Regulation of the Expression of Cell Wall Stress Stimulon Member Gene msrA1 in Methicillin-Susceptible or -Resistant Staphylococcus aureus

Abstract

Genome-wide transcriptional profiling studies of the response of Staphylococcus aureus to cell wall-active antibiotics have led to the discovery of a cell wall stress stimulon of genes induced by these agents. msrA1, encoding methionine sulfoxide reductase, is a highly induced member gene of this stimulon. In the present study we show that msrA1 induction by oxacillin is common to all methicillin-susceptible strains studied but did not occur in two homogeneous and two heterogeneous methicillin-resistant strains. However, msrA1 was induced by vancomycin and/or d-cycloserine in methicillin-resistant strains. Lysozyme and lysostaphin treatment did not induce msrA1 expression. Oxacillin-induced msrA1 expression was enhanced by ca. 30% in a SigB⁺ derivative (SH1000) of the SigB-defective RN450 (NCTC 8325-4) strain. msrA1 expression was not affected in mutants in the global regulatory systems agr and sar. Glycerol monolaurate, an inhibitor of signal transduction, inhibited the oxacillin-induced transcription of msrA1 and other cell wall stress stimulon member genes, vraS and dnaK. These observations suggest that the cell wall stress stimulon is induced by inhibition of the process of peptidoglycan biosynthesis, and the inhibitory effects of glycerol monolaurate indicate that gene expression is dependent on a signal transduction pathway.

Staphylococcus aureus is a versatile pathogen that is a leading cause of both nosocomial and community-onset infections worldwide (2). In addition to its virulence, S. aureus has acquired resistance to a majority of the commonly used antibiotics at an alarming rate. In recent years, S. aureus has shown resistance to the glycopeptide antibiotic vancomycin (3, 7, 18). This is of notable concern since vancomycin has been the only antibiotic to which there has been uniform susceptibility in multidrug-resistant methicillin-resistant S. aureus (MRSA).

It is well accepted that cell wall-active antibiotics inhibit bacterial growth by inhibiting peptidoglycan biosynthesis (49). It is becoming clear that upon treatment with cell wall-active antibiotics, S. aureus and other bacteria undergo an extensive program of gene and protein expression. Using a proteomic approach, Singh et al. (43) showed that a set of at least nine proteins was induced upon challenge of a mid-exponential- phase S. aureus culture with oxacillin and other cell wall-active antibiotics. One of the most strongly induced proteins was MsrA1, methionine sulfoxide reductase.

Recently, we used DNA microarray technology to capture a genome-wide picture of changes within the S. aureus transcriptome in response to challenge with cell wall-active antibiotics (48). A large number of genes in various functional categories were upregulated by oxacillin, bacitracin, and d-cycloserine. Several cell wall-related genes were induced by each of the three antibiotics, including pbpB, the gene that encodes the essential penicillin-binding protein 2 (PBP2). murZ was also upregulated; this gene encodes the enzyme UDP-N-acetylglucosamine-1 carboxylvinyl transferase-2, which catalyzes the first step unique to peptidoglycan biosynthesis. The cell appears to respond to cell wall-active antibiotics by attempting to increase the rate of peptidoglycan biosynthesis. The cell also appears to attempt to regulate cell wall-active antibiotic-induced autolysis by increasing the transcription of the regulators of autolysis, fmt and lytR, and decreasing the transcription of atl (48), which encodes the major S. aureus autolysin (32). The cell wall-related genes fmt, tca, and vraR were also shown to be upregulated by cell wall-active antibiotics. Interestingly, they have previously been encountered in the context of methicillin resistance (fmt) (21) and glycopeptide resistance (tca and vraR) (4, 23). vraSR encodes a putative two-component sensor response regulator pair that may be involved in signal transduction and control of gene expression.

