Role of CovR phosphorylation in gene transcription in Streptococcus mutans

Abstract

Streptococcus mutans, the primary aetiological agent of dental caries, is one of the major bacteria of the human oral cavity. The pathogenicity of this bacterium is attributed not only to the expression of virulence factors, but also to its ability to respond and adapt rapidly to the ever-changing conditions of the oral cavity. The two-component signal transduction system (TCS) CovR/S plays a crucial role in virulence and stress response in many streptococci. Surprisingly, in S. mutans the response regulator CovR appears to be an orphan, as the cognate sensor kinase, CovS, is absent in all the strains. We found that acetyl phosphate, an intracellular phosphodonor molecule known to act in signalling, might play a role in CovR phosphorylation in vivo. We also found that in vitro, upon phosphorylation by potassium phosphoramide (a high-energy phophodonor) CovR formed a dimer and showed altered electrophoretic mobility. As expected, we found that the conserved aspartic acid residue at position 53 (D53) was the site of phosphorylation, since neither phosphorylation nor dimerization was seen when an alanine-substituted CovR mutant (D53A) was used. Surprisingly, we found that the ability of CovR to act as a transcriptional regulator does not depend upon its phosphorylation status, since the D53A mutant behaved similarly to the wild-type protein in both in vivo and in vitro DNA-binding assays. This unique phosphorylation-mediated inhibition of CovR function in S. mutans sheds light on an unconventional mechanism of the signal transduction pathway.

Keywords: CovR; Streptococcus mutans; Two component regulator.

Fig. 1.

Phosphorylation of CovR occurs at Asp53 (D53). CovR, CovR D53A and CovR D53E proteins were purified from E. coli and ~5 µg each protein was incubated with or without potassium phosphoramidate (PAM; 20 mM) for 30 min at room temperature for phosphorylation. The samples were then loaded on a native 8 % polyacrylamide gel under non-denaturing conditions and run at 120 V for 3 h 30 min. The gels were washed and stained with PageBlue (Thermo Scientific). The experiments were repeated at least twice and a representative gel is shown. Samples denoted as ‘P’ are PAM-treated (lanes 2, 4 and 6).

Fig. 2.

Dimerization of CovR. (a) Wild-type (WT) S. mutans CovR protein was purified from E. coli and then phosphorylated with PAM. Phosphorylated CovR protein (~5 µg) was then either heat treated at 90 °C for 3 min or directly incubated with 1 % formaldehyde without heat treatment. The samples were then separated on a 10 % SDS-polyacrylamide gel. (b) S. mutans D53A CovR protein (~5 µg) was purified from E. coli after incubation with either PAM or PAM plus 1 % formaldehyde. The samples were separated on a 10 % SDS-polyacrylamide gel. Experiments were repeated at least thrice and a representative gel is shown.

Fig. 3.

Detection of phosphorylated CovR in vivo. Western blot using an anti-CovR antibody of lysate wild-type (UA159), ΔcovR (IBS10), ΔackA (IBST12), Δpta (IBST6) and ΔackA Δpta (IBST16) samples separated on a 10 % phos-tag-SDS-polyacrylamide gel. Prior to loading, lysates derived from cultures grown to mid-exponential phase were incubated with β–mercaptoethanol and 3× SDS sample buffer (New England Biolabs) on ice. CovR-specific bands are indicated on the right. Note that a CovR-specific band (indicated by an asterisk, *) accumulated in the ΔackA and Δpta single mutants. The experiments were repeated at least thrice and a representative gel is shown.

Fig. 4.

Differential gene expression by CovR. Semi-quantitative RT-PCR analysis of gbpC, SMU.1882 and bacA in the wild-type (UA159/pIB184Km), ΔcovR (IBS10/pIB184Km) and various covR complementing strains (IBS10/Smu CovR, IBS10/Smu CovR D53A, IBS10/Smu CovR D53E, IBS10/GAS CovR and IBS10/GAS CovR D53A). RNA was isolated at the mid-exponential phase of growth and sQRT-PCR was performed as described in the text. The gyrA gene was included as an internal control to ensure that equal amounts of RNA were loaded per lane. The experiments were repeated at least twice, with two independent RNA isolations. (a) Lane 1, UA159/pIB184Km; lane 2, IBS10/pIB184Km; lane 3, IBS10/Smu CovR; lane 4, IBS10/Smu CovR D53A; lane 5, IBS10/Smu CovR D53E. (b) Lane 1, UA159/pIB184Km; lane 2, IBS10/pIB184Km; lane 3, IBS10/Smu CovR; lane 4, IBS10/GAS CovR; lane 5, IBS10/GAS CovR D53A. Note that lanes 4 and 5 contain CovR from GAS.

Fig. 5.

CovR-mediated gene expression in the AckA–Pta pathway mutant. Expression of P_gbpC (a) and P_SMU.1882 (b) in the wild-type (UA159), ∆covR (IBS10) and ∆ackA∆pta (IBST16) strains. The cultures were grown in THY broth at 37 °C and harvested at the mid-exponential phase, while Gus activity was measured as described in the text. The values shown are units of glucuronidase activity (with standard errors of the mean of experiments repeated at least twice).

Fig. 6.

Differential binding of wild-type and mutant CovR. Biotinylated promoter regions of gbpC and SMU.1882 were immobilized on streptavidin biosensors and then exposed to 0.5 µM CovR, CovR D53A and CovR D53E proteins purified from E. coli as described in the text. The reactions were carried out in binding buffer [20 mM Tris, 100 mM NaCl, 0.01 mM DTT, 5 % glycerol (v/v), 1 mM EDTA, 0.01 mg ml⁻¹ BSA, 5 mM MgCl₂ and 10 µg ml⁻¹ poly (dI-dC) at pH 7.5] for a period of 5 min to allow association followed by 2 min exposure to binding buffer to allow dissociation.

Fig. 7.

Putative structure of the receiver domain (REC) of S. mutans CovR. The N-terminal REC domain of S. mutans CovR was superimposed on the GAS CovR crystal structure (PDB ID: 3RJP). The structures were computed with I-TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) using the full-length sequences of CovR proteins. The key residues that constitute the ‘acidic triad’ (Glu9, Asp10 and the phosphor-accepting Asp53) are shown. Also shown are the ‘Y/T-coupling’ residues (Th74 and Try99). The residues shown in magenta belong to S. mutans CovR, while the residues shown in cyan are the GAS CovR.