ABSTRACTThecontrolofvirulence two-component gene regulatory system (CovRS) is critical to the pathogenesis of many medically important streptococci. Inemm1group A streptococci (GAS), CovR directly binds the promoters of numerous GAS virulence factor-encoding genes. Elimination of CovS phosphatase activity increases CovR phosphorylation (CovR~P) levels and abrogates GAS virulence. Given theemmtype-specific diversity of CovRS function, in this study we used chromatin immunoprecipitation sequencing (ChIP-seq) to define global CovR DNA occupancy in the wild-typeemm3strain MGAS10870 (medium CovR~P) and its CovS phosphatase-negative derivative 10870-CovS-T284A (high CovR~P). In the wild-typeemm3strain, 89% of the previously identifiedemm1CovR binding sites present in theemm3genome 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 CovR~P levels. Differential motif searches using sequences enriched at high versus low CovR~P levels revealed two distinct binding patterns. At high CovR~P, 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 isolatedATTARAmotifs suggesting an interaction with a monomer. These data extend understanding of global CovR DNA occupancy beyondemm1GAS and provide a mechanism for previous observations regarding hypovirulence induced by CovS phosphatase abrogation.IMPORTANCEGiven 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 inemm1to a non-emm1strain, which is important considering the known inter-emm-type heterogeneity in GAS CovRS function. Our data provide mechanistic understanding for variation in CovRS function betweenemmtypes 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.