Another functional category of upregulated genes was genes involved in posttranslational modification, in protein turnover, and as chaperones. msrA1 belongs to this category. Gene fusion analysis, Northern blotting, and transcriptional profiling have revealed that msrA1 is highly induced by cell wall-active antibiotics (43, 44, 48). Methionine sulfoxide reductases are oxidative defense proteins that reduce methionine sulfoxide residues in proteins to methionine and hence restore protein function (5, 26). S. aureus MsrA1 specifically reduces the S-enantiomer of methionine sulfoxide (27). MsrA is believed to contribute to the virulence of a variety of bacterial pathogens (10, 17, 50). It is not clear why msrA1 is induced in S. aureus specifically by cell wall-active antibiotics. Possibly, treatment with cell wall-active antibiotics results in oxidative stress to proteins. There are multiple genes encoding methionine sulfoxide reductase activity in S. aureus, and their regulation is complex (44, 46). Although an msrA knockout mutant showed increased susceptibility to hydrogen peroxide, an MRSA msrA1 mutant did not show increased oxacillin susceptibility (44).

In the present study we have studied the regulation of the expression of msrA1 in greater detail than before. We report here on the inducibility of msrA1 in a variety of methicillin-susceptible, methicillin-resistant, and mutant strains and on the role of SigB and signal transduction in msrA1 expression. In addition to yielding information specific to msrA1, the results carry implications for the regulation of expression of the cell wall stress stimulon as a whole.

MATERIALS AND METHODS

Strains and growth conditions.

All strains used in the present study and their relevant characteristics are described in Table 1. The strains were stored at −80°C in 30% (vol/vol) glycerol and periodically streaked out onto tryptic soy agar (Difco Laboratories, Detroit, Mich.) to provide working plates that were stored at 4°C. S. aureus and Escherichia coli cells were grown in or on tryptic soy broth (TSB) or tryptic soy agar and Luria-Bertani broth or agar, respectively, at 37°C. Liquid cultures were grown in a shaking incubator (250 rpm). When needed, ampicillin (50 μg ml⁻¹), erythromycin (20 μg ml⁻¹), tetracycline (10 μg ml⁻¹), and X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; 40 μg ml⁻¹) were added to the growth medium.

TABLE 1.

Bacterial strains and plasmids used in this study

S. aureus strains	Characteristicsa	Source or reference
RN450 (NCTC 8325-4)	Laboratory strain of S. aureus cured of all the prophages	29
RN450PmsrA1::lacZ	S. aureus RN450; a reporter strain for the msrA1 promoter (Ermr)	43
RN4220	Restriction minus derivative of S. aureus RN450	22
COL	Methicillin-resistant (ho) S. aureus	35
COL::msrA1	S. aureus COL with mutation in the msrA1 gene (Kanr)	This study
SH1000	Functional rsbU derivative of S. aureus RN450	20
SH1000PmsrA1::lacZ	Reporter strain for the msrA1 promoter (Ermr)	This study
BB270	Methicillin-resistant (he) S. aureus	14
BB270PmsrA1::lacZ	Methicillin-resistant msrA1 promoter reporter strain (Ermr)	This study
13136p−m+	Methicillin-resistant (he) S. aureus	35
DU4916	Methicillin-resistant (ho) S. aureus	14
RN6911agr	agr mutant of S. aureus RN6390 (Tetr)	41
RN6911PmsrA1::lacZ	agr msrA1 mutant promoter reporter strain (Tetr Ermr)	This study
ALC136	sar mutant of S. aureus RN6390 (Ermr)	41
H	Laboratory strain of S. aureus
Wood 46	Laboratory strain of S. aureus
RN7497	Laboratory strain of S. aureus	This study

^aKan^r, kanamycin resistant; Erm^r, erythromycin resistant; Tet^r, tetracycline resistant; he and ho, hetero- and homogeneous, respectively.

Determination of msrA1 promoter activity.

The construction of an msrA1 promoter-lacZ (PmsrA1::lacZ) fusion strain has been previously described (43). β-Galactosidase activity in the reporter strains was determined by using o-nitrophenyl-β-d-galactopyranoside (ONPG) as the substrate (25).

