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Journal of Bacteriology, May 2005, p. 3455-3464, Vol. 187, No. 10
0021-9193/05/$08.00+0     doi:10.1128/JB.187.10.3455-3464.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Role of mutS and mutL Genes in Hypermutability and Recombination in Staphylococcus aureus

Anne-Laure Prunier and Roland Leclercq*

Service de Microbiologie and EA 2128 Relations hôte et microorganismes des épithéliums, Hôpital Côte de Nacre, Université de Caen, 14033 Caen cedex, France

Received 8 December 2004/ Accepted 11 February 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The mutator phenotype has been linked in several bacterial genera to a defect in the methyl-mismatch repair system, in which the major components are MutS and MutL. This system is involved both in mismatch repair and in prevention of recombination between homeologous fragments in Escherichia coli and has been shown to play an important role in the adaptation of bacterial populations in changing and stressful environments. In this report we describe the molecular analysis of the mutS and mutL genes of Staphylococcus aureus. A genetic analysis of the mutSL region was performed in S. aureus RN4220. Reverse transcriptase PCR experiments confirmed the operon structure already reported in other gram-positive organisms. Insertional inactivation of mutS and mutL genes and complementation showed the role of both genes in hypermutability in this species. We also designed an in vitro model to study the role of MutS and MutL in homeologous recombination in S. aureus. For this purpose, we constructed a bank of S. aureus RN4220 and mutS and mutL mutants containing the integrative thermosensitive vector pBT1 in which fragments with various levels of identity (74% to 100%) to the S. aureus sodA gene were cloned. MutS and MutL proteins seemed to have a limited effect on the control of homeologous recombination. Sequence of mutS and mutL genes was analyzed in 11 hypermutable S. aureus clinical isolates. In four of five isolates with mutated or deleted mutS or mutL genes, a relationship between alterations and mutator phenotypes could be established by negative complementation of the mutS or mutL mutants.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Hypermutable strains display higher mutation frequencies than their wild-type (wt) counterparts (22). They have been shown to play an important role in the adaptation of bacterial populations in changing and stressful environments (42). Adaptation abilities are particularly relevant in the case of pathogenic bacteria like Staphylococcus aureus, for which conditions inside the host can be extreme and subjected to rapid changes. The mutator phenotype has been linked in several bacterial genera to a defect in the methyl-mismatch repair (MMR) system (9). The primary role of this system is to correct mismatched or unpaired bases that escaped the proofreading exonuclease of the replicating DNA polymerase (16). In Escherichia coli, initiation of mismatch repair requires MutS, MutL, and MutH, which are able to recognize the mismatch and to cleave the transiently unmethylated lagging strand of DNA immediately after replication, allowing the classical DNA repair enzymes (helicase, exonucleases, DNA polymerase, and ligase) to fix the mismatch (18). Gram-positive bacteria and eukaryotes have an MMR system that is functionally equivalent to that of E. coli, although there are some differences. First, they lack a MutH equivalent; furthermore, the discrimination between the leading strand and lagging strand that needs to be repaired is not based on the transient state of DNA hemimethylation but probably on the presence of single-strand breaks in the newly synthesized strand (23).

Previous work with E. coli and Salmonella proved that the MMR system acts as a barrier to the recombination between divergent sequences which occurs during genetic exchanges, such as conjugational crosses, transduction with phages, or transformation (19, 35): recombination between DNA substrates containing mismatches occurs much less frequently than between identical sequences. The frequency of recombination between homeologous sequences (partially divergent DNA sequences) is often dramatically increased in MMR-defective bacteria (50-52). In fact, recombination frequency decreases exponentially with increasing sequence divergence (46). This observation holds true in Streptococcus pneumoniae, where the hexAB system is functionally equivalent to the MMR system in E. coli (15).

For S. aureus, little is known about the genetic basis of hypermutability. A previous study has shown that inactivation of mutS in S. aureus RN4220 led to a hypermutator phenotype (28). It has also been suggested that hypermutable S. aureus might exist in naturally occurring populations (5, 40). Although the study by O'Neill and Chopra (28) failed to detect any strong mutator strain in a large collection of clinical isolates, we recently detected a high proportion of hypermutable S. aureus among strains isolated from cystic fibrosis (CF) patients (33).

In this report we describe the molecular analysis of mutS and mutL genes in S. aureus. A genetic analysis of the mutSL region was performed, confirming the operon structure already reported in other gram-positive organisms (6, 21, 47). Sequence of both genes was analyzed in hypermutable S. aureus clinical strains isolated in our previous study. Implication of the mutS and mutL mutations in hypermutability of the strains was investigated. We also designed an in vitro model to study the role of MutS and MutL in homeologous recombination in S. aureus.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains and plasmids. E. coli DH10B and S. aureus RN4220 were used as hosts for cloning of the different plasmids, listed in Table 1.


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  		TABLE 1. Plasmids used in this study

 
Transformation procedures. Electrocompetent cells were prepared as described by Schenk and Laddaga (39) and Sambrook and Russel (36) for S. aureus and E. coli, respectively. Plasmid transformants were selected on brain heart infusion (BHI) agar medium (Difco Laboratories, Detroit, MI) supplemented with antibiotics at the following concentrations: amoxicillin, 100 µg/ml; chloramphenicol, 20 µg/ml; erythromycin, 500 µg/ml for E. coli and 5 µg/ml for S. aureus; gentamicin, 10 µg/ml for E. coli and 30 µg/ml for S. aureus; kanamycin, 50 µg/ml.

DNA techniques. PCRs were performed with the primers listed in Table 2, generally using EurobioTaqII (Eurobio, Les Ulis, France). Plasmids were extracted from E. coli transformants with the Sigma GenElute plasmid miniprep kit (St. Louis, MO).


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  		TABLE 2. Primers used in this study

 
For sequencing, fragments obtained from PCR with specific primers were purified using ExoSAP-IT (USB, Cleveland, OH), labeled with the DNA sequencing kit from Applied Biosystems (Foster City, CA), and sequenced in an ABI Prism 377 automated device (Applied Biosystems).

Reverse transcriptase PCR (RT-PCR) was performed on S. aureus RN4220 and mutS1 (RN4220 inactivated in the mutS gene by insertion of the whole pBT1 plasmid; see below). Total RNA of the strains was extracted from overnight cultures in BHI broth at 37°C using the High Pure RNA isolation kit (Roche, Indianapolis, IN), which includes a DNase treatment. A control PCR was performed on RNA extracts to control the absence of DNA, and a second DNase treatment was performed on extracts when necessary, followed by the subsequent steps of the High Pure RNA isolation kit protocol. The QIAGEN OneStep RT-PCR kit (Valencia, CA) was used, using primers shown in Fig. 1A.



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  		FIG. 1. Genetic organization of the mutSL region in S. aureus and results of the transcriptional analysis by RT-PCR. (A) Schematic organization of the mutSL locus in S. aureus determined by in silico analysis. The hairpin symbol indicates a putative transcription terminator. The size of each intergenic region is indicated. Localization of the pBT1 insertion in the mutS gene of the S. aureus mutS1 strain is indicated. The black arrows flanking the dotted lines indicate the positions of the primers used in the RT-PCR analysis. Numbers in parentheses indicate the sizes of the amplified fragments. (B) Gel electrophoresis of the fragments obtained by RT-PCR. C, negative control without RNA; 4220, amplification obtained with S. aureus RN4220 total RNA extract; mutS, amplification obtained with S. aureus mutS1 total RNA extract. Primers used for each amplification are indicated.

 
Construction of strains disrupted for the mutS or mutL gene. A strain disrupted for the mutS gene (S. aureus mutS1) was constructed by insertional inactivation of the gene using the pBT1 thermosensitive shuttle plasmid (4). A 278-bp mutS fragment was amplified using the SBamHI- and SEcoRI-modified primers (Table 2) and subsequently cloned into the corresponding sites of the pBT1 plasmid. The resulting plasmid was electroporated into S. aureus RN4220, and the recombination between the construct and the homologous fragment in the chromosome was forced by submitting the strains to three successive subcultures at 42°C in BHI broth containing chloramphenicol.

The S. aureus mutS2 strain was obtained as shown in Fig. 2. Basically, a kanamycin resistance cassette was cloned between two adjacent fragments of the mutS gene in the pBT1 thermosensitive shuttle plasmid. The construct was then introduced into S. aureus RN4220, and the double-recombination event was obtained by submitting the strain to several subcultures at 42°C with the selective pressure of kanamycin, as previously described (4). The recombinant S. aureus mutS2 strain was finally retrieved among Kanr Chls colonies (recognized by counterselection), indicating successful replacement recombination. Construction of the S. aureus mutL strain was performed following the same procedure with the mutL gene inactivation primers (Table 2), resulting in the construction of the pBT1mutL inactivation vector. Efficient insertions were verified by PCR.



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  		FIG. 2. Construction of the S. aureus mutS2 strain. Two fragments of mutS from S. aureus RN4220 (PCR 1, SNheIU5'/SSalIL5'; PCR 2, SKpnIU3'/SPvuIIL3') and the totality of the Tn1545 kanamycin resistance cassette (PCR 3, KSalIU/KKpnIL) were amplified, restricted with the appropriate enzymes, and ligated. The construct obtained was amplified (PCR 4, SNheIU5'/SPvuIIL3'), and a NheI/PvuII restriction was performed on the resulting fragment and on the pBT1 thermosensitive vector (bla, ß-lactamase gene; cat, chloramphenicol acetyltransferase gene; Ts, thermosensitivity; MCS, multiple cloning site). Both were ligated together, and the resulting construct (pBT1mutS2) was introduced into S. aureus RN4220. The double-recombination event was obtained at 42°C as previously described (4), using kanamycin (12.5 µg/ml) as the selective marker.

 
Cloning of the different mutS and mutL genes. PCR was performed with specific oligonucleotides (modified to contain restriction sites) using the Platinum Pfx DNA polymerase (Invitrogen, Carlsbad, CA) in a final volume of 50 µl containing 5 µl of DNA template extracted with Instagene Matrix (Bio-Rad, Marne-La-Coquette, France), 10 pmol of deoxynucleoside triphosphates, 20 pmol of each primer, 1 µl of supplied MgSO4, 10 µl of supplied 10x buffer, 5 µl of supplied PCR Enhancer 10X, and 0.5 µl of the Pfx DNA polymerase. The PCR mixture was denatured (5 min at 94°C) and then subjected to 40 cycles of amplification (30 s of denaturation at 94°C, 30 s of annealing at 40°C, 3 min of elongation at 68°C). The fragments obtained were purified using the MicroSpin S-400 HR columns (Amersham Biosciences, Piscataway, NJ) and restricted with the appropriate enzymes (Table 2) from New England Biolabs (Beverly, MA). The T4 DNA ligase from New England Biolabs was used for ligations. Effectiveness of the cloning was checked by determining the sequence of the cloned fragments. Constructs were finally electroporated into electrocompetent E. coli cells.

Determination of mutation frequencies. Mutation frequencies were determined as previously described (28) on BHI agar plates supplemented with 100 µg/ml of rifampin. For every strain, the experiment was repeated at least four times, and results are indicated as a mean with calculated standard deviation.

Determination of recombination frequencies. sodA fragments with various percentages of identity with the corresponding S. aureus RN4220 sodA fragment were amplified from S. aureus RN4220 (100% identity), Staphylococcus epidermidis RP62A (87.5% identity), Staphylococcus auricularis UCN44 (82.3% identity), and Staphylococcus sciuri UCN45 (74% identity) with the degenerated staphylococcal sodA primers (Table 2), as described previously (30). The nucleotide substitutions observed in these fragments were homogeneously distributed within the DNA sequences compared to the S. aureus RN4220 sodA fragment. To obtain additional DNA fragments displaying 95 and 93% identity with the wt S. aureus sodA gene with a random distribution of the mutations, MgCl2 was replaced by 400 µM MnCl2 in PCR experiments (denaturation: 5 min at 94°C, 50 cycles of amplification [30 s of denaturation at 94°C, 30 s of annealing at 50°C, 5 min of elongation at 72°C]; final elongation, 10 min at 72°C) using specific S. aureus sodA primers (Table 2). The resulting fragments were directly cloned into pGEM-T Easy vector system I (Promega, Madison, WI) before sequencing with the universal M13 primers and subcloning into pBT1. The recombinant plasmids were introduced into S. aureus RN4220, mutS2, and mutL strains by electroporation as previously described (39).

To estimate the frequency at which the different pBT1SodA plasmids recombine with the wt chromosomal sodA in the S. aureus RN4220, mutS2, and mutL strains, each strain was submitted to a first overnight culture at 37°C. Then, three subsequent overnight subcultures inoculated with 1/100 dilutions were carried out at 42°C in a shacked water bath under the selective pressure of chloramphenicol (and kanamycin for the mutS2 and mutL strains), so that only the recombinant derivatives can survive. These conditions were chosen since after three subcultures at 42°C with chloramphenicol, S. aureus RN4220 containing the pBT1 thermosensitive plasmid did not survive. The initial inoculum for each strain was measured by plating dilutions of an overnight 37°C culture on BHI plates containing chloramphenicol (and kanamycin for the mutS2 and mutL strains). The 42°C subcultures were numbered each day by the same technique. For every strain, the experiment was repeated between three and six times, and results are indicated as the mean ratio of the logarithm of survival after three subcultures (total count at the third day) to the logarithm of the corresponding initial inoculum with calculated standard deviation. Variance analysis was carried out using the F test.

Homology searches, genome analysis, and multiple sequence alignments. Sequences of the mutS, mutL, and sodA genes of S. aureus were retrieved from the NIH GenBank database (www.ncbi.nlm.nih.gov). Alignments were performed using the Clustalw program (43), and identity percentages were retrieved using the Align program (24).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Genetic organization of the mutSL region. The in silico analysis of the seven S. aureus genomic complete sequences available at the NIH website (www.ncbi.nlm.nih.gov) revealed that, as already described for Bacillus subtilis (6), Listeria monocytogenes (21), and Enterococcus faecium (47), mutS and mutL are adjacent and in the same orientation in the S. aureus chromosome (Fig. 1A). The 302-bp intergenic region which precedes the mutS gene comprises a putative transcription terminator for the open reading frame (ORF) SA1136, forming a hairpin loop ({Delta}G = –17.3 kJ/mol) and a potential transcription initiation sequence. The mutL gene starts 12 bp after the stop codon of mutS. As in L. monocytogenes, mutL is immediately followed (14 bp after the stop codon) by a third ORF putatively encoding a GlpP protein (regulator for the glycerol uptake operon). The downstream sequence differs in S. aureus by the presence 36 bp downstream of glpP of a fourth 90-bp ORF encoding a hypothetical protein, followed by a large 348-bp non coding region containing another putative transcription terminator ({Delta}G = –22.4 kJ/mol) and another ORF which encodes GlpF (glycerol uptake facilitator).

Deduced amino acid sequences of mutSL genes in the seven S. aureus genomic complete sequences were submitted to multiple alignments. We found that the MutS and MutL sequences were well conserved. There is one amino acid exchange in the mutS gene of S. aureus 8325 (E840K), and there are five exchanges in the mutS gene of S. aureus MRSA252 (N181H, V239A, T415M, Q431H, and L811S). There are also several substitutions in the central part of MutL in some strains. On the basis of these alignments, we designed 10 pairs of deoxynucleotide primers that allowed amplification of overlapping fragments in mutS/L genes. The sequence of the genes was determined in S. aureus RN4220 and did not reveal any additional substitution.

We performed a transcriptional analysis of the mutSL locus by RT-PCR on the wt strain (RN4220) and on the mutS strain in order to confirm the hypothesis of an operon structure for mutSL and to rule out the possibility that the mutS gene insertional inactivation could generate a polarity effect on mutL and glpP gene expression. Using primers designed to amplify overlapping fragments of mutS, mutL, and glpP genes, we found that the mutS, mutL, and glpP genes were cotranscribed, confirming that the three genes belong to the same operon (Fig. 1). Moreover, the mutL and glpP genes were transcribed in the S. aureus mutS1 strain, demonstrating that insertional inactivation of mutS with pBT1 had no major polarity effect on their transcription.

Role of MutS and MutL in hypermutability. We used the mutS and mutL primers to determine the totality of mutS and mutL sequence for 11 of the hypermutable strains reported in reference 33. We confirmed the absence of mutations in the deduced MutS/L amino acid sequences in four strains; in addition, we could not confirm mutations that we previously reported in UCN28 and UCN31. For five other strains, mutations were found in MutS, MutL, or both (Table 3). The mutSL genes of the hypermutable strain UCN22 could be only partially amplified, which was explained after further analysis by inverse PCR by a large 5,060-bp deletion (including 1,900 bp in the 3' end of the mutS gene, the totality of the mutL and glpP genes, and 107 bp in the beginning of the glpF gene). Strain 1A had a large 41-amino-acid deletion, from F488 to R528, in the middle part of the protein, which has been shown in E. coli to participate in the mismatch binding and in the interaction between the two monomers (clamp domain) (11, 48), and a point T192A mutation of unknown significance. In UCN29, various mutations of unknown significance were found. N373D and T415M are located in the central part responsible for the mismatch binding of the protein (48). N588D is in a key region, the ATPase domain of the protein, implicated in interaction with MutL and dimerization (11, 48). Finally, L811S and S814C are located in the C-terminal region of MutS, which is also important for dimerization (48). A clinical S. aureus small-colony variant mutator strain was reported previously to bear exactly the same substitutions (38). Intriguingly, three of these exchanges (N181H, T415M, and L811S) are also found in the sequence of S. aureus MRSA252 available in the databases. Four strains displayed mutations in MutL. As already mentioned, UCN22 had a total deletion of the mutL gene. UCN27 had a small five-amino-acid deletion, not located in a particularly conserved region of the protein, although altering the N-terminal fragment described in E. coli as the most functionally relevant part of the protein (ATPase domain) (2, 3). In UCN28 and UCN29, various mutations of unknown significance were found, located in the very middle of the protein sequence, on well-conserved amino acids, but not known to have a particular importance for the functions of the protein.


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  		TABLE 3. Alterations of MutS and MutL in S. aureus hypermutable strains

 
The mutS or mutL gene was amplified from four strains, S. aureus RN4220, 1A, UCN27, and UCN29, with specific primers modified to contain restriction sites (Table 2). The various mutL and mutS genes were cloned into pAT392 and pORI23, respectively. The roles of MutS and MutL in mutation avoidance were evaluated by comparing the spontaneous mutation frequencies of S. aureus RN4220 with those of the S. aureus mutS2 and mutL strains complemented or not with the wt mutS and mutL genes or their mutated counterparts. The growth curves of S. aureus RN4220 and the mutS2, mutL, mutS2/pORI23S, and mutL/pAT392L strains were similar, showing that mutS or mutL gene inactivation or introduction of exogenous plasmids into the strains did not result in lower fitness (data not shown). Mutation frequencies are shown in Table 4. The wt strains, containing or not containing the different plasmid vectors, displayed mutation frequencies around 10–7 to 10–8, which is similar to those previously reported (29). Alteration of MutS is responsible for hypermutability in S. aureus, since the S. aureus mutS2 strain showed a higher mutation frequency than S. aureus RN4220, whereas complementation of the strain (mutS2/pORI23S) restored a low mutation frequency. These results confirmed those reported by O'Neill and Chopra using another integrative vector (28). Introduction of plasmids with the mutated mutS genes from strains 1A and UCN29 in S. aureus mutS2 (mutS2/pORI23S1A and mutS2/pORI23S29) failed to restore a low mutation frequency, which suggested that these mutations are responsible for the hypermutability of the clinical strains (upper part of Table 4).


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  		TABLE 4. Effects of alterations of mutS and mutL genes on mutation frequencies

 
MutL also seems to be involved in hypermutability, since the S. aureus mutL strain showed a higher mutation frequency than S. aureus RN4220. The increase in frequency was much greater than that observed with mutS2. We have no explanation for this observation. However, after introduction of the control plasmid pAT392 (mutL/pAT392), the mutation frequencies were similar to that of mutS2/pORI23. As expected, mutation frequencies were markedly reduced after complementation with the wt mutL gene borne by pAT392 (mutL/pAT392L). Analysis of the effects of MutL mutants showed that UCN29 mutations did not seem to account for hypermutability of the strain, while the five-amino-acid deletion identified in UCN27 could be responsible for the elevated mutation frequency of the strain (lower part of Table 4).

Unfortunately, we failed to complement clinical strains by electroporation, which is consistent with the notion that the presence of a restriction system (which is absent in S. aureus RN4220 but present in S. aureus clinical strains) in the recipient is a major barrier to plasmid transfer from E. coli to S. aureus, both by mobilization (45) and by electroporation (44). The experiments failed even after introduction of plasmids into S. aureus RN4220 as an intermediate host.

Role of mutS and mutL in recombination. The ability of the two genes to prevent recombination between partially divergent sequences was investigated. We designed a bank of S. aureus RN4220, mutS2, and mutL strains containing integrative thermosensitive vectors in which fragments with various levels of identity to the S. aureus sodA gene were cloned. The survival of each strain after three consecutive subcultures at 42°C with chloramphenicol was taken as an estimation of the recombination frequency. Figure 3 shows the results obtained after calculating the mean ratio of the logarithm of survival after three subcultures (total count at the third day) to the logarithm of the corresponding initial inoculum with calculated standard deviation. Survival of the control strains containing only the pBT1 plasmid without an inserted sodA fragment was either nil (for RN4220/pBT1) or markedly reduced (8% and 13% of the initial inoculum for the mutS2/pBT1 and mutL/pBT1 strains, respectively). Overall, survival of bacteria was directly affected by sequence divergence: the less divergent was the insert, the more the strain survived. This observation can be correlated with previous work, as it has been shown in E. coli and in B. subtilis that the frequency of recombination decreases exponentially with the degree of DNA sequence divergence between donor and recipient, which leads to a log-linear relationship between the two parameters (46, 53). Inactivation of the mutS and mutL genes did not seem to have any significant effect on the recombination process, since the variance analysis for each pair of strains did not show any statistical significant difference using the F test. Of note, for some strains, mostly S. aureus RN4220 wt, mutS, and mutL strains containing pBT1SodA93 and for S. aureus RN4220 wt and mutS strains containing pBT1SodA87, the experiment had to be repeated six times and showed a great heterogeneity in the results obtained: the strains could fully survive or die independently of the initial inoculum size, resulting in the large error bars shown in the graph. This could be related to the implication of the SOS system in homeologous recombination in bacteria. Indeed, it is known that a randomly limited fraction of a population submitted to interspecies recombination activates this system (20). The lower correlation coefficients observed in the graphs in Fig. 3B and C could account for a partial effect of MutS and MutL in preventing recombination between nonhomologous DNA fragments. This effect was particularly noticed for MutL, since bacterial survival was consistently observed with the pBT1Sod87 strain compared to that for the S. aureus RN4220 and mutS2 strains.



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  		FIG. 3. Estimation of the recombination frequencies in balance with sequence identity in S. aureus wt (RN4220) (A), mutS2 (B), and mutL (C) strains. Survival of the strains containing the different constructs after three subcultures at 42°C with chloramphenicol (and kanamycin for the mutS2 and mutL strains) was taken as an indicator of recombination frequencies. Points represent the mean ratio of the log of survival after three subcultures to the log of the corresponding initial inoculum. Bars indicate standard deviation. Regression lines and their equations are shown, as well as their correlation coefficients.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The MMR system has been extensively studied with bacteria, particularly with gram-negative bacteria, such as E. coli, but very little is known about the role of this system in gram-positive organisms. The hexAB system in S. pneumoniae has been shown to be implicated in mutation avoidance and control of the recombination rate (10, 32), the mutSL operon has been studied with B. subtilis (6, 41), L. monocytogenes (21), and E. faecium (47), and the implication of the mutS gene in the S. aureus mutator phenotype has already been reported (28). We have reported the first hypermutable S. aureus clinical strains with alterations of the mutS gene (33). Here we report the complete analysis of the mutSL operon in these strains. Alterations of the mutS or mutL gene were found in 5 out of 11 hypermutable strains studied. Interestingly, the 123-bp deletion resulting in a 41-amino-acid deletion in the MutS protein of strain 1A occurred between two 10-bp direct repeats that are also present in the wt mutS gene, suggesting the existence of a possible recombinational "hot spot" inside the gene. Similar observations have already been reported in mutS mutator P. aeruginosa (27) and E. coli (12) natural isolates. The fact that we found exactly the same alterations in UCN29 as those reported previously by Schaaff et al. in another clinical S. aureus mutator isolate (38) reinforces the notion that these mutations are important for hypermutability. The functionality of the gene products was tested for the three strains containing the most altered structures. The 41-amino-acid deletion in strain 1A and the five-amino-acid deletion in strain UCN27 accounted for hypermutability of the strains, since the mutated MutS and MutL alleles failed to restore a low mutation frequency in the S. aureus mutS2 and mutL strains, respectively. In S. aureus UCN29, it seemed that the amino acid substitutions in MutS were also responsible for the mutator phenotype of the strain, while the amino acid substitutions in MutL seemed to be unrelated to the hypermutability of the strain, since the MutL allele successfully complemented the S. aureus mutL strain. These MutL mutations could only be a consequence of the hypermutable phenotype of the strain. These results are consistent with the structure-function relationship established in previous studies on the MutS enzymes of E. coli (11) and Thermus aquaticus (26) and on the 40-kDa N-terminal fragment (designed LN40) of E. coli MutL (3). However, the significance of the amino acid substitutions or deletions remains speculative, since, as yet, no structure-function relationship has been identified for MutS or MutL homologues in gram-positive bacteria.

Therefore, in 4 of the 11 hypermutator isolates that we have studied, hypermutability could be explained by alterations of mutSL, confirming the importance of this system in S. aureus. In the other hypermutable strains with no mutations in the mutSL operon, alteration of other mutator genes might occur. Other putative mutator genes have already been identified in the B. subtilis chromosome (37), and some have homologues in the S. aureus chromosome.

Interestingly, we showed that the mutS and mutL genes were cotranscribed, as in other gram-positive species (6, 21, 47), and contrary to the case with E. coli. The operon structure has been suggested to be advantageous to the cell, leading to the coordinate expression of the two proteins involved in mismatch repair (6). This seems also to be the case for S. pneumoniae, since although hexA and hexB are not adjacent, a similar putative regulatory sequence upstream from the genes might allow a coordinate expression (31).

In a previous study, we found that a majority of the macrolide-resistant S. aureus isolates from CF patients displayed ribosomal mutations, particularly in the rrl gene encoding the 23S rRNA. These results were probably related to the high proportion of hypermutable isolates in CF patients (33). The rrl gene can be found in five or six copies in the S. aureus chromosome, and a minimum of three copies need to be mutated to get a significant level of resistance to macrolides. We sequenced each copy of the rrl gene in macrolide-resistant mutated strains where a relevant mutation (A2058G) was associated with a mutation not known to confer macrolide resistance (A2207T), and we found that both were always associated (data not shown). Taken together, these data gave a relevant clue as to the link between hypermutability and hyperrecombination in S. aureus. The hypothesis was that in the presence of a macrolide, mutation of a first rrn operon in the rrl gene of the mutator strain conferred a low level of resistance to the antibiotic and that the mutation was then spread all over the chromosome in the other operons to get higher levels of resistance. It is known that rRNA operons can be homogenized by gene conversion (7), and this kind of phenomenon has already been hypothesized to account for linezolid resistance in Enterococcus faecium and Enterococcus faecalis (17). These observations lead us to design a model for the in vitro study of the role of mutSL in recombination in S. aureus. The results that we report here tend to suggest a more important role for MutL than for MutS in preventing homeologous recombination, although both displayed only a very limited effect. However, no known mechanism could account for this putative effect of MutL in preventing homeologous recombination. These results could therefore reflect only an experimental artifact and the weakness of our model to cope with subtle mechanisms implicated in the prevention of recombination in S. aureus. Our model studies bacterial populations rather than individual cells and therefore cannot identify genetic events at the cellular level.

However, it has been shown for B. subtilis that the mismatch repair is only marginally efficient in preventing homeologous recombination (14), and this seems to be also the case for S. aureus.

It was recently shown that S. aureus strains can display very high divergences and heterogeneity in their chromosomal structures, leading to the rise of particularly dangerous strains bearing multiple antibiotic resistances and virulence characteristics thanks to the high number of repeats that can generate genetic reorganizations (8, 13). This problem could be particularly relevant in a context of illegitimate recombination favored by a mutator phenotype. The study of the genomic evolution of pathogenic hypermutable and nonhypermutable strains could give more information on the possible relationship between the two phenomenons.


   ACKNOWLEDGMENTS
 
This work was supported by grants from Conseil Régional de Basse-Normandie and the association "Vaincre la Mucoviscidose."


   FOOTNOTES
 
* Corresponding author. Mailing address: CHU de Caen, Service de Microbiologie, Avenue Côte de Nacre, 14033 Caen Cedex, France. Phone: (33) 02 31 06 45 72. Fax: (33) 02 31 06 45 73. E-mail: leclercq-r{at}chu-caen.fr. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Bacteriology, May 2005, p. 3455-3464, Vol. 187, No. 10
0021-9193/05/$08.00+0     doi:10.1128/JB.187.10.3455-3464.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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