Hyperphosphorylation of the Group A Streptococcal Control of Virulence Regulator Increases Promoter Occupancy Specifically at Virulence Factor-Encoding Genes.

The ontrol f irulence two-component gene regulatory system (CovRS) is critical to the pathogenesis of many medically important streptococci. In group A streptococci (GAS), CovR directly binds the promoters of numerous GAS virulence factor-encoding genes. Elimination of CovS phosphatase activity increases CovR phosphorylation (CovRP) levels and abrogates GAS virulence. Given the type-specific diversity of CovRS function, in this study we used chromatin immunoprecipitation sequencing (ChIP-seq) to define global CovR DNA occupancy in the wild-type strain MGAS10870 (medium CovRP) and its CovS phosphatase-negative derivative 10870-CovS-T284A (high CovRP). In the wild-type strain, 89% of the previously identified CovR binding sites present in the genome were also enriched; additionally, we ascertained unique CovR binding, primarily to genes in mobile genetic elements and other sites of interstrain chromosomal differences. Elimination of CovS phosphatase activity specifically increased CovR occupancy at the promoters of a broad array of CovR repressed virulence factor-encoding genes, including those encoding the key GAS regulator Mga and M protein. However, a limited number of promoters had augmented enrichment at low CovRP levels. Differential motif searches using sequences enriched at high versus low CovRP levels revealed two distinct binding patterns. At high CovRP, a pseudopalindromic AT-rich consensus sequence (WTWTTATAAWAAAAWNATDA) consistent with CovR binding as a dimer was determined. Conversely, sequences specifically enriched at low CovR~P contained isolated ATTARA motifs suggesting an interaction with a monomer. These data extend understanding of global CovR DNA occupancy beyond GAS and provide a mechanism for previous observations regarding hypovirulence induced by CovS phosphatase abrogation. Given its key role in pathogenesis of Gram-positive bacteria, CovR is one of the most important members of the OmpR/PhoB family of transcriptional regulators. Herein we extend recent GAS CovR global binding analyses done in to a non- strain, which is important considering the known inter--type heterogeneity in GAS CovRS function. Our data provide mechanistic understanding for variation in CovRS function between types and the profound hypovirulence of CovS phosphatase-negative strains in addition to indicating differential targeting by phosphorylated and nonphosphorylated CovR isoforms at specific CovR binding sites. These findings advance knowledge regarding how a key bacterial virulence regulator impacts pathogenesis and add to the growing appreciation of the function of nonphosphorylated OmpR/PhoB family members.