Transduction of the msrA1 promoter::lacZ fusion construct into various S. aureus strains.

The fusion construct (PmsrA1::lacZ) was transduced into various S. aureus strains through phage 80 alpha transduction as described by Novick et al. (29). For preparation of phage 80 alpha lysate, RN4220 harboring the fusion construct was grown in TSB for 12 h and then diluted 100-fold in LK medium (tryptone, yeast extract, KCl) containing 10 mM CaCl₂. After incubation at 37°C for 1.5 h, 1 ml of phage lysate propagated in RN4220 was added to the culture, which was then kept at 30°C until completely lysed (∼10 h). The lysate was then filtered through a 0.45-μm-pore-size Millipore filter and stored at 4°C. Overnight-grown cultures of the desired strains were diluted 100-fold and incubated at 37°C for 1.5 h. Cells were harvested, washed once with 5.0 ml of TSB, and resuspended in 0.95 ml of TSB containing 5 mM CaCl₂. Cells were infected with 50 μl of phage lysate prepared on RN450 (NCTC 8325-4) containing PmsrA1::lacZ and incubated at room temperature for 10 min and then at 30°C for 35 min. The cells were harvested, washed once with TSB, and resuspended in 7 ml of TSB at 37°C for 1.5 h. The resulting culture was centrifuged and resuspended in 1 ml of TSB, and transductants were selected on tryptic soy agar containing erythromycin (20 μg ml⁻¹).

RNA isolation and Northern blot analysis.

RNA isolation and Northern blot analysis were carried out as described earlier (44). Overnight-grown S. aureus cultures were diluted 100-fold in 2.5 ml of fresh TSB and were grown to an optical density at 600 nm (OD₆₀₀) of 0.3. At this point, 1.2 μg of oxacillin or 300 μg of d-cycloserine or other antibiotics (selected concentrations) ml⁻¹ was added, and the cells, including antibiotic-free control cultures, were allowed to grow for 1.5 h. Cells were harvested by centrifugation, washed once in 20 mM Tris-HCl buffer (pH 7.5) containing 145 mM NaCl, and lysed in Tris-HCl buffer (pH 7.5) containing lysostaphin (50 μg ml⁻¹) for 10 min at 37°C. Total RNA was subsequently isolated by using a Qiagen RNeasy minikit (Qiagen). Equal amounts of RNA samples (10 μg) from each condition were run on a 1.2% denaturing agarose gel and then transferred to a nitrocellulose membrane. The radiolabeled msrA1 or other probes were prepared by using the Prime-a-Gene labeling system (Promega) in the presence of [α-³²P]dCTP (specific activity, >3,000 Ci mmol⁻¹; ICN Pharmaceuticals, Inc., Irvine, Calif.) and used to probe the membrane.

Molecular genetic procedures.

Plasmid and chromosomal DNA isolation, DNA manipulations, digestion of DNA with restriction enzymes, DNA ligation, oligolabeling, PCRs, and Northern blotting were performed as described by Novick (30) and Sambrook et al. (40). All enzymes were used as directed by the manufacturer.

RESULTS

Induction of msrA1 in various S. aureus strains.

Since the msrA1 induction phenomenon has only been demonstrated in strain RN450 (43), Northern blot analysis was performed to determine whether cell wall-active antibiotics induced a similar response in various other S. aureus strains. Total RNA was isolated from control and treated cultures and electrophoresed, blotted, and probed with msrA1. As shown in Fig. 1A, the expression of msrA1 was significantly higher in cells treated with oxacillin (Fig. 1A, lanes 2, 4, 6, and 8) or d-cycloserine (Fig. 1B, lanes 2, 4, 6, and 8) than in untreated cells of methicillin-susceptible strains RN450, SH1000, RN7497, and H (Fig. 1A and B, lanes 1, 3, and 5). However, msrA1 was not induced by oxacillin in MRSA strain COL (Fig. 1A, lane 10) but was induced by d-cycloserine (Fig. 1B, lane 10). This phenomenon was also observed in another homogeneous MRSA strain, DU4916, and in the heterogeneous MRSA strains 13136 and BB270. msrA1 was not induced by oxacillin (Fig. 1C, lanes 2, 5, and 8) but was induced by d-cycloserine (Fig. 1C, lanes 3, 6, and 9) in both hetero- and homogeneous MRSA strains.

