesLO28 and clpB mutant ranging between 5 x 10⁵ and 5 x 10⁸ bacteria. Mortality was observed over a 14-day period . The 50% lethaldose was determined by the Probit method . Mice were killed bycervical dislocation in accordance with the policies of theAnimal Welfare Committee of the Faculté Necker [Paris,France].

Gel mobility shift DNA-binding assays. A 189-base-pair EcoRI/BamHI DNA fragment corresponding to thepromoter region of clpB was generated by PCR using oligonucleotides ID73 and ID74 . Radiolabeling, DNA-binding, and gel shift experiments were performed as previously described [10].

DNase I footprinting. A 229-base-pair DNA fragment used for DNase I footprinting wasprepared by PCR using Pfu DNA polymerase [Stratagene, La Jolla,Calif.] and oligonucleotides ID75 [-147] [5'-AAATTCAGAAGATCTGCCAACC-3']and ID76 [+82] [5'-CTTATGTTCTGATGCAATAGC-3'] . Labeling and DNaseI treatment were performed as previously described [10].

RNA extraction and primer extension. L . monocytogenes strains were grown in BHI medium at 37°Cwith aeration until the OD₆₀₀ reached 0.5; half of the culturewas then shifted to 42°C, and incubation was undertakenfor another 10 min . Cells were pelleted and frozen immediately,and RNA extraction and primer extension were then performedas previously described [5] using radiolabeled oligonucleotideAC209 [+22] [5'-GTGTAAATTTTTGTAAATCCATTC-3'] . Radioactive gelswere exposed to storage phosphor screens and scanned with aMolecular Dynamics Storm 860 optical scanner . Quantitation ofprimer extension products was performed using the ImageQuant5.1 software package [Molecular Dynamics].

 
Genome sequence analysis reveals ClpB ATPase in L . monocytogenes. Analysis of the complete genome of L . monocytogenes EGDe reveals a protein with a deduced 63% amino acid sequence identity with ClpB of Lactococcus lactis and 52% with that of E . coli [Fig. 1] . The ATG initiation codon of clpB is preceded by a classicalribosome binding site [RBS], tAAGGAGG, at a suitable distance,and this sequence encodes a predicted protein of 866 amino acidresidues with a calculated molecular mass of 97.5 kDa . We note that a GTG codon is located 151 codons downstream from the ATG codon and is also preceded by a typical RBS, [AgAGGAGG], atan appropriate distance of 7 bp . This potential internal translation initiation site suggests the existence, as shown for E . coli, of a smaller form of ClpB, with 716 amino acid residues anda theoretical molecular mass of 80.6 kDa [47, 62].

 
Analysis of the amino acid sequence of the protein revealedtwo typical Clp signature motifs [60, 61] . Indeed, two ATP-bindingsites are present, one with a single Walker A and two WalkerB motifs and the other presenting only one of each Walker motif,a finding that is characteristic of HSP100 proteins [Fig . 1].There are also two repeated Clp amino-terminal domain motifs[ClpN], which are typical of ClpA and ClpB proteins [3] butare also present in most ClpC proteins.

This Clp ATPase also presents a long central domain separatingthe two ATP-binding sites, approximately 130 amino acids inlength, which is characteristic of ClpB proteins . This domaincontains a predicted coiled-coil motif [37; http://smart.embl-heidelberg.de], which may be involved in multimerization . Analysis of the carboxy-terminal domain, between the second ATP-binding site and the PDZ-like sensor and substrate discrimination domain, revealed the absenceof the IGF loop required for interaction with the ClpP proteolytic subunit [30] . This suggests that, as in E . coli, ClpB functionin L . monocytogenes is restricted to chaperone activity withoutany interaction with ClpP.

clpB is a novel member of the L . monocytogenes CtsR regulon. Three clp genes of L . monocytogenes have been shown to be controlledby the CtsR repressor of stress response genes [42], and manyclpB genes are known to be heat shock-induced genes . To investigatea potential mechanism of transcriptional regulation of clpB,we analyzed the sequence of the promoter region, thereby revealingthe presence of a potential binding site for CtsR [GGTCAAA AAAGGTCAgA] [see Fig . 3B], suggesting that ClpB may be a novelmember of the L . monocytogenes CtsR regulon.

