The synthesis of virulence factors in Staphylococcus aureus is controlled by a regulatory RNA molecule, RNAIII, encoded by the agr locus. Transcription of genes coding for secreted toxins and enzymes is stimulated, while transcription of cell‐surface protein genes is repressed by RNAIII. In the case of staphylococcal alpha‐toxin, RNAIII also seems to stimulate translation by an independent mechanism. In this report we show that in a mutant lacking RNAIII the rate of alpha‐toxin (hla) production relative to the cellular concentration of hla mRNA was reduced 10‐fold as compared with the wild‐type strain. A 75% complementarity between the 5′ end of RNAIII and the 5′ untranslated region of the hla transcript suggests a direct interaction between the RNAs. A complex of RNAIII and hla mRNA was demonstrated in extracts of total RNA from the wild‐type strain, and also with in vitro synthesized RNAs. Ribonuclease T1 digestion experiments revealed that the ribosome binding site of the hla transcript is blocked by intramolecular base‐pairing. Hybridization with RNAIII prevents this intramolecular base‐pairing and makes the hla mRNA accessible for translation initiation. This is, to our knowledge, the first example of an ‘antisense RNA’ that stimulates translation of the target mRNA.

Staphylococcus aureus produces a wide array of cell surface and extracellular proteins involved in virulence. Expression of these virulence factors is tightly controlled by numerous regulatory loci, including agr, sar, sigB, sae, and arl, as well as by a number of proteins with homology to SarA. Rot (repressor of toxins), a SarA homologue, was previously identified in a library of transposon-induced mutants created in an agr-negative strain by screening for restored protease and alpha-toxin. To date, all of the SarA homologues have been shown to act as global regulators of virulence genes. Therefore, we investigated the extent of transcriptional regulation of staphylococcal genes by Rot. We compared the transcriptional profile of a rot agr double mutant to that of its agr parental strain by using custom-made Affymetrix GeneChips. Our findings indicate that Rot is not only a repressor but a global regulator with both positive and negative effects on the expression of S. aureus genes. Our data also indicate that Rot and agr have opposing effects on select target genes. These results provide further insight into the role of Rot in the regulatory cascade of S. aureus virulence gene expression.

The synthesis of at least 14 extracellular toxins and enzymes in Staphylococcus aureus is regulated by a set of trans‐acting elements from the agr (accessory gene regulator) locus. We have shown that the delta‐lysin gene (hld) that is transcribed from a promoter immediately upstream of the agr locus, and which is positively controlled by agr, is part of this regulatory system. Deletion replacement mutagenesis of the chromosomal hld gene had the same pleiotropic effect on the synthesis of several virulence factors as agrA mutations. Characteristically, these mutants had an almost complete block in the synthesis of alpha‐toxin, serin‐ and metalloprotease, whereas synthesis of protein A was greater than 10‐fold higher than in the parental strain. Corresponding changes in the levels of alpha‐toxin and protein A mRNAs were demonstrated by northern blotting experiments. The effects of the hld deletion mutation could be fully complemented by the hld gene on a plasmid. A plasmid insertion mutation in the 3′ non‐coding region of hld had a similar effect on exoprotein synthesis, indicating a role of the hld transcript in the regulation of exoprotein synthesis. This was confirmed by the finding that the effects of alpha‐toxin and protein A synthesis by the hld deletion replacement mutation could be fully complemented by a hld allele in which we had introduced an early stop codon in the delta‐lysin structural gene. However, the mutant hld allele could not complement the defect in production of extracellular proteases, indicating that delta‐lysin may act in conjunction with its mRNA to regulate the expression of some exoprotein genes.

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.