rRNA regulation during growth and under stringent conditions in Staphylococcus aureus.

The control of rRNA synthesis and, thereby, translation is vital for adapting to changing environmental conditions. The decrease of rRNA is a common feature of the stringent response, which is elicited by the rapid synthesis of (p)ppGpp. Here we analysed the properties and regulation of one representative rRNA operon of Staphylococcus aureus under stringent conditions and during growth. The promoters, P1 and P2, are severely downregulated at low intracellular guanosine triphosphate (GTP) concentrations either imposed by stringent conditions or in a guanine auxotroph guaBA mutant. In a (p)ppGpp(0) strain, the GTP level increased under stringent conditions, and rRNA transcription was upregulated. The correlation of the intracellular GTP levels and rRNA promoter activity could be linked to GTP nucleotides in the initiation region of both promoters at positions between +1 and +4. This indicates that not only transcriptional initiation, but also the first steps of elongation, requires high concentrations of free nucleotides. However, the severe downregulation of rRNA in post-exponential growth phase is independent of (p)ppGpp, the composition of the initiation region and the intracellular nucleotide pool. In summary, rRNA transcription in S. aureus is only partially and presumably indirectly controlled by (p)ppGpp.

Methionine biosynthesis in Staphylococcus aureus is tightly controlled by a hierarchical network involving an initiator tRNA-specific T-box riboswitch.

In line with the key role of methionine in protein biosynthesis initiation and many cellular processes most microorganisms have evolved mechanisms to synthesize methionine de novo. Here we demonstrate that, in the bacterial pathogen Staphylococcus aureus, a rare combination of stringent response-controlled CodY activity, T-box riboswitch and mRNA decay mechanisms regulate the synthesis and stability of methionine biosynthesis metICFE-mdh mRNA. In contrast to other Bacillales which employ S-box riboswitches to control methionine biosynthesis, the S. aureus metICFE-mdh mRNA is preceded by a 5'-untranslated met leader RNA harboring a T-box riboswitch. Interestingly, this T-box riboswitch is revealed to specifically interact with uncharged initiator formylmethionyl-tRNA (tRNAi(fMet)) while binding of elongator tRNA(Met) proved to be weak, suggesting a putative additional function of the system in translation initiation control. met leader RNA/metICFE-mdh operon expression is under the control of the repressor CodY which binds upstream of the met leader RNA promoter. As part of the metabolic emergency circuit of the stringent response, methionine depletion activates RelA-dependent (p)ppGpp alarmone synthesis, releasing CodY from its binding site and thereby activating the met leader promoter. Our data further suggest that subsequent steps in metICFE-mdh transcription are tightly controlled by the 5' met leader-associated T-box riboswitch which mediates premature transcription termination when methionine is present. If methionine supply is limited, and hence tRNAi(fMet) becomes uncharged, full-length met leader/metICFE-mdh mRNA is transcribed which is rapidly degraded by nucleases involving RNase J2. Together, the data demonstrate that staphylococci have evolved special mechanisms to prevent the accumulation of excess methionine. We hypothesize that this strict control might reflect the limited metabolic capacities of staphylococci to reuse methionine as, other than Bacillus, staphylococci lack both the methionine salvage and polyamine synthesis pathways. Thus, methionine metabolism might represent a metabolic Achilles' heel making the pathway an interesting target for future anti-staphylococcal drug development.

Two small (p)ppGpp synthases in Staphylococcus aureus mediate tolerance against cell envelope stress conditions.

The stringent response is a conserved global regulatory mechanism that is related to the synthesis of (p)ppGpp nucleotides. Gram-positive bacteria, such as Staphylococcus aureus, possess three (p)ppGpp synthases: the bifunctional RSH (RelA/SpoT homolog) protein, which consists of a (p)ppGpp synthase and a (p)ppGpp hydrolase domain, and two truncated (p)ppGpp synthases, designated RelP and RelQ. Here, we characterized these two small (p)ppGpp synthases. Biochemical analyses of purified proteins and in vivo studies revealed a stronger synthetic activity for RelP than for RelQ. However, both enzymes prefer GDP over GTP as the pyrophosphate recipient to synthesize ppGpp. Each of the enzymes was shown to be responsible for the essentiality of the (p)ppGpp hydrolase domain of the RSH protein. The staphylococcal RSH-hydrolase is an efficient enzyme that prevents the toxic accumulation of (p)ppGpp. Expression of (p)ppGpp synthases in a hydrolase-negative background leads not only to growth arrest but also to cell death. Transcriptional analyses showed that relP and relQ are strongly induced upon vancomycin and ampicillin treatments. Accordingly, mutants lacking relP and relQ showed a significantly reduced survival rate upon treatments with cell wall-active antibiotics. Thus, RelP and RelQ are active (p)ppGpp synthases in S. aureus that are induced under cell envelope stress to mediate tolerance against these conditions.