 
We used B . subtilis as a heterologous host to test whether CtsR plays a role in controlling clpB expression . For this purpose, a transcriptional fusion was constructed between the L . monocytogenes clpB promoter region and the bgaB gene of B . stearothermophilus,which encodes a thermostable ß-galactosidase [25][see Materials and Methods] . The fusion was integrated as asingle copy at the amyE locus of B . subtilis strain QB4991,in which the endogenous ctsR gene is deleted [10] . The L . monocytogenesctsR gene was then integrated as a single copy at the thrC locus, under the control of the PxylA xylose-inducible promoter, by using plasmid pxyl59/60 [42], thus leading to strain QB8060.

Strain QB8060 was grown at 37°C in the presence or absenceof xylose, and ß-galactosidase activities were assayed.As shown in Fig. 2, clpB'-bgaB was expressed up to approximately 550 Miller units · mg^-1 of protein at 37°C in the absence of the repressor [without xylose] . This expression was repressed 15-fold [35 Miller units · mg^-1 of protein]in the presence of CtsR of L . monocytogenes [with xylose] . However, when the culture was shifted to high-temperature conditions [48°C] instead of 37°C, expression was induced up to47-fold [1,650 Miller units · mg^-1 of protein] in thepresence of CtsR [with xylose; data not shown] . These resultsdemonstrate that clpB of L . monocytogenes is under negativeregulation by CtsR and that this repression is thermosensitive.

 
clpB is expressed from a ^A-dependent heat-inducible promoter. In order to demonstrate the thermoinducibility of clpB and arole for CtsR in its regulation, an analysis of clpB transcriptionin L . monocytogenes was performed by using primer extensionexperiments . First, the transcription initiation site was determinedby using RNA from L . monocytogenes cells grown in BHI at 37°Cand harvested in mid-exponential phase [see Materials and Methods].This procedure revealed a single transcriptional start site45 bp upstream from the ATG start codon of clpB [Fig . 3A and B].Consensus -10 and -35 sequences recognized by the E^A RNApolymerase holoenzyme were identified upstream from the transcriptionalstart site, suggesting a ^A-dependent promoter [Fig . 3B] . A comparativetranscriptional analysis of RNA expression at 37 and 42°Cwas performed by primer extension . As shown in Fig . 3A, clpBwas expressed at a low basal level during growth in BHI at 37°C[Fig. 3A, lane 1], and transcription was increased fourfold when the culture was shifted to 42°C [Fig . 3A, lane 3], a finding which is consistent with a thermosensitive transcriptional regulation.

Repression of clpB by CtsR was examined in vivo in L . monocytogenes. The LM2001 ctsR mutant strain was constructed by deleting theentire ctsR coding sequence and replacing it with the aphA3kanamycin resistance gene . This resistance cassette was deprivedof its transcription initiation and termination signals in orderto rule out any polar effects on expression of the downstreamgenes . Expression of clpB at 37°C in the wild-type [L028][Fig . 3A, lane 1] and ctsR [LM2001] [Fig . 3A, lane 2] strainswas followed by primer extension analysis, which revealed increasedtranscription of clpB [6.5-fold] in the absence of CtsR . Itis interesting that clpB derepression at 42°C is only partialsince expression levels are higher in the ctsR deletion mutantat 37°C [Fig. 3A, lane 2] than in the wild-type strain at42°C [Fig . 3A, lane 3], thus suggesting a limited inactivation of CtsR at this temperature . In conclusion, the in vivo evidence indicates that clpB expression is repressed by CtsR and is heat shock inducible.

