Molecular Insights into the Control of Transcription Initiation at the Staphylococcus aureus agr operon

Abstract

The accessory gene regulatory (agr) operon of the opportunistic human pathogen Staphylococcus aureus is a prime pathogenesis factor in this bacterium. The agr operon consists of two transcription units, RNAII and RNAIII, which are transcribed from divergent promoters, P2 and P3, respectively. RNAII encodes a quorum-sensing system, including AgrA, the master transcription activator of the agr operon. RNAIII is the effector RNA molecule that regulates the expression of many virulence genes. Owing to the atypical spacer lengths of P2 and P3, it is widely considered that transcription from P2 and P3 only occurs in a strictly AgrA-dependent manner. Here, using a fully native S. aureus in vitro transcription system, we provide the first molecular and mechanistic characterisation of the regulation of transcription initiation at the agr operon. Surprisingly, the results demonstrate that RNA polymerase (RNAp) can interact with P2 and P3 equally well in the absence of AgrA. However, formation of a transcription-competent open promoter complex (RPo) occurs more readily at P2 than at P3 when AgrA is absent. Reducing the atypical P3 spacer region length to the optimal length of 17 nucleotides significantly improves promoter activity by facilitating the isomerisation of the initial RNAp-P3 complexes to RPo, and the extended -10-like element of P3 is required for optimal promoter activity. AgrA increases the occupancy of both promoters by RNAp and thereby increases the amount of transcription initiated at P2 and P3. However, the AgrA-mediated effect on transcription initiation is more prominent at P3 that at P2. The effect of AgrA at P2 and P3 appears to be restricted to events leading to the formation of RPo. The relevance of AgrA-independent and AgrA-dependent transcription initiation at P2 and P3 is presented in the context of our current understanding of the role of the agr operon in the pathobiology of S. aureus.

Organizational Requirements of the SaeR Binding Sites for a Functional P1 Promoter of the sae Operon in Staphylococcus aureus

“…Although not analyzed by MS, the last protein band is presumed to be the subunit (Fig. 3A) (45). When used for an in vitro transcription assay for the gyrB promoter, a SaeRindependent constitutive promoter, the purified RNAP produced transcripts in a concentration-dependent manner (Fig.…”

“…*, P Ͻ 0.05; **, P Ͻ 0.005; ns, not significant. 20-bp spacing of the agr P3 promoter was reduced to 17 bp, the resulting mutant promoter showed improved promoter activity (45). To examine whether the spacing between promoter elements plays any role in SaeR-mediated transcriptional activation, we altered the spacing of both promoters to 17 bp (P1 Ϫ1 and Pcoa ϩ1 in Fig.…”

“…Recently, staphylococcal RNAP has been purified and used to study the molecular mechanism of transcription regulators (44,45). Reynolds and Wigneshweraraj purified core RNAP from S. aureus NCTC 8325 by affinity chromatography and formed a holoenzyme by adding the vegetative sigma factor A that was expressed and purified from E. coli (45).…”

Influence of the AgrC-AgrA Complex on the Response Time of Staphylococcus aureus Quorum Sensing

“…These observations suggest that the sequence of events that precede the AgrA-induced phenotypic switch from persistent to virulent bacteria is more elaborate than hitherto anticipated. In this context, it is worth noting that phosphorylation dictates AgrA-DNA interactions (42). Phosphorylated AgrA binds the P3 promoter that governs RNAIII expression.…”

Novel Phenol-soluble Modulin Derivatives in Community-associated Methicillin-resistant Staphylococcus aureus Identified through Imaging Mass Spectrometry

“…Downstream to these PSMs are proteins annotated as NADH dehydrogenase, PAP2 superfamily phosphatase, carboxyesterase, or hypothetical proteins. The gene encoding PSM␥ is distant from the PSM␣1 gene, contained within the agr gene cluster, and embedded within rnaIII (29). Because no gene immediately adjacent to the PSMs showed clear homology to known proteinases, we explored the potential contribution of the well studied zinc-dependent S. aureus metalloprotease aureolysin.…”