Coordinated Regulation by AgrA, SarA, and SarR To Control agr Expression in Staphylococcus aureus

Abstract

ABSTRACT The agr locus of Staphylococcus aureus is composed of two divergent transcripts (RNAII and RNAIII) driven by the P2 and P3 promoters. The P2-P3 intergenic region comprises the SarA/SarR binding sites and the four AgrA boxes to which AgrA binds. We reported here the role of AgrA, SarA, and SarR on agr P2 and P3 transcription. Using real-time reverse transcription (RT)-PCR and promoter fusion studies with selected single, double, triple, and complemented mutants, we showed that AgrA is indispensable to agr P2 and P3 transcription, whereas SarA activates and SarR represses P2 transcription. In vitro runoff transcription assays revealed that AgrA alone promoted transcription from the agr P2 promoter, with SarA enhancing it and SarR inhibiting agr P2 transcription in the presence of AgrA or with SarA and AgrA. Electrophoretic mobility shift assay (EMSA) analysis disclosed that SarR binds more avidly to the agr promoter than SarA and displaces SarA from the agr promoter. Additionally, SarA and AgrA bend the agr P2 promoter, whereas SarR does not. Collectively, these data indicated that AgrA activates agr P2 and P3 promoters while SarA activates the P2 promoter, presumably via bending of promoter DNA to bring together AgrA dimers to facilitate engagement of RNA polymerase (RNAP) to initiate transcription.