FIG. 1.

Northern blot hybridization of the msrA gene with the total RNA isolated from various S. aureus strains treated with oxacillin (A) or d-cycloserine (B) and then probed with the radiolabeled msrA gene. S. aureus strains treated with oxacillin (A) were as follows: RN450 (lanes 1 and 2), SH1000 (lanes 3 and 4), RN7497 (lanes 5 and 6), H (lanes 7 and 8), and COL (lanes 9 and 10). Strains are the same for d-cycloserine treatment (B), except that Wood 46 rather than RN7497 is shown in lanes 5 and 6. The first lane is RNA from untreated cells, and the second is RNA from the antibiotic-treated cells. (C) Northern blot hybridization of msrA with total RNA isolated from S. aureus strains 13136 (lanes 1 to 3), DU4916 (lanes 4 to 6), and BB270 (lanes 6 to 9) and then probed with the radiolabeled msrA gene. Lanes 1, 4, and 7 are RNA from untreated cells; lanes 2, 5, and 8 are RNA from cells treated with oxacillin; and lanes 3, 6, and 9 are RNA from cells treated with d-cycloserine.

To further confirm that msrA1 in MRSA strains was not induced by oxacillin, the PmsrA1::lacZ fusion construct was transduced into the MRSA COL strain, and the β-galactosidase activity was determined in response to cell wall-active antibiotic treatment. As shown in Fig. 2A, no significant induction of msrA1 was detected in the presence of increasing oxacillin concentrations. Peptidoglycan synthesis probably continues in the presence of β-lactam antibiotics in MRSA strains due to the presence of PBP2a. However, induction of msrA1 was observed in response to the other cell wall-active antibiotics, vancomycin (Fig. 2B) and d-cycloserine (Fig. 2C), to which COL is susceptible. Slightly higher concentrations of vancomycin and d-cycloserine were needed for induction of MRSA strains than of susceptible strains. The peptidoglycan-degrading enzymes lysostaphin and lysozyme were unable to induce msrA1 expression (data not shown). The results suggest that inhibition of the process of peptidoglycan biosynthesis is necessary for msrA1 induction.

FIG. 2.

Effect of cell wall-active antibiotics on the expression of β-galactosidase in the S. aureus COL PmsrA1::lacZ reporter strain. At 2 h after antibiotic treatment, cells were harvested and the β-galactosidase activity was determined. Error bars represent the standard deviations of triplicate experiments.

Influence of SigB on msrA1 induction.

S. aureus strain RN450 (8325-4) has an 11-bp deletion mutation in rsbU, which regulates SigB activity (13). SigB is an alternative sigma factor shown to be involved in S. aureus stress responses (12). Horsburgh et al. (20) have constructed SH1000, a SigB⁺ RN450 derivative, and this strain was used to evaluate the influence of SigB on msrA1 induction. Interestingly, oxacillin-induced expression of msrA1 in SH1000 was significantly higher than that in RN450 (Fig. 1, lanes 2 and 4). To further confirm that SigB was involved in msrA1 induction, a PmsrA1::lacZ fusion was transduced in SH1000 to compare the level of expression of msrA1 between RN450 and SH1000 (sigB⁺) by measuring the β-galactosidase activity. As shown in Fig. 3, β-galactosidase activity in SH1000 was induced at least 30% more than in RN450(8325-4) in response to oxacillin, d-cycloserine and vancomycin at all of the concentrations tested. These results suggest that SigB, a transcription factor known to be a global regulator of various stress response genes, may have an enhancing effect on msrA1 induction, although sigma B is not necessary for induction.

FIG. 3.

Analysis of transcription from PmsrA1::lacZ fusions in S. aureus RN450 (▪) and SH1000 (□) in response to oxacillin, d-cycloserine, and vancomycin. At 2 h after antibiotic treatment, cells were harvested and the β-galactosidase activity was determined. Error bars represent the standard deviations of triplicate experiments.

Effect of sar and agr mutations on the expression of msrA1.

The expression of several genes in S. aureus is governed by the global regulatory elements agr and sar (9, 11, 33). To investigate the possibility for a role of these regulatory systems in the induction of msrA1, Northern blot analysis was performed. RNA was isolated from mutant strains grown in the presence or absence of oxacillin and d-cycloserine and probed with the radiolabeled msrA1 gene. As shown in Fig. 4, the levels of msrA1 expression were similar in the agr and sar mutants and the parent strains. Similar results were obtained by using agr or sar mutants containing the PmsrA1::lacZ fusion construct (data not shown). The results suggest that Agr and Sar are not involved in the cell wall-active antibiotic msrA1 induction phenomenon.

FIG. 4.

Northern blot hybridization of the msrA gene with the total RNA isolated from the S. aureus RN6390 agr and ALC136 sarA mutant strains treated with oxacillin (lanes 2 and 5) and d-cycloserine (lanes 3 and 6) and then probed with radiolabeled msrA1 probe. Lanes 1 and 5 are RNA from untreated cells.

Induction of msrA1 transcription is specific to cell wall-active antibiotics.

Previous studies with the PmsrA1::lacZ fusion strain RN450 (43, 44) and the present studies of strain SH1000 indicate that induction of msrA1 is specific to cell wall-active antibiotics. However, it is conceivable that potential msrA1 induction by inhibitors of translation could be masked in the fusion strain, where protein synthesis is necessary for synthesis of LacZ, which is used to monitor msrA1 transcription in this case. Hence, Northern blot analysis was performed to monitor msrA1 expression at the transcriptional level and to rule out the possibility of induction of msrA1 by translation inhibitors that was masked by the effect of the antibiotics on the synthesis of the fusion gene product. As shown in Fig. 5, no induction was detected by any of the non-cell-wall-active antibiotics, such as streptomycin, tetracycline, and chloramphenicol (Fig. 5, lanes 3, 4, and 6). The response has thus far shown specificity to cell wall-active antibiotics and may be part of a stress response to cell wall damage initiated by these antibiotics.

FIG. 5.

Northern blot analysis of S. aureus SH1000 incubated with various antibiotics. RNA (10 μg) separated on a 1.2% denaturing agarose gel was analyzed with the radiolabeled msrA probe. Lane 1, control untreated; lane 2, oxacillin (1.2 μg ml⁻¹); lane 3, streptomycin (5 μg ml⁻¹); lane 4, tetracycline (2 μg ml⁻¹); lane 5, vancomycin (2.5 μg ml⁻¹); lane 6, chloramphenicol (2 μg ml⁻¹).

Effect of environmental stressing agents on msrA1 expression.

Various environmental stressing agents were tested for their effects on msrA1 expression in SH1000. Actively growing cultures were exposed to various pH levels, temperatures, levels of osmotic stress (NaCl, KCl, and sucrose), and the presence of various ions (a2 mM concentration each of Fe²⁺, Zn²⁺, Co²⁺, Cu²⁺, Mn²⁺, and Ni²⁺). msrA1 expression remained comparable to levels in control cultures stressed by different temperatures, pH values, and NaCl concentrations (data not shown). None of the ions tested were able to induce msrA1 to a significantly higher level as tested by using the fusion construct (data not shown). Lipase and proteases were also unable to induce msrA1 expression (data not shown).

Inhibition of msrA1 transcription by glycerol monolaurate (GML), an agent that interferes with signal transduction.

