Phosphorylation of the Group A Streptococcal CovR Response Regulator Causes Dimerization and Promoter-Specific Recruitment by RNA Polymerase

Abstract

The group A streptococcus (GAS), Streptococcus pyogenes, is an important human pathogen that causes infections ranging in severity from self-limiting pharyngitis to severe invasive diseases that are associated with significant morbidity and mortality. The pathogenic effects of GAS are mediated by the expression of virulence factors, one of which is the hyaluronic acid capsule (encoded by genes in the has operon). The expression of these virulence factors is controlled by the CovR/S (CsrR/S) two-component regulatory system of GAS which regulates, directly or indirectly, the expression of about 15% of the genome. CovR is a member of the OmpR/PhoB family of transcriptional regulators. Here we show that phosphorylation by acetyl phosphate results in dimerization of CovR. Dimerization was not observed using a D53A mutant of CovR, indicating that D53 is the site of phosphorylation in CovR. Phosphorylation stimulated binding of CovR to a DNA fragment containing the promoter of the has operon (Phas) approximately twofold. Binding of CovR D53A mutant protein to Phas was indistinguishable from the binding of wild-type unphosphorylated CovR. In vitro transcription, using purified GAS RNA polymerase, showed that wild-type CovR repressed transcription, and repression was stimulated more than sixfold by phosphorylation. In the presence of RNA polymerase, binding at Phas of phosphorylated, but not unphosphorylated, CovR was stimulated about fourfold, which accounts for the difference in the effect of phosphorylation on repression versus DNA binding. Thus, regulation of Phas by CovR is direct, and the degree of repression of Phas is controlled by the phosphorylation of CovR.

Unraveling the Regulatory Network in Streptococcus pyogenes : the Global Response Regulator CovR Represses rivR Directly

“…Because changing the double thymine residues to double guanine residues in these ATTARA motifs prevents binding of CovR, this motif was suggested to be a consensus binding sequence (7). Transcription from Phas is repressed in vitro by CovR in the absence of other factors, showing that this repression is direct (14). Similar results were reported for the cov promoter (Pcov) (15).…”

“…The ability of CovR to bind to promoter regions and the role of this binding have been characterized for several genes that are repressed by CovR (1,7,10,14,15,17). At the has promoter (Phas), which directs transcription of genes required for synthesis of the hyaluronic acid capsule, CovR binds to five sites, each of which contains a single ATTARA motif (7).…”

“…Comparison of the transcriptome of a ⌬covR strain to that of its wild-type parent showed that CovR represses expression of several proven and putative transcriptional regulators (3,11). CovR also represses its own transcription directly (14). To begin an investigation of the CovR network, we have started to explore this regulatory cascade by focusing on CovR regulation of a potential transcriptional regulator gene found in microarray analyses to be repressed by CovR, SPy0216 (3,11), which encodes Ralp4.…”

Two-component signal transduction systems in oral bacteria

“…There is evidence that CovS Spy is a bifunctional sensor protein, switching kinase or phosphatase activities depending on the environmental stimuli. Under growth conditions, CovS Spy acts as a kinase that phosphorylates CovR at conserved apartate (D-53) of the receiver domain, thus increasing its affinity for target gene promoters leading to repression of transcription [163]. Under environmental stress conditions including high temperature (40°C), low pH (≈6.0), and high salt concentration, CovS Spy acts as a phosphatase that dephosphorylates CovR Spy , which derepresses transcription of target genes [164].…”

Modulation ofcovRExpression inStreptococcus mutansUA159

“…A consensus binding sequence (CBS), ATTARA, was identified in the promoter sequences regulated by CovR in GAS, including the promoters for covR, has, and dppA (18,(23)(24)(25). However, while the ATTARA sequence appeared to be essential for binding under in vitro conditions, the CBS did not appear to be required under in vivo conditions, since expres- 4486 CHONG ET AL.…”