Identification of group A Streptococcus genes directly regulated by CsrRS and novel intermediate regulators [ChIP-seq]

Adaptation of group A Streptococcus (GAS) to its human host is mediated by two component systems that transduce external stimuli to regulate bacterial physiology. Among such systems, CsrRS (also known as CovRS) is the most extensively characterized for its role in regulating ~10% of the GAS genome including several virulence genes. Here we show that extracellular magnesium and the human antimicrobial peptide LL-37 have opposing effects on phosphorylation of the response regulator CsrR by the receptor kinase CsrS. Genetic inactivation of CsrS phosphatase or kinase activity, respectively, had similar, but more pronounced effects on CsrR phosphorylation compared to growth in magnesium or LL-37. These changes in CsrR phosphorylation were correlated with repression or activation of CsrR-regulated genes as assessed by NanoString analysis. Chromatin immunoprecipitation and DNA sequencing (ChIP-seq) revealed CsrR binding at CsrRS-regulated promoters and lower affinity binding at many other locations on the GAS chromosome. Because ChIP-seq did not detect CsrR binding at promoters associated with some CsrR-regulated genes, we investigated whether these genes might be controlled indirectly by intermediate regulators whose expression is modulated by CsrR. Transcriptional profiling of mutant strains deficient in expression of either of two previously uncharacterized transcriptional regulators in the CsrR regulon indicated that one or both proteins participated in regulation of 22 of the 42 CsrR-regulated promoters for which no CsrR binding was detected by ChIP-seq. Taken together, these results illuminate CsrRS-mediated regulation of GAS gene expression through modulation of CsrR phosphorylation, CsrR binding to regulated promoters, and control of intermediate transcriptional regulators. Identification of DNA associated with the GAS protein CsrR