Phosphorylation at the D53 but Not the T65 Residue of CovR Determines the Repression of rgg and speB Transcription in emm 1- and emm 49-Type Group A Streptococci

Abstract

CovR/CovS is a two-component regulatory system in group A Streptococcus and primarily acts as a transcriptional repressor. The D53 residue of CovR (CovR_D53) is phosphorylated by the sensor kinase CovS, and the phosphorylated CovR_D53 protein binds to the intergenic region of rgg-speB to inhibit speB transcription. Nonetheless, the transcription of rgg and speB is suppressed in covS mutants. The T65 residue of CovR is phosphorylated in a CovS-independent manner, and phosphorylation at the D53 and T65 residues of CovR is mutually exclusive. Therefore, how phosphorylation at the D53 and T65 residues of CovR contributes to the regulation of rgg and speB expression was elucidated. The transcription of rgg and speB was suppressed in the strain that cannot phosphorylate the D53 residue of CovR (CovR_D53A mutant) but restored to levels similar to those of the wild-type strain in the CovR_T65A mutant. Nonetheless, inactivation of the T65 residue phosphorylation in the CovR_D53A mutant cannot derepress the rgg and speB transcription, indicating that phosphorylation at the T65 residue of CovR is not required for repressing rgg and speB transcription. Furthermore, trans complementation of the CovR_D53A protein in the strain that expresses the phosphorylated CovR_D53 resulted in the repression of rgg and speB transcription. Unlike the direct binding of the phosphorylated CovR_D53 protein and its inhibition of speB transcription demonstrated previously, the present study showed that inactivation of phosphorylation at the D53 residue of CovR contributes dominantly in suppressing rgg and speB transcription.IMPORTANCE CovR/CovS is a two-component regulatory system in group A Streptococcus (GAS). The D53 residue of CovR is phosphorylated by CovS, and the phosphorylated CovR_D53 binds to the rgg-speB intergenic region and acts as the transcriptional repressor. Nonetheless, the transcription of rgg and Rgg-controlled speB is upregulated in the covR mutant but inhibited in the covS mutant. The present study showed that nonphosphorylated CovR_D53 protein inhibits rgg and speB transcription in the presence of the phosphorylated CovR_D53in vivo, indicating that nonphosphorylated CovR_D53 has a dominant role in suppressing rgg transcription. These results reveal the roles of nonphosphorylated CovR_D53 in regulating rgg transcription, which could contribute significantly to invasive phenotypes of covS mutants.

Keywords: CovR/CovS; Rgg; SpeB; group A Streptococcus.

FIG 1

Phosphorylation levels of CovR and the expression of CovR, rgg, and speB in the wild-type A20 strain (emm1 type) and its CovR_D53A and CovR_T65A mutants. Bacterial strains were cultured to the exponential (for detecting CovR) and stationary (for detecting rgg and speB) phases of growth. RNAs, culture supernatants, and total proteins were collected for quantitative real-time PCR (RT-qPCR), Western blotting, and Phos-tag Western blot analyses. (A) Levels of phosphorylation and expression of CovR protein in A20, CovR_D53A mutant, and CovR_T65A mutant. CovR∼P, phosphorylated CovR; CovR, nonphosphorylated CovR. Total protein serves as the loading control. (B) Transcription of rgg in A20, CovR_D53A mutant, and CovR_T65A mutant. (C) The transcription of speB and secretion of SpeB protein in A20, CovR_D53A mutant, and CovR_T65A mutant. Biological replicate experiments were performed using three independent preparations. The expression of rgg and speB was normalized to that of gyrA. Wt, wild-type strain; *, P < 0.05.

FIG 2

Phosphorylation levels of CovR and the expression of CovR, rgg, and speB in the wild-type NZ131 strain (emm49 type) and its CovR_D53A and CovR_T65A mutants. Bacterial strains were cultured to the exponential (for detecting CovR) and stationary (for detecting rgg and speB) phases of growth. RNAs, culture supernatants, and total proteins were collected for RT-qPCR, Western blotting, and Phos-tag Western blot analyses. (A) Levels of phosphorylation and expression of CovR protein in NZ131, CovR_D53A mutant, and CovR_T65A mutant. CovR∼P, phosphorylated CovR; CovR, nonphosphorylated CovR. Total protein serves as the loading control. (B) Transcription of rgg in NZ131, CovR_D53A mutant, and CovR_T65A mutant. (C) Transcription of speB and secretion of SpeB protein in NZ131, CovR_D53A mutant, and CovR_T65A mutant. Biological replicate experiments were performed using three independent preparations. The expression of rgg and speB was normalized to that of gyrA. Wt, wild-type strain; *, P < 0.05.

FIG 3

Phosphorylation levels of CovR and the expression of CovR, rgg, and speB in emm1-type A20 and its CovR_T65A and CovR_D53A-T65A mutants. Bacterial strains were cultured to the exponential (for detecting CovR) and stationary (for detecting rgg and speB) phases of growth. RNAs, culture supernatants, and total proteins were collected for RT-qPCR, Western blotting, and Phos-tag Western blot analyses. (A) Levels of phosphorylation and expression of CovR protein in A20, CovR_D53A mutant, and CovR_D53A-T65A mutant. CovR∼P, phosphorylated CovR; CovR, nonphosphorylated CovR. Total protein serves as the loading control. (B) Transcription of rgg in A20, CovR_T65A mutant, and CovR_D53A-T65A mutant. (C) Transcription of speB and secretion of SpeB protein in A20, CovR_T65A mutant, and CovR_D53A-T65A mutant. Biological replicate experiments were performed using three independent preparations. The expression of rgg and speB was normalized to that of gyrA. Wt, wild-type strain; *, P < 0.05.

FIG 4

Expression of rgg and speB in CovR_T65A mutants and their vector control and CovR_D53A trans-complementary strains. Bacterial strains were cultured to the stationary phase of growth. RNAs and culture supernatants were collected for RT-qPCR and Western blot analyses. (A and B) Transcription of rgg and the transcriptional and translational levels of SpeB expression in the emm1-type A20 strain, CovR_T65A mutant, and its vector control (Vector) and CovR_D53A trans-complementary (pCovR_D53A) strains. (C and D) Transcription of rgg and the transcriptional and translational levels of SpeB expression in the emm49-type NZ131 strain, CovR_T65A mutant, and its vector control and CovR_D53A trans-complementary strains. Biological replicate experiments were performed using three independent preparations. The expression of rgg and speB was normalized to that of gyrA. Wt, wild-type strain; *, P < 0.05.

FIG 5

Expression of rgg and speB in CovR_D53A mutants and their vector control and CovR_T65A trans-complementary strains. Bacterial strains were cultured to the stationary phase of growth. RNAs and culture supernatants were collected for RT-qPCR and Western blot analyses. (A and B) Transcription of rgg and the transcriptional and translational levels of SpeB expression in the emm1-type A20 strain, CovR_D53A mutant, and its vector control (Vector) and CovR_T65A trans-complementary (pCovR_T65A) strains. (C and D) Transcription of rgg and the transcriptional and translational levels of SpeB expression in the emm49-type NZ131 strain, CovR_D53A mutant, and its vector control and CovR_T65A trans-complementary strains. Biological replicate experiments were performed using three independent preparations. The expression of rgg and speB was normalized to that of gyrA. Wt, wild-type strain; *, P < 0.05.