Among various compounds screened for their ability to affect oxacillin-induced msrA1 transcription, one, the surfactant GML, was shown to inhibit the transcription of msrA1 at low concentrations. GML has previously been shown to inhibit the production of various staphylococcal proteins and virulence factors at the transcriptional level (36). GML was added in increasing concentrations to actively growing cultures with oxacillin, and inhibition of msrA1 expression was monitored by Northern blotting (Fig. 6A and B). Concentrations of GML as low as 25 μg ml⁻¹ added to an actively growing culture 1 h prior to addition of 1.2 μg of oxacillin ml⁻¹ significantly inhibited the transcription of msrA1 (Fig. 6, lane 3). However, if GML was added 1 h later than the oxacillin to the actively growing culture, the transcription of msrA1 was not affected (Fig. 6C), thus indicating that induction of msrA1 had already occurred, and the transcript was therefore unaffected by GML addition. Addition of GML and oxacillin at the same time also inhibited the transcription of msrA1 (Fig. 6B). Similar results were also seen in gene fusion analyses (Fig. 7). When GML was added to an actively growing culture 1 h prior to addition of 1.2 μg of oxacillin ml⁻¹, it significantly inhibited the β-galactosidase activity (Fig. 7B). However, if GML was added 1 h later than the oxacillin (1.2 μg/ml) to the actively growing culture, the β-galactosidase activity was almost unaffected (Fig. 7A), thus indicating that induction of msrA1 had already occurred, and the transcript was therefore unaffected by GML addition. Inhibition of msrA1 induction by GML indicates that an unidentified signal transduction pathway initiated by exposure to oxacillin was responsible for msrA1 induction.

FIG. 6.

Effect of GML on the cell wall-active antibiotic-induced expression of msrA by Northern blot analysis. RNA (10 μg) separated on a 1.2% denaturing agarose gel was analyzed with the radiolabeled msrA probe. (A) S. aureus SH1000 cultures at an OD₆₀₀ of 0.3 were treated with various concentrations of GML for 1 h and then with 1.2 μg of oxacillin ml⁻¹ for 1 h. (B) Cultures were simultaneously treated with oxacillin (1.2 μg ml⁻¹) and GML (50 μg ml⁻¹) for 1 h. (C) Cultures at an OD₆₀₀ of 0.3 were treated with oxacillin (1.2 μg ml⁻¹) for 1 h and then various concentrations of GML for 1 h. The GML concentrations were as follows: lane 2, 0 μg ml⁻¹; lane 3, 25 μg ml⁻¹; lane 4, 50 μg ml⁻¹; lane 5, 100 μg ml⁻¹; lane 6, 200 μg ml⁻¹; lane 7, 300 μg ml⁻¹; and lane 8, 400 μg ml⁻¹. Lane 1, not treated with oxacillin or GML.

FIG. 7.

Inhibition of the cell wall-active antibiotic-induced expression of PmsrA1::lacZ by GML. After treatment, cells were harvested, and the β-galactosidase activity was determined. Error bars represent the standard deviations of triplicate experiments. (A) S. aureus SH1000 cultures at an OD₆₀₀ of 0.3 were treated with oxacillin (1.2 μg ml⁻¹) for 1 h and then various concentrations of GML for 1 h. (B) S. aureus SH1000 cultures at an OD₆₀₀ of 0.3 were treated with various concentrations of GML for 1 h and then with 1.2 μg of oxacillin ml⁻¹ for 1 h. GML concentrations were as follows: lane 2, 0 μg ml⁻¹; lane 3, 25 μg ml⁻¹; lane 4, 50 μg ml⁻¹; lane 5, 100 μg ml⁻¹; lane 6, 200 μg ml⁻¹; lane 7, 300 μg ml⁻¹; and lane 8, 400 μg ml⁻¹. Lane 1, not treated with oxacillin or GML.

GML inhibits the induction of the other cell wall stress stimulon genes vraS and dnaK.

To investigate whether GML specifically inhibits the oxacillin inducibility of msrA1 alone, its effect on the expression of other cell wall stress stimulon genes, such as dnaK and vraS, was studied. The transcription of dnaK and vraS was induced by oxacillin treatment. However, the addition of GML completely inhibited the expression of these genes (data not shown). These results further indicate that a signaling system involved in the cell wall stress stimulon is disrupted by GML.

DISCUSSION

msrA1 has previously been shown to be induced by cell wall-active antibiotics through proteomic studies (43), Northern blotting and gene fusion analysis (44), and microarray transcription profiling (48) in the methicillin-susceptible strain RN450. This gene is believed to be a member gene of what has been referred to as a cell wall stress stimulon (48). A total of 105 genes were upregulated in their expression in response to cell wall-active antibiotics (48). In the present study, oxacillin challenge of several methicillin-susceptible strains indicated that msrA1 induction is common to S. aureus strains in general. Recently, Chan et al. (P. F. Chan, R. Gagnon, M. Lonetto, R. Javed, R. Boyle, S. O'Brien, D. Lunsford, and D. Jaworski, Abstr. 103rd Gen. Meet. Am. Soc. Microbiol. 2003, abstr. A165, 2003) also reported that msrA was induced by different cell wall-active antibiotics in a different S. aureus strain. Northern blot analysis confirmed that msrA1 was not induced by antibiotics acting against targets other than the cell wall and was specific to cell wall-active antibiotics. This removes any uncertainty that antibiotics inhibiting protein synthesis may appear falsely negative for msrA1 induction, when detection of induction is dependent on protein synthesis (43).

S. aureus contains three msrA genes (msrA1, msrA2, and msrA3) and msrB. msrA1 and msrA2 have been shown to have methionine reductase activity specific for the S-enantiomer of Met-O, and MsrB is specific for the R-enantiomer of Met-O (27, 46).

msrA1 is a member gene of the cell wall stress stimulon, and findings on the regulation of msrA1 probably also apply more generally to the cell wall stress stimulon. msrA1 was not induced by low concentrations of oxacillin in a homogeneous strain and in two heterogeneous MRSA strains but was induced by vancomycin or d-cycloserine, cell wall-active antibiotics that inhibit different targets in peptidoglycan biosynthesis than oxacillin does (49). Interestingly, somewhat higher concentrations of vancomycin and d-cycloserine were needed to induce msrA1 in strain COL than in methicillin-susceptible strains. The reason for this is not clear. Possibly, peptidoglycan biosynthesis is activated in MRSA, for which there is some evidence (47), as is known to be the situation in some glycopeptide-intermediate S. aureus strains (15), and higher concentrations of antibiotic are needed for inhibition. Lysostaphin and lysozyme, which degrade mature peptidoglycan, did not result in msrA1 induction. Evidence for induction of a likely cell wall stress stimulon by cell wall-active antibiotics has been provided for Bacillus subtilis (6) and Streptomyces coelicolor (19). Alland et al. (1) have reported that the Mycobacterium tuberculosis promoter iniBAC is induced by various agents that inhibit cell wall synthesis. Induction was not simply limited to inhibitors of peptidoglycan biosynthesis but included inhibitors of the biosynthesis of other wall polymers as well. iniBAC was not induced by cell wall-degrading enzymes.

Failure to induce msrA1 in both homogeneous and heterogeneous MRSA strains implies that msrA1 and the cell wall stress stimulon is not being induced by the cell wall-active antibiotic molecules per se but is being induced as a result of the inhibition of the process of peptidoglycan biosynthesis. MRSA strains continue to make peptidoglycan in the presence of β-lactam antibiotics (47). The nature of the signal caused by inhibition of peptidoglycan biosynthesis and its sensing mechanism is unclear. A subset of the S. aureus cell wall stress stimulon genes are controlled by the two-component system VraSR (24). In gram-negative bacteria there is a clearly defined example of the regulation of chromosomal β-lactamase by muropeptide peptidoglycan breakdown products (16).

Recently, Rossi et al. (37) reported on msrR, which is believed to belong to the LytR-CpsA-Psr family of cell envelope- related transcriptional attenuators. msrR was discovered in a transposon insertion mutant showing increased susceptibility to β-lactam antibiotics. This gene was separated from msrA2 by 136 nucleotides, and msrR and msrA2 are transcribed divergently. msrR is clearly a member gene of the cell wall stress stimulon (24, 48). Rossi et al. (37) showed that the transcription of msrR was increased by cell wall-active antibiotics and by lysostaphin. msrR was proposed to be a sensor of cell wall damage and to influence sarA and agr transcription.

SigB mutants express less resistance to β-lactam and glycopeptide antibiotics than SigB-intact parent strains (42, 45, 51). Oxacillin-induced msrA1 transcription was ca. 30% higher in the SigB⁺ derivative of strain RN450 (NCTC 8325-4), i.e., SH1000. SigB is an alternative sigma factor that is involved in various aspects of S. aureus physiology and pathogenicity (8, 12). Clearly, SigB has an enhancing effect on the expression of msrA1, and probably the entire cell wall stress stimulon as well, but is not required for induction. msrA1 induction was not markedly diminished in agr and sarA mutants, implying that these global regulator operons do not play a major role in msrA1 expression or in expression of the cell wall stress stimulon.

The oxacillin-induced expression of msrA1 was inhibited by GML. GML is a surfactant that inhibits the synthesis of many S. aureus exoproteins, including toxins, at the level of transcription (36). In Enterococcus faecalis, GML inhibits the induction of the VanS-VanR pathway necessary for vancomycin resistance (38). GML is considered to inhibit transcription at the level of signal transduction. The inhibition of msrA1 induction by GML suggests that a signal transduction pathway is involved between the inhibition of peptidoglycan biosynthesis and the expression of the cell wall stress stimulon.

The two-component system genes vraSR are member genes of the cell wall stress stimulon (24, 48; Chan et al., Abstr. 103rd Gen. Meet. Am. Soc. Microbiol.). Previously, vraSR has been shown to be upregulated in two clinical glycopeptide-intermediate S. aureus strains compared to glycopeptide-susceptible strains (23). Recently, Chan et al. (Abstr. 103rd Gen. Meet. Am. Soc. Microbiol.), Kuroda et al. (24), and Utaida et al. (48) have shown that vraSR is induced by cell wall-active antibiotics. Microarray analysis showed that vancomycin induced the transcription of 139 genes in MRSA strain N315 (24). Forty-six of these genes appeared to be under the control of vraSR because they were not induced by vancomycin in a vraSR-null mutant.

The reason for the induction of msrA1, which is part of a polycistronic message including msrB, and a gene specifying enzyme IIA of the phosphotransferase system (44), by cell wall-active antibiotic is not yet clear. Methionine sulfoxide reductases are believed to play a role in defense against oxidative stress (5) and in the maintenance of cell surface molecules in various pathogens (31, 50). In S. aureus several cell surface proteins, such as protein A, are covalently linked to cell wall peptidoglycan, and such proteins bear a C-terminal cell wall sorting signal containing an LPXTG motif (28). The peptide bond between the threonine and the glycine residues of the LPXTG motif is cleaved, and the carboxyl group of the threonine is amide linked to the amino group of the pentaglycine cross-bridge in the lipid II precursor molecule of peptidoglycan biosynthesis (34). The cell surface protein is then incorporated into the cell wall by transglycosylation and transpeptidation reactions. Inhibition of peptidoglycan biosynthesis by cell wall-active antibiotics is expected to interfere with the incorporation of cell surface proteins into the cell wall. Perhaps unincorporated cell surface proteins accumulate, and this leads to the induction of various genes encoding proteins involved in posttranslational modification, protein turnover, and chaperones such as msrA1, prsA, htrA, and htrO, which are part of the cell wall stress stimulon (48). Induction of msrA1 and msrB may be related to a disturbance in the processing and incorporation of cell surface proteins caused by cell wall-active antibiotics.