CtsR binds specifically to the clpB promoter region. An in vitro approach was used to demonstrate a direct interaction between CtsR and the clpB promoter region . Histidine-tagged CtsR of L . monocytogenes, presenting a carboxy-terminal extension of six histidine residues, was overproduced and purified by using a Ni-nitrilotriacetic acid agarose column [42] . This recombinantprotein was used in gel mobility shift DNA-binding assays witha 189-bp radiolabeled PCR-generated DNA fragment corresponding to the clpB promoter region . This DNA fragment, extending from positions -193 to -5 relative to the translation initiation codon, was incubated with increasing amounts of purified CtsRin the presence of nonspecific competitor DNA [poly-[dI-dC]].As shown in Fig . 4A, CtsR bound specifically to the radiolabeled fragment, leading to progressive displacement of the probe to the single higher-molecular-weight protein/DNA complex . Althoughan incomplete displacement was observed even at the highestCtsR concentrations, the single DNA/protein complex suggeststhe presence of only one CtsR-binding site in this promoter.These results demonstrate that CtsR of L . monocytogenes repressesclpB expression by binding directly to the promoter region.

 
DNase I footprinting assays were performed for L . monocytogenes DNA fragments corresponding to the clpB promoter region in order to precisely determine the location of the CtsR-binding site. When the nontemplate strand of clpB DNA was end labeled, CtsR protected a region extending from positions -42 to -20 [Fig. 4B and D] . When the template strand was end labeled, the protectedregion extended from positions -46 to -24 [Fig. 4C and D] . Allpositions given are relative to the respective translationalstart site.

A single region within the clpB promoter is protected from DNase I cleavage, a finding which is in agreement with the single protein/DNA complex observed in the gel mobility shift DNA-binding assay [Fig . 4A] . This protected region overlaps the transcriptionalstart site of clpB and contains the predicted CtsR direct-repeatrecognition sequence [GGTCAAA AAA GGTCAGA] [Fig. 4D] . Theseresults indicate that CtsR negatively regulates clpB expressionby directly binding to its operator sequence in the promoterregion.

In conclusion, using both in vitro and in vivo approaches, wehave shown that L . monocytogenes clpB is a heat shock gene thatis under the negative regulation of CtsR, extending the L . monocytogenes CtsR regulon.

ClpB is involved in virulence of L . monocytogenes. Since ClpP, ClpC, and ClpE of L . monocytogenes have been shown to play a role in virulence [16, 43, 54], we therefore examinedthe virulence of an L . monocytogenes clpB mutant in a murinemodel.

For this purpose, we constructed the LM2000 mutant strain ofL . monocytogenes, in which the entire coding sequence of clpB was deleted [see Materials and Methods] . Virulence of the clpB strain was assayed by intravenous inoculation as described in Materials and Methods and was compared to that of the wild-typeL028 strain . The 50% lethal dose of the clpB mutant was 5.4x 10^6.3 bacteria, whereas that of L028 was 5.4 x 10^4.2 bacteria.The clpB mutant thus displays a significant decrease in virulence[100-fold].

We monitored the survival of mice for 12 days after an inoculation of 5.4 x 10⁵ bacteria . Mice infected with the wild-type strainbegan to die after 5 days, and all were dead after the 10thday, whereas all animals infected by strain LM2000 [clpB] werestill alive after 12 days [Fig . 5] . These results clearly showthat ClpB plays a significant role in the pathogenicity of L.monocytogenes.

 
In order to determine whether ClpB of Listeria monocytogenes affects expression of virulence genes, primer extension experiments were carried out for the wild-type and for the clpB and ctsR mutants in order to compare expression of the L . monocytogenes hly gene, which encodes listeriolysin O, a major virulence determinant.Expression was identical for all three strains [data not shown],a finding which indicated that the major virulence PrfA regulonis not controlled by ClpB or CtsR and that the role for ClpB in virulence is most likely due to its chaperone activity rather than to a regulatory role in virulence gene expression.

