CovS Inactivation Reduces CovR Promoter Binding at Diverse Virulence Factor Encoding Genes in Group A Streptococcus

We performed CovR ChIP-seq analysis in the emm1 strain MGAS2221 and its CovS kinase deficient derivative strain 2221-CovS-E281A. We identified that CovR bound in the promoter regions of nearly all virulence factor encoding genes in the CovR regulon. Additionally, direct CovR binding was observed for numerous genes encoding proteins involved in amino acid metabolism, but we found limited direct CovR binding to genes encoding other transcriptional regulators. The consensus sequence AATRANAAAARVABTAAA was present in the promoters of genes directly regulated by CovR, and mutations of highly conserved positions within this motif relieved CovR repression of the hasA and M2221_0187 promoters. Analysis of strain 2221-CovS-E281A revealed that binding of CovR at repressed, but not activated, promoters is highly dependent on CovR~P state. CovR repressed  virulence factor encoding genes could be grouped dependent on how variation in CovR~P differentially impacted DNA binding and gene transcript levels. Comparison of WT (MGAS2221), CovS kinase-deficient mutant (E281A) Please note that CovR-specific antibody was custom made (polyclonal, raised in rabbits against purified untagged CovR protein) and is not commercially available but has been used/validated in previous publications (PMIDs: 24788524, 25561708, 28289082, and 30379939)