GENE REGULATION IN S MUTANS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Streptococcus mutans is considered as the main etiological agent for human dental caries, a global problem in oral health. S. mutans is also a leading cause of infective endocarditis. S. mutans produces a wide range of virulence factors that are needed for the biofilm formation, commonly known as dental plaque. These virulence factors are regulated by several two-component systems (TCS). A typical TCS consists of a membrane- associated sensor kinase protein, which responