Molecular insights into the control of transcription initiation at the Staphylococcus aureus agr operon

Abstract

Central to the regulation of the diverse array of virulence factors encoded by the opportunistic human pathogen Staphylococcus aureus is the accessory gene regulator (agr) operon. The agr operon contains two transcription units; RNAII, encoding a quorum-sensing system including the master transcriptional activator AgrA, and RNAIII, the effector RNA molecule that regulates virulence gene expression. RNAII and RNAIII are transcribed from the divergent promoters P2 and P3, respectively. Due to the sub-optimal spacer length of the P3 promoter, it is widely believed that transcription from P3 is dependent on AgrA. A fully native S. aureus in vitro transcription system was setup to provide the first insight into the molecular and mechanistic characterisation of the regulation of transcription initiation at the agr operon. Surprisingly, results revealed that, in the absence of AgrA, RNA polymerase (RNAp) can interact with P2 and P3 equally well, however the transcription-competent open promoter complex (RPo) forms more readily at P2 than P3. AgrA was demonstrated to increase the occupancy of P2 and P3 by RNAp, to increase transcription initiation at both promoters, with a more pronounced effect at P3. The P3 promoter is unusual as it has a genuine extended -10 motif and a near to consensus -35 promoter element. "Scrambling" of the -35 element or shortening of the sub-optimal spacer to the optimal 17 nucleotides both significantly increased transcription initiation at P3, by facilitating the rate of isomerisation of the initial RNAp-P3 complexes to the RPo, in a strictly extended -10 promoter element dependent manner. Therefore, two possible mutually inclusive mechanisms by which AgrA activates transcription at the agr operon P3 promoter are proposed. In addition, the involvement of the transcription factor SarA on transcription from p2 and P3 was investigated. Overall, this study provides the first molecular insights into how transcription is regulated at the agr operon in S. aureus.