Antagonistic Rgg Regulators Mediate Quorum Sensing via Competitive DNA Binding in Streptococcus pyogenes

Recent studies have established the fact that multiple members of the Rgg family of transcriptional regulators serve as key components of quorum sensing (QS) pathways that utilize peptides as intercellular signaling molecules. We previously described a novel QS system in Streptococcus pyogenes which utilizes two Rgg-family regulators (Rgg2 and Rgg3) that respond to neighboring signaling peptides (SHP2 and SHP3) to control gene expression and biofilm formation. We have shown that Rgg2 is a transcriptional activator of target genes, whereas Rgg3 represses expression of these genes, and that SHPs function to activate the QS system. The mechanisms by which Rgg proteins regulate both QS-dependent and QS-independent processes remain poorly defined; thus, we sought to further elucidate how Rgg2 and Rgg3 mediate gene regulation. Here we provide evidence that S. pyogenes employs a unique mechanism of direct competition between the antagonistic, peptide-responsive proteins Rgg2 and Rgg3 for binding at target promoters. The highly conserved, shared binding sites for Rgg2 and Rgg3 are located proximal to the 35 nucleotide in the target promoters, and the direct competition between the two regulators results in concentrationdependent, exclusive occupation of the target promoters that can be skewed in favor of Rgg2 in vitro by the presence of SHP. These results suggest that exclusionary binding of target promoters by Rgg3 may prevent Rgg2 binding under SHP-limiting conditions, thereby preventing premature induction of the quorum sensing circuit.