ClpB is not required for general stress response but is necessary for heat shock-induced thermotolerance. Since several Clp proteins are involved in virulence and becausemany are also essential for resistance to various stress conditions,one might argue that Clp protein effects on pathogenicity maybe indirect consequences of generally lowered cell fitness,thus leading to increased sensitivity to stress when invadingthe host.

A functional analysis of ClpB was undertaken, during which survival of the clpB mutant was examined under different stress conditions.The LM2000 [clpB] mutant strain had no obvious phenotype, sincethe mutant cells showed no morphological defects and becausethe growth curve in BHI at 37°C was identical to that ofthe LO28 reference strain [data not shown].

The stress resistance of the clpB strain was evaluated undervarious conditions, such as heat stress, treatment with puromycin,the presence of salt, and SDS-induced stress, all of which areknown to require the activity of other Clp proteins . Wild-typeand mutant strains were grown in BHI medium until an OD₆₀₀ of0.3 was reached, cultures were divided into two parts, and onepart was subjected to stress conditions [see Materials and Methods].The results presented in Fig . 6 summarize data obtained fortypical growth curves for each stress condition . As shown inFig . 6, growth of the wild-type and mutant strains was affectedwhen the temperature was equal to or greater than 42°C,when the concentration of puromycin was greater than 30 µg/ml,or in the presence of 0.01% SDS . However, no difference wasobserved between the clpB and the L028 reference strain, sincethe two strains grew equally well under all conditions tested.In conclusion, L . monocytogenes ClpB is not required for generalstress adaptation, a finding that is contrary to the situationfor gram-negative bacteria such as E . coli [29], Brucella suis[12], or Helicobacter pylori [1].

 
A recent study reports the induction of L . monocytogenes clpB during growth at low temperature [36], a condition which seemsto induce the activity of most of the general stress proteins; for the cyanobacterium Synechococcus sp., ClpB was shown to be involved in cold adaptation [49] . Growth at low temperatureis an important part of the L . monocytogenes life cycle andis one which favors food contamination and outbreaks of food-bornedisease . The role of ClpB in adaptation of L . monocytogenesto cold stress was tested . An overnight culture grown at roomtemperature was diluted and placed at 5°C for 4 days or was first grown to the mid-exponential phase at 37°C before shifting the culture to a temperature of 5°C . In both cases,the growth rate of the clpB mutant at 5°C was the same asthat of the parental strain [data not shown], suggesting thatClpB of L . monocytogenes is not involved with adaptation tocold stress.

It was previously shown that L . monocytogenes has a higher survival rate to lethal temperatures following previous exposure to a sublethal temperature [46, 56] . This phenomenon is known asinduced thermotolerance, a characteristic which stands in contrastto basal thermotolerance and has been described as occurringin many bacteria . ClpB was shown to be required for inducedthermotolerance in the cyanobacterium Synechococcus sp . [13]and in the eukaryote Saccharomyces cerevisiae [58] . In orderto test the involvement of ClpB in induced thermotolerance ofL . monocytogenes, we incubated both wild-type L028 and clpB mutant strains in liquid BHI medium at 37°C until an OD₆₀₀ of 0.3 was reached . The cultures were divided into two parts; one half was maintained at 37°C, and the other was preincubatedat 48°C for 20 min . Both cultures were then subjected toheat treatment at 60°C . As shown in Fig . 7, preincubatedwild-type cells presented an increased resistance to lethalheat shock, since after 5 min of incubation at 60°C, thesurvival rate was approximately 100-fold higher than that foruntreated cells . In contrast, no induced thermotolerance couldbe observed for the clpB strain, which remained as sensitiveto lethal temperatures as were the untreated cells . Consequently,contrary to the situation for wild-type L028, a preincubationat 48°C did not protect clpB cells, thus revealing a rolefor ClpB in induced thermotolerance.

