Regulation of the Glucosyltransferase ( gtfBC ) Operon by CovR in Streptococcus mutans

Abstract

Streptococcus mutans is an important etiological agent of dental caries in humans. The extracellular polysaccharides synthesized by cell-associated glucosyltransferases (encoded by gtfBC) from sucrose have been recognized as one of the important virulence factors that promote cell aggregation and adherence to teeth, leading to dental plaque formation. In this study, we have characterized the effect of CovR, a global response regulator, on glucosyltransferase expression. Inactivation of covR in strain UA159 resulted in a marked increase in the GtfB and GtfC proteins, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With the use of a transcriptional reporter system of a single chromosomal copy of the PgtfB-gusA and PgtfC-gusA fusions, we confirmed the transcriptional regulation of these promoters by CovR. By in vitro electrophoretic mobility shift assays with purified CovR protein, we showed that CovR regulates these promoters directly. DNase I footprinting analyses suggest that CovR binds to large regions on these promoters near the transcription start sites. Taken together, our results indicate that CovR negatively regulates the expression of the gtfB and gtfC genes by directly binding to the promoter region.

Two-component signal transduction systems in oral bacteria

“…The overlap includes genes for cell-wall division and biofilm formation ( gbpB , lysM, gtfB , gtfC ), with VicR positively regulating these genes and CovR acting as a repressor [107,141,142]. In EMSA assays, it was observed that VicR Sm VicR and CovR co-bind the promoter regions of lysM , gbpB , and gtfC [107].…”

“…Later studies revealed that CovR Sm negatively or positively regulates about 6.5% of the S. mutans genome [142]. More importantly, CovR Sm directly represses a panel of genes required for the synthesis of water-soluble and water-insoluble glucan ( gtfB , gtfC ) and other exopolysaccharides derived or not from sucrose ( epsC and ftf ), as well as surface proteins GbpC and GbpB that bind to glucan [107,141,142]. Deletion of covR Sm reduced S. mutans cariogenicity in rat models by mechanisms not completely understood, but likely by affecting biofilm structure [162].…”

“…On the other hand, only the D-53 site is conserved in S. mutans , while neither the D-53 nor T-65 phosphorylation sites are present in CovR orthologues of streptococci of the Mitis and Sanguinus groups (Figure 4), implying different mechanisms of CovR activation between streptococcal species. In S. mutans , it was reported that in vitro phosphorylation of CovR Sm does not increase the binding affinity to target promoters [141]. It was proposed that CovR Sm -mediated regulation is dependent on the amounts of CovR Sm present in cells regardless of the phosphorylation state; i.e.…”

“…It was proposed that CovR Sm -mediated regulation is dependent on the amounts of CovR Sm present in cells regardless of the phosphorylation state; i.e. CovR Sm would be always active and/or likely phosphorylated by small phosphodonors such as AcP [141,172]. The absence of CovS protein in S. mutans and amino acid differences between CovR Spy and CovR Smu [142] (Figure 4) account for this proposal.…”

Degradation of SsrA-tagged proteins in streptococci

“…All the constructs were sequenced. For chromosomal expression of GFP or its derivatives, DNA fragments carrying gfp genes with the P 23 promoter were amplified from pIB184 derivatives and cloned into pIB107, a plasmid used for integration at the SMU.1405 locus (Biswas & Biswas, 2006). The resulting plasmids were linearized by BglI and transformed into S. mutans to obtain the desired strains.…”