The stringent response of Staphylococcus aureus and its impact on survival after phagocytosis through the induction of intracellular PSMs expression.

The stringent response is initiated by rapid (p)ppGpp synthesis, which leads to a profound reprogramming of gene expression in most bacteria. The stringent phenotype seems to be species specific and may be mediated by fundamentally different molecular mechanisms. In Staphylococcus aureus, (p)ppGpp synthesis upon amino acid deprivation is achieved through the synthase domain of the bifunctional enzyme RSH (RelA/SpoT homolog). In several firmicutes, a direct link between stringent response and the CodY regulon was proposed. Wild-type strain HG001, rsh(Syn), codY and rsh(Syn), codY double mutants were analyzed by transcriptome analysis to delineate different consequences of RSH-dependent (p)ppGpp synthesis after induction of the stringent response by amino-acid deprivation. Under these conditions genes coding for major components of the protein synthesis machinery and nucleotide metabolism were down-regulated only in rsh positive strains. Genes which became activated upon (p)ppGpp induction are mostly regulated indirectly via de-repression of the GTP-responsive repressor CodY. Only seven genes, including those coding for the cytotoxic phenol-soluble modulins (PSMs), were found to be up-regulated via RSH independently of CodY. qtRT-PCR analyses of hallmark genes of the stringent response indicate that an RSH activating stringent condition is induced after uptake of S. aureus in human polymorphonuclear neutrophils (PMNs). The RSH activity in turn is crucial for intracellular expression of psms. Accordingly, rsh(Syn) and rsh(Syn), codY mutants were less able to survive after phagocytosis similar to psm mutants. Intraphagosomal induction of psmα1-4 and/or psmß1,2 could complement the survival of the rsh(Syn) mutant. Thus, an active RSH synthase is required for intracellular psm expression which contributes to survival after phagocytosis.

RNase Y of Staphylococcus aureus and its role in the activation of virulence genes.

RNase Y of Bacillus subtilis is a key member of the degradosome and important for bulk mRNA turnover. In contrast to B. subtilis, the RNase Y homologue (rny/cvfA) of Staphylococcus aureus is not essential for growth. Here we found that RNase Y plays a major role in virulence gene regulation. Accordingly, rny deletion mutants demonstrated impaired virulence in a murine bacteraemia model. RNase Y is important for the processing and stabilization of the immature transcript of the global virulence regulator system SaePQRS. Moreover, RNase Y is involved in the activation of virulence gene expression at the promoter level. This control is independent of both the virulence regulator agr and the saePQRS processing and may be mediated by small RNAs some of which were shown to be degraded by RNase Y. Besides this regulatory effect, mRNA levels of several operons were significantly increased in the rny mutant and the half-life of one of these operons was shown to be extremely extended. However, the half-life of many mRNA species was not significantly altered. Thus, RNase Y in S. aureus influences mRNA expression in a tightly controlled regulatory manner and is essential for coordinated activation of virulence genes.

Diversification of clonal complex 5 methicillin-resistant Staphylococcus aureus strains (Rhine-Hesse clone) within Germany.

Since 1995, a methicillin-resistant Staphylococcus aureus (MRSA) clone has spread in southern Germany. The strain was assigned to the Rhine-Hesse pulsed-field gel electrophoresis (PFGE) type by the staphylococcal reference center and was highly similar to epidemic clones known to belong to clonal complex 5 (CC5; USA100) based on multilocus sequence typing (MLST). Here we analyzed a defined collection of strains assigned to the Rhine-Hesse/USA100 PFGE type. Using sequence-based typing methods (MLST, spa), the isolates were divided into two distinct clusters, ST5 and its single-locus variant ST225. These two lineages are not distinguishable by PFGE or phage typing. Most of the ST5 isolates were derived from patients and volunteers from the Tübingen area in southwest Germany, whereas the ST225 isolates were mostly from other locations in Germany. The locally restricted ST5 isolates were shown to contain different SSCmec islands and exhibited different antibiotic resistance profiles. In contrast, the ST225 isolates form a highly homogenous group and are emerging all over Germany. The two lineages are clearly distinguishable by their phage content and spa type: ST5 strains from Tübingen are characterized by a Sa7int phage that carries the virulence gene sak, which codes for staphylokinase, and ST225 isolates are characterized by a Sa1int phage. In conclusion, based on sequence typing and phage content, CC5 strains can be subdivided into two distinct lineages with different epidemicities.

