Identification of the P3 Promoter and Distinct Roles of the Two Promoters of the SaeRS Two-Component System in Staphylococcus aureus

In Staphylococcus aureus, the SaeRS two-component system (TCS) encoded by the saePQRS operon controls expression of major virulence factors, such as coagulase and alpha-hemolysin. The saePQRS operon has two promoters: P1 and P3. The P1 promoter, a strong promoter, is autoinduced and can transcribe all four genes. Compared with P1, P3 shows fairly low but constitutive promoter activity, and it transcribes only saeR and saeS, the two genes encoding response regulator SaeR and sensor kinase SaeS. However, the role of each promoter in sae signaling has not been rigorously defined. In this study, we found that the genuine transcription start site (TSS) of P3 is located 78 nucleotides downstream of the previously reported TSS. Subsequently, the P3 promoter sequence was identified and validated by mutagenesis analyses. Deletion of the saePQ region including the P1 promoter did not significantly alter the expression patterns of coagulase and alpha-hemolysin, two well-known sae target genes. Due to its L18P substitution in a transmembrane domain, SaeS in strain Newman has a constitutive kinase activity. Interestingly, the mutation also rendered the protein unstable, but the protein stability was restored by SaeQ, suggesting a possible SaeQ-SaeS interaction. Ironically, the same mutation seems to increase mRNA stability. SaeR appears to be stabilized by SaeS, possibly by a protein-protein interaction. Chromosomal mutation of P1 did not affect the expression pattern of coagulase and alpha-hemolysin. Based on these results, we conclude that transcription of saeRS from P3 is sufficient for target gene activation and that P1 is not involved in the activation.