 
Clp-HSP100 proteins make up a ubiquitous family of ATPases thatact both as chaperones and as ATPase subunits for the Clp ATP-dependent protease . Most of them are induced by stress and are implicatedin stress tolerance . Moreover, Clp proteins are involved incrucial steps of the infectious process for many gram-positiveand gram-negative bacteria as well as in lower eukaryotes . InL . monocytogenes, ClpC, ClpP, and ClpE are involved in virulenceand are required for thermotolerance and resistance to saltstress [16, 43, 44, 54] . Their expression is thermoinducibleand is under the negative control of CtsR, a repressor thatbinds to a heptad operator sequence in the promoter region [42].

Analysis of the complete sequence of L . monocytogenes EGDe [18] revealed several new clp genes . Just as for its closest relative, B . subtilis, we noted the presence of genes encoding orthologs to ATPase subunits ClpY [65.5% identity] and ClpX [81% identity] and to the proteolytic subunit ClpQ [78.5% identity] . Surprisingly, and contrary to the situation for B . subtilis, there are two additional clp genes, both of which are preceded by potential operator sites for the CtsR repressor in their promoter regions. One of the encoded proteins shared 40% amino acid sequence identity with the ClpP proteolytic subunit of L . monocytogenes and is now referred to as ClpP2.

The second new clp gene revealed during our analysis has no ortholog in the low-G+C gram-positive model bacterium B . subtilis but shares strong similarities with clpB of L . lactis . We performeda systematic search for ClpB homologs to determine the extentof its distribution among low-G+C gram-positive bacterial genomes.Unlike the situation for B . subtilis, ClpB orthologs are foundin all staphylococci, clostridia, and enterococci, as well asin L . lactis, Listeria innocua, and most bacilli . Streptococciseem to be the only group without this homolog, despite thepresence in S . mutans of a ClpB-like protein presenting thecharacteristically long spacer region between both ATP-binding sites [34] . However, the very divergent sequence places thisparalog far from all the other known eubacterial clpB genes,suggesting a recent acquisition by way of horizontal transfer. Consequently, ClpB ATPases are well represented among low-G+C gram-positive bacteria.

Examination of the clpB promoter sequence revealed a typical ^A promoter and a potential CtsR-binding site . In this work,we showed that CtsR represses the expression of clpB in L . monocytogenes. We showed in vitro that CtsR binds directly to the clpB promoter region . The gel mobility shift experiments reveal a single protein/DNA complex . This was confirmed by DNase I footprints in which only one protected region was observed, a finding which correspondedto the predicted CtsR box and overlapping the transcriptionalstart site.

A detailed DNA motif analysis of the complete genome of L . monocytogenes allowed us to determine that there appear to be only five members of the CtsR regulon: the ctsR-clpC operon, clpP, clpE, clpB,and potentially clpP2 . Interestingly, although the dnaK operonis also preceded by a canonical CtsR operator, it does not seemto be controlled by this regulator, although the repressor canbind to this sequence in vitro [4].

Stress induction of clp genes is generally correlated with a role in stress resistance . Data from E . coli suggest that, unlike the other Clp ATPases, ClpB does not associate with the ClpP proteolytic subunit and has no effect on protease activity [68]; consequently, ClpB acts exclusively as a chaperone.

In agreement with this activity, ClpB has been shown to havethree closely related functions in several gram-negative bacteriaand in eukaryotes: [i] resistance to high-temperature stressin H . pylori, B . suis, and E . coli [1, 12, 29, 62]; [ii] cold acclimatization in Synechococcus sp.; and [iii] induced thermotolerance to lethal stress in the cyanobacterium Synechococcus sp . [13] and in the eukaryote S . cerevisiae [35, 58] . HSP101, a memberof the Clp/HSP100 family that is present in plants, has alsobeen shown to be implicated in induced thermotolerance in bothArabidopsis thaliana [53] and maize [45] . However, until now,only two clpB genes have been described for gram-positive bacteria,those of Streptomyces albus G [20] and L . lactis [27], and noobvious phenotype was associated with the respective mutants. Indeed, the clpB mutant of L . lactis was still resistant totemperature, salt, and puromycin stresses [27], and no thermosensitivity was observed for the S . albus mutant [C . Grandvalet, personal communication], even though both genes were shown to be thermoinducible. Here, we demonstrate that ClpB is required for induced thermotolerance of L . monocytogenes, which allows for better survival of lethal conditions when cells have been exposed to a nonlethal stress.

ClpB is required for induced thermotolerance of L . monocytogenes; this fact may contribute to the persistence of this bacterium and the health hazard it constitutes . Indeed, this bacteriumhas the ability to grow in a wide range of temperatures, evenduring the refrigeration process or after high-temperature short-time pasteurization and is considered one of the most thermotolerant bacteria among non-spore-forming food-borne pathogens [11, 14].It is clear that the ability of L . monocytogenes to grow athigh temperatures is an important problem for food processing,and our results suggest that ClpB may be partially responsiblefor this adaptation faculty.

A deletion of clpB is associated with a reduction in virulence of several eukaryotes and gram-negative bacteria, such as Leishmania major [26], Leishmania donovani [6, 31, 32], S . enterica serovar Typhimurium [64], Y . enterocolitica [2], and F . novicida [21].However, the exact function of ClpB—and more generally,that of Clp homologs—in virulence is still unclear becausetheir targets have not yet been discovered . Clp proteins, becauseof their central role in protein folding, are important factorsfor efficient growth and cell fitness . ClpP proteolytic subunits,for example, have pleiotropic roles, and their deletion, evenin optimum conditions, greatly affects growth [5, 40] . In mostcases, Clp proteins involved in virulence are also requiredfor stress survival, and since infection is one of the moststressful conditions encountered by bacteria, one can arguethat effects observed in a clp deletion mutant are due to adeficiency in cell fitness . We have shown here that ClpB is not required for general stress survival of L . monocytogenes, with the exception of induced thermotolerance at 60°C . Thus,it is tempting to speculate that the significant reduction inL . monocytogenes virulence of the clpB mutant might not be dueto a reduction in survival ability or in adaptation to stressfulconditions but rather to a specific alteration in a key processfor pathogenic development, where ClpB probably acts as a chaperone.This speculation is supported by the fact that expression ofthe hly gene, a major virulence determinant belonging to thePrfA regulon, is not modified in the clpB or ctsR mutants.

In conclusion, our results demonstrate a role for ClpB in induced thermotolerance and present the first evidence for a role forClpB in virulence of L . monocytogenes; our work constitutesthe first description of phenotypes for a clpB gene in gram-positive bacteria.

 
We are grateful to G . Rapoport for critical reading of the manuscript, and we thank P . Berche, in whose laboratory part of this work was carried out.

This work was supported by research funds from the Institut Pasteur, Centre National de Recherche Scientifique, UniversitéParis 7, European Commission [grant number QLG2-CT-1999-01455],Ministčre de la Défense [DélégationGénérale pour l'Armement, grant number 0034069004707501],and the Programme de Recherche Fondamentale en Microbiologie,Maladies Infectieuses et Parasitaires of the Ministčre de la Recherche . I.D . and A.C . were the recipients of a fellowship from the Ministčre de l'Education Nationale de la Rechercheet de la Technologie, and A.C . was the recipient of a fellowshipfrom the Fondation pour la Recherche Médicale [FRM] andthe CANAM [Caisse Nationale d'Assurance Maladie et Maternitédes Travailleurs Non Salariés des Professions Non Agricoles].

 
* Corresponding author . Mailing address: Unité de Biochimie Microbienne, Institut Pasteur, 25 rue du Dr . Roux, 75724 Paris Cedex 15, France . Phone: 33-1-45-68-88-09 . Fax: 33-1-45-68-89-38 . E-mail: tmsadek@pasteur.fr .

A.C . and I.D . contributed equally to this report.

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