Opposing effects of aminocoumarins and fluoroquinolones on the SOS response and adaptability in Staphylococcus aureus.

OBJECTIVES: RecA is the key enzyme involved in DNA repair, recombination and induction of the SOS response and is central to the development of antibiotic resistance. Here we assessed the interaction of two different gyrase inhibitors, ciprofloxacin (a fluoroquinolone) and novobiocin (an aminocoumarin), on RecA activity and the SOS response in Staphylococcus aureus. METHODS: The influence of different gyrase inhibitors on the SOS response of S. aureus (including recA and lexA mutants) was analysed by northern blot analysis, real-time RT-PCR, western blot analysis and promoter activity assays. Recombination as well as mutation frequencies were determined for the different antibiotic combinations. RESULTS: We verified that ciprofloxacin leads to RecA activation and therefore induction of the SOS response. In contrast, novobiocin treatment resulted in an inhibition of recA transcription independent of LexA. When novobiocin and ciprofloxacin were added simultaneously, recA was reduced to the same level as with novobiocin alone. In combination, novobiocin also partially reduces the ciprofloxacin-mediated induction of the LexA target gene umuC (error-prone polymerase). Apart from reducing recA and umuC expression, novobiocin also inhibited the frequency of recombination, mutation and the formation of non-haemolytic variants. CONCLUSION: In summary, aminocoumarins inhibit recA expression in S. aureus and probably delay the process of developing antibiotic resistance and gene transfer. A clinical re-evaluation of these compounds as well as designing more applicable derivatives should be considered.

Production of capsular polysaccharide does not influence Staphylococcus aureus vancomycin susceptibility.

BACKGROUND: Diverse mechanisms (increased cell wall thickness, low cross linking, decreased autolysis, etc.) have been reported for Staphylococcus aureus strains with intermediate vancomycin susceptibility (VISA). This study was conducted to identify common mechanisms responsible for decreased vancomycin susceptibility in a VISA strain pair. RESULTS: Transcriptional profiling of the clinical heterogeneous VISA isolate SA137/93A and its spontaneous homogeneous mutant strain SA137/93G pointed to an increased capsule production in the strain pair compared to a susceptible control. Furthermore, transcript quantification of the gene cap5E, which is essential for capsule biosynthesis, revealed elevated levels in the VISA strains SA137/93A, SA137/93G and Mu50 in comparison with susceptible strains Reynolds, Newman and SA1450/94. The increased expression was observed in bacteria from exponential as well as stationary growth phase. However, suppression of type 5 capsule formation by expression of antisense RNA did not increase vancomycin susceptibility in the VISA strain SA137/93G. Likewise, construction of inducible mutants of S. aureus Newman or repair of capsule biosynthesis of S. aureus HG001 and S. aureus 1450/94 did not influence resistance to vancomycin. Furthermore, purified type 5 polysaccharide did not protect indicator strains from the action of vancomycin. CONCLUSIONS: The VISA strain tested in this study displayed an increased production of type 5 capsular polysaccharide. However, the production of capsule material did not protect strain SA137/93G and three vancomycin sensitive strains in the presence of vancomycin and thus is not part of the resistance mechanism; however it may represent a by-product of VISA life style that is often characterized by a high sigma factor B activity.

Wall teichoic Acid-dependent adsorption of staphylococcal siphovirus and myovirus.

The molecular interactions between staphylococcal phages and host cell surfaces are poorly understood. Employing Staphylococcus aureus teichoic acid mutants, we demonstrate that wall teichoic acid (WTA), but not lipoteichoic acid, serves as a receptor for staphylococcal siphovirus and myovirus, while only the siphovirus requires glycosylated WTA.

Vectors for improved Tet repressor-dependent gradual gene induction or silencing in Staphylococcus aureus.

