The global regulators agr (accessory gene regulator) and sarA (staphylococcal accessory regulator) have been reported to be both activators and repressors of virulence gene expression in Staphylococcus aureus. How the effector of the agr system, RNAIII, interacts with target gene promoters is unknown. SarA, on the other hand, is a DNA‐binding protein, which binds to conserved DNA motifs immediately upstream of both positively and negatively regulated promoters. Here, we searched for additional regulators that could explain the differential effects of RNAIII and SarA. Four differently regulated genes (hla, alpha‐toxin; hld, RNAIII; spa, protein A; ssp, serine protease) were analysed for binding of potential regulatory proteins to the corresponding promoter DNA fragments, linked to magnetic beads. One protein (29 kDa), with affinity for all four promoters, showed a high degree of similarity to SarA and was named SarH1 (Sar homologue 1). Expression of sarH1 was strongly repressed by sarA and agr. Analysis of hla, hld, ssp and spa mRNAs in sarH1, sarA and agr mutants, and in sarA/sarH1 and agr/sarH1 double mutants, revealed that sarH1 has a strong repressive effect on hla and an activating effect on spa transcription. SDS–PAGE analysis of secreted proteins from the different mutants showed that the production of several other exoproteins was affected by sarH1. In conclusion, we show that both the agr‐dependent suppression of protein A production and the sarA‐dependent stimulation of alpha‐toxin production is mediated via a new regulator, SarH1, which belongs to a family of Sar homologues.

Many of the genes coding for extracellular toxins, enzymes and cell-surface proteins in Staphylococcus aureus are regulated by a 510 nt RNA molecule, RNAIII. Expression of the RNAIII gene is positively controlled by the closely linked agr operon, which encodes a multicomponent signal-transduction system, and by an unlinked operon called sar. We have analysed the 120 bp region that separates the RNAIII promoter, P3, from the divergent agr promoter, P2. By transcription analysis, it was shown that P3 can function in trans of the agr operon. A stretch of 53 bp upstream of P3, containing an interrupted repeat of 7 bp, was found to be required for the agr-dependent expression of RNAIII. A single cytoplasmic protein was shown to bind to at least two sites within this regulatory region. The protein, which was absent in extracts from a sarA mutant, was identified as the sarA product by N-terminal amino acid sequencing. A DNA fragment from the P2 region, encompassing an almost identical repeated DNA motif, competed for the same protein. No interaction between the regulatory DNA sequence and any agr-dependent products could be demonstrated. The results of this study suggest that P3 and P2 are controlled by a mechanism involving the binding of the SarA protein to multiple sites within the regulatory regions immediately upstream of each promoter, and the as yet unknown activity of AgrA.

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Data have been presented indicating thatStaphylococcus aureus produces several cell surface proteins which bind specifically to different host extracellular matrix proteins and plasma proteins (12,13,32). For many of the cell surface proteins a role in colonization and virulence has been demonstrated in animal models of infection (17,23,27,33). Two highly homologous fibronectin-binding proteins (FnBPA and FnBPB), encoded by fnbA and fnbB, have been characterized (14, 21, 25, 41) and shown to be involved in adherence to damaged heart valves (23) and to promote internalization of S. aureus by epithelial cells (9). Although S. aureus is primarily considered to be an extracellular pathogen, the intracellular niche could promote long-term colonization and maintenance of chronic infections.Protein A (Spa), which binds immunoglobulin G (IgG) by the Fc segment, is a major surface protein present in virtually all strains of S. aureus (10, 11). Strains of S. aureus with a high content of Spa are more resistant to phagocytosis by human neutrophils in vitro than strains with less Spa (34). Reduced virulence of a spa mutant compared to that of the corresponding wild type was demonstrated in a mouse intraperitoneal infection (31).We have recently shown that transcription of the fnbA and fnbB genes is negatively regulated by agr and by an agr-independent mechanism that restricts fnb mRNA synthesis to the early exponential phase of growth (38). A similar temporal control of fnb transcription was also found in another strain of S. aureus (Newman) (43). However, only fnbA appeared to be regulated by agr in this strain. It was also found that fnbA, but not fnbB, was positively regulated by sarA. As for fnbA and fnbB, transcription of spa is negatively regulated by agr (20). However, unlike for fnbA, transcription of spa is negatively controlled by sarA (3,42).Data from recent studies indicate that both FnBPs and protein A may be degraded by extracellular proteases (3, 26, 42). Four major extracellular proteases are produced by S. aureus (1): staphylococcal serine protease (V8 protease) (SspA), a metalloprotease named aureolysin (Aur), a cysteine protease (Scp) named staphopain (18), and a second cysteine protease (SspB) encoded within the same operon as SspA (2, 36).

Many of the genes coding for extracellular toxins, enzymes, and cell surface proteins in Staphylococcus aureus are regulated by a 510-nucleotide (nt) RNA molecule, RNAIII. Transcription of genes encoding secreted toxins and enzymes, includinghla (alpha-toxin), saeB (enterotoxin B),tst (toxic shock syndrome toxin 1), and ssp(serine protease), is stimulated, while transcription of genes encoding cell surface proteins, like spa (protein A) andfnb (fibronectin binding proteins), is repressed. Besides being a regulator, RNAIII is also an mRNA coding for staphylococcal delta-lysin. We have identified RNAIII homologs in three different coagulase-negative staphylococci (CoNS), i.e., Staphylococcus epidermidis, Staphylococcus simulans, andStaphylococcus warneri. RNAIII from these CoNS turned out to be very similar to that of S. aureus and contained open reading frames encoding delta-lysin homologs. Though a number of big insertions and/or deletions have occurred, mainly in the 5′ half of the molecules, the sequences show a high degree of identity, especially in the first 50 and last 150 nt. The CoNS RNAIII had the ability to completely repress transcription of protein A in an RNAIII-deficientS. aureus mutant and the ability to stimulate transcription of the alpha-toxin and serine protease genes. However, the stimulatory effect was impaired compared to that of S. aureus RNAIII, suggesting that these regulatory functions are independent. By creating S. epidermidis-S. aureus RNAIII hybrids, we could also show that both the 5′ and 3′ halves of the RNAIII molecule are involved in the transcriptional regulation of alpha-toxin and serine protease mRNAs in S. aureus.