The RofA Binding Site inStreptococcus pyogenesIs Utilized in Multiple Transcriptional Pathways

Understanding the regulation of adhesins defines a pathogenic bacterium's interaction with the local environment within the host. In certain strains ofStreptococcus pyogenes , transcription ofprtF , the gene which encodes the fibronectin-binding adhesin protein F, is activated by RofA under anaerobic conditions. RofA binds specifically to DNA in its target promoters and autoregulates its own expression. In this study, we have used DNase I protection assays to further investigate the interaction of RofA with its target promoters. In the region betweenrofA and the gene which encodes protein F (prtF ), RofA binds to two distinct sites: a smaller site (17 bp) adjacent to therofA promoter, and a larger site (40 bp) adjacent to theprtF promoter. Analysis of fusions to a novel reporter gene whose product consists of the fusion of the N-terminal secretion domain of protein F with the C-terminal enzymatic domain of the enterococcal alkaline phosphatase (PhoZ) revealed that the small RofA binding site had no direct role in control ofprtF transcription but contributed to regulation ofrofA . Comparison in several strains representing different patterns ofprtF expression indicated that the larger site was required for activation ofrofA and ofprtF in all strains by both RofA-dependent and -independent pathways. Thus, it would appear that a common recognition sequence provides separate entries to a final common pathway inS. pyogenes virulence gene expression. The identification of multiple RofA-like proteins and promoters with RofA binding sites implies the existence of a widespread interacting regulatory network.