A set of vectors for improved tetracycline-dependent gene regulation in Staphylococcus aureus is presented. Plasmid pRAB11 was generated from pRMC2 by adding a second tet operator within the TetR-regulated promoter P(xyl/tet). Pronounced repression was observed in the absence of anhydrotetracycline (ATc) combined with high induction in the presence of the drug, as demonstrated for pRAB11 bearing staphylococcal nuclease nuc1, lacZ or gfp. Also, in plasmid pCG261, the pRAB11 tetR-P(xyl/tet) regulatory architecture permitted tight repression and a stepwise increase in transcript amounts of the target gene rny (putative RNase) correlated with rising ATc concentrations. Additionally, pRAB11-derived vectors harbouring semi-rationally designed P(xyl/tet)-like fragments, mutated at up to six defined positions, were constructed. Sixteen mutant sequences with single to quadruple exchanges were analysed for transcriptional strength and ATc-dependent inducibility. A set of promoters with gradually decreased activities and improved repression is presented. Finally, the implementation of reverse TetR revtetR-r2, which exhibits three amino acid exchanges and binds to tetO in the presence of ATc, yielded an efficiently co-repressible vector within the pRAB11 system. Intriguingly, revtetR was found to contain a fourth mutation only after propagation in S. aureus. We predict that the described vectors constitute valuable tools for staphylococcal genetics.

Temporal expression of adhesion factors and activity of global regulators during establishment of Staphylococcus aureus nasal colonization.

The human pathogen Staphylococcus aureus successfully colonizes its primary reservoir, the nasal cavity, most likely by regulatory adaptation to the nose environment. Cotton rats represent an excellent model for the study of bacterial gene expression in the initial phases of colonization. To gain insight into the expression profile necessary for the establishment of colonization, we performed direct transcript analysis by quantitative real-time reverse-transcription polymerase chain reaction on cotton rat noses removed from euthanized animals on days 1, 4, or 10 after instillation of 2 human S. aureus nose isolates. Global virulence regulators (agr, sae) were not active in this early phase, but the essential 2-component regulatory system WalKR seems to play an important role. Accordingly, an elevated expression of walKR target genes (sak, sceD) could be detected. In agreement with previous studies that demonstrated the essential role played by wall teichoic acid (WTA) polymers in nasal colonization, we detected a strongly increased expression of WTA-biosynthetic genes. The expression profile switched to production of the adhesive proteins ClfB and IsdA at later stages of the colonization process. These data underscore the temporal differences in the roles of WTA and surface proteins in nasal colonization, and they provide the first evidence for a regulation of WTA biosynthesis in vivo.

Differential target gene activation by the Staphylococcus aureus two-component system saeRS.

The saePQRS system of Staphylococcus aureus controls the expression of major virulence factors and encodes a histidine kinase (SaeS), a response regulator (SaeR), a membrane protein (SaeQ), and a lipoprotein (SaeP). The widely used strain Newman is characterized by a single amino acid change in the sensory domain of SaeS (Pro18 in strain Newman [SaeS(P)], compared with Leu18 in other strains [SaeS(L)]). SaeS(P) determines activation of the class I sae target genes (coa, fnbA, eap, sib, efb, fib, sae), which are highly expressed in strain Newman. In contrast, class II target genes (hla, hlb, cap) are not sensitive to the SaeS polymorphism. The SaeS(L) allele (saeS(L)) is dominant over the SaeS(P) allele, as shown by single-copy integration of saePQRS(L) in strain Newman, which results in severe repression of class I target genes. The differential effect on target gene expression is explained by different requirements for SaeR phosphorylation. From an analysis of saeS deletion strains and strains with mutated SaeR phosphorylation sites, we concluded that a high level of SaeR phosphorylation is required for activation of class I target genes. However, a low level of SaeR phosphorylation, which can occur independent of SaeS, is sufficient to activate class II target genes. Using inducible saeRS constructs, we showed that the expression of both types of target genes is independent of the saeRS dosage and that the typical growth phase-dependent gene expression pattern is not driven by SaeRS.

Role of the (p)ppGpp synthase RSH, a RelA/SpoT homolog, in stringent response and virulence of Staphylococcus aureus.

In most bacteria, nutrient limitations provoke the stringent control through the rapid synthesis of the alarmones pppGpp and ppGpp. Little is known about the stringent control in the human pathogen Staphylococcus aureus, partly due to the essentiality of the major (p)ppGpp synthase/hydrolase enzyme RSH (RelA/SpoT homolog). Here, we show that mutants defective only in the synthase domain of RSH (rsh(syn)) are not impaired in growth under nutrient-rich conditions. However, these mutants were more sensitive toward mupirocin and were impaired in survival when essential amino acids were depleted from the medium. RSH is the major enzyme responsible for (p)ppGpp synthesis in response to amino acid deprivation (lack of Leu/Val) or mupirocin treatment. Transcriptional analysis showed that the RSH-dependent stringent control in S. aureus is characterized by repression of genes whose products are predicted to be involved in the translation machinery and by upregulation of genes coding for enzymes involved in amino acid metabolism and transport which are controlled by the repressor CodY. Amino acid starvation also provoked stabilization of the RNAs coding for major virulence regulators, such as SaeRS and SarA, independently of RSH. In an animal model, the rsh(syn) mutant was shown to be less virulent than the wild type. Virulence could be restored by the introduction of a codY mutation into the rsh(syn) mutant. These results indicate that stringent conditions are present during infection and that RSH-dependent derepression of CodY-regulated genes is essential for virulence in S. aureus.

Multilocus PCR typing strategy for differentiation of Staphylococcus aureus siphoviruses reflecting their modular genome structure.

Given the great biological importance and high diversity of temperate Staphylococcus aureus bacteriophages, a method is needed for the description of their genomic structure. Here we have updated a multiplex PCR strategy for the complex characterization of S. aureus phages of the family Siphoviridae. Based on the comparative genomic analysis of the available phage sequences, a multilocus PCR strategy for typing the major modules of the phage genome was designed. The genomic modules were classified on the basis of the genes for integrase (10 types), anti-repressor (five types), replication proteins polA, dnaC and dnaD (four types), dUTPase (four types), portal protein (eight types), tail appendices (four types) and endolysin (four types) corresponding to the integrase locus, lysogeny control region, and modules for DNA replication, transcription regulation, packaging, tail appendices and lysis respectively. The nine PCR assays designed for the above sequences were shown to be capable to identify the bacteriophage gene pool present both in the phage and bacterial genomes and their extensive mosaic structure. The established multiplex PCR-based multilocus diagnostic scheme is convenient for rapid and reliable phage and prophage classification and for the study of bacteriophage evolution.

Association of recurrent furunculosis with Panton-Valentine leukocidin and the genetic background of Staphylococcus aureus.

Staphylococcus aureus is a major cause of skin and soft tissue infections, such as furuncles, carbuncles, and abscesses, but it also frequently colonizes the human skin and mucosa without causing clinical symptoms. Panton-Valentine leukocidin (PVL) is a pore-forming toxin that has been associated with soft tissue infections and necrotizing pneumonia. We have compared the genotypes, virulence gene repertoires, and phage patterns of 74 furunculosis isolates with those of 108 control strains from healthy nasal carriers. The large majority of furunculosis strains were methicillin sensitive. Clonal cluster (CC) 121 (CC121) and CC22 accounted for 70% of the furunculosis strains but for only 8% of the nasal isolates. The PVL-encoding genes luk-PV were detected in 85% of furunculosis strains, while their prevalence among colonizing S. aureus strains was below 1%. luk-PV genes were distributed over several lineages (CCs 5, 8, 22, 30, and 121 and sequence type 59). Even within the same lineages, luk-PV-positive phages characterized furunculosis strains, while their luk-PV-negative variants were frequent among nasal strains. The very tight epidemiological linkage between luk-PV and furunculosis, which could be separated from the genetic background of the S. aureus strain as well as from the gene makeup of the luk-PV-transducing phage, lends support to the notion of an important role for PVL in human furunculosis. These results make a case for the determination of luk-PV in recurrent soft tissue infections with methicillin-sensitive as well as methicillin-resistant S. aureus.

Adaptation of Staphylococcus aureus to the cystic fibrosis lung.

Staphylococcus aureus colonizes the lungs of cystic fibrosis (CF) patients and despite treatment with antibiotics results in recurrent and relapsing infections. With increasing duration of the infection, the bacterial population is exposed more and more to changing selective pressures exerted by the host immune system, to frequent therapeutic interventions, and to interference with other microorganisms. S. aureus has evolved a variety of strategies to adapt to these challenges: Recombination and mutation provide the population with a preselected heterogeneity, resulting in an inheritable shift of phenotypic traits. This includes the emergence of isolates with mutations in metabolic (e.g. small-colony variants) and regulatory (e.g. agr mutants) genes. Additionally, phages become mobilized with a higher frequency during infection, raising the propensity for recombination. On the other hand, S. aureus can also adapt to the CF lung using regulatory mechanisms which are not well understood in this context. The quorum-sensing system agr is not activated during lung infection in CF, which is consistent with a proposed biofilm mode of growth in the lungs and also with the observation that in CF patients the organism usually remains localized in the lungs without systemic manifestation. Altogether, adaptive processes result in the generation of a heterogeneous S. aureus population in the CF lung which is highly protected against antibiotic therapy, expressing factors necessary for persistence rather than virulence.

The synthesis and function of the alarmone (p)ppGpp in firmicutes.

In most bacteria, nutrient limitations provoke the stringent control by the rapid synthesis of the alarmones pppGpp and ppGpp. The most prominent and highly conserved reaction is the repression of rRNA synthesis. Additionally, (p)ppGpp synthesis is also linked to many other physiological changes involving gene activation/repression but also protein translation, enzyme activation and replication. Whereas much of the basic research was performed with Escherichia coli there is now growing evidence that in gram-positive bacteria there are fundamental differences in (p)ppGpp synthesis, regulation and molecular function. Here we will focus on basic differences between firmicutes and proteobacteria, particularly E. coli.

CodY in Staphylococcus aureus: a regulatory link between metabolism and virulence gene expression.

The repressor CodY is reported to inhibit metabolic genes mainly involved in nitrogen metabolism. We analyzed codY mutants from three unrelated Staphylococcus aureus strains (Newman, UAMS-1, and RN1HG). The mutants grew more slowly than their parent strains in a chemically defined medium. However, only codY mutants were able to grow in medium lacking threonine. An excess of isoleucine resulted in growth inhibition in the wild type but not in the codY mutants, indicating that isoleucine plays a role in CodY-dependent repression. Prototypic CodY-repressed genes including the virulence regulator agr are repressed after up-shift with isoleucine. The CodY-dependent repression of agr is consistent with the concomitant influence of CodY on typical agr-regulated genes such as cap, spa, fnbA, and coa. However, some of these virulence genes (e.g., cap, fnbA, and spa) were also regulated by CodY in an agr-negative background. Microarray analysis revealed that the large majority of CodY-repressed genes were involved in amino acid metabolism; CodY-activated genes were mainly involved in nucleotide metabolism or virulence. In summary, CodY in S. aureus not only acts as a repressor for genes involved in nitrogen metabolism but also contributes to virulence gene regulation by supporting as well as substituting for agr function.

Diversity of prophages in dominant Staphylococcus aureus clonal lineages.

Temperate bacteriophages play an important role in the pathogenicity of Staphylococcus aureus, for instance, by mediating the horizontal gene transfer of virulence factors. Here we established a classification scheme for staphylococcal prophages of the major Siphoviridae family based on integrase gene polymorphism. Seventy-one published genome sequences of staphylococcal phages were clustered into distinct integrase groups which were related to the chromosomal integration site and to the encoded virulence gene content. Analysis of three marker modules (lysogeny, tail, and lysis) for phage functional units revealed that these phages exhibit different degrees of genome mosaicism. The prevalence of prophages in a representative S. aureus strain collection consisting of 386 isolates of diverse origin was determined. By linking the phage content to dominant S. aureus clonal complexes we could show that the distribution of bacteriophages varied remarkably between lineages, indicating restriction-based barriers. A comparison of colonizing and invasive S. aureus strain populations revealed that hlb-converting phages were significantly more frequent in colonizing strains.

Functional characterization of the sigmaB-dependent yabJ-spoVG operon in Staphylococcus aureus: role in methicillin and glycopeptide resistance.

The alternative sigma factor sigma(B) of Staphylococcus aureus controls the expression of multiple genes, including virulence determinants and global regulators; promotes capsule production; and increases the resistance levels of methicillin-resistant S. aureus (MRSA) and glycopeptide-intermediate-resistant S. aureus (GISA) strains. We show here that deletion of the sigma(B)-controlled yabJ-spoVG operon, which codes for potential downstream regulators of sigma(B), abolished capsule synthesis and reduced resistance in MRSA and GISA to the same extent that sigma(B) inactivation did. Introduction of the yabJ-spoVG operon in trans restored the original phenotype. By genetic manipulations, we show that SpoVG but not YabJ is required for complementation. We therefore postulate that SpoVG is the major factor of the yabJ-spoVG operon required in S. aureus for capsule formation and antibiotic resistance.