The Streptococcus mutans serine/threonine kinase, PknB, regulates competence development, bacteriocin production, and cell wall metabolism

Abstract

Bacteria can detect, transmit, and react to signals from the outside world by using two-component systems (TCS) and serine-threonine kinases and phosphatases. Streptococcus mutans contains one serine-threonine kinase, encoded by pknB. A gene encoding a serine-threonine phosphatase, pppL, is located upstream of pknB. In this study, the phenotypes of pknB and pppL single mutants and a pknB pppL double mutant were characterized. All mutants exhibited a reduction in genetic transformability and biofilm formation, showed abnormal cell shapes, grew slower than the wild-type strain in several complex media, and exhibited reduced acid tolerance. The mutants had reduced cariogenic capacity but no significant defects in colonization in a rat caries model. Whole-genome transcriptome analysis revealed that a pknB mutant showed reduced expression of genes involved in bacteriocin production and genetic competence. Among the genes that were differentially regulated in the pknB mutant, several were likely to be involved in cell wall metabolism. One such gene, SMU.2146c, and two genes encoding bacteriocins were shown to also be downregulated in a vicK mutant, which encodes a sensor kinase involved in the response to oxidative stress. Collectively, the results lead us to speculate that PknB may modulate the activity of the two-component signal transduction systems VicKR and ComDE. Real-time reverse transcriptase PCR (RT-PCR) showed that genes downregulated in the pknB mutant were upregulated in the pppL mutant, indicating that PppL serves to counteract PknB.

FIG. 1.

Transmission electron microscopy of S. mutans UA159 and the mutants EA72, EA74, and EA75 (see Table 1) at magnifications of ×2,500 (A) and ×12,000 (B). The dashed lines indicate planes of division which are in an angle. wt, wild type.

FIG. 2.

Growth of mutants (see Table 1) at pH 7.0 and pH 5.0.

FIG. 3.

Inhibition of E. faecalis CG110 by S. mutans. wt, wild type.

FIG. 4.

(A) Comparison of microarray results and real-time RT-PCR results. Filled bars, results from microarray analysis; open bars, results from real-time RT-PCR analysis. (B) Correlation between gene expression data obtained from microarray analysis and real-time PCR analysis.

FIG. 5.

Expression of SMU.2146c, bsmH, and bsmA in hk11 (OMZ 955), vicK (OMZ 953), comDE (OMZ 1111), and ciaRH (OMZ 1110) mutants relative to that in the wild-type strain (UA159).

FIG. 6.

Real-time PCR analysis of expression of selected genes in the pppL mutant strain EA74 relative to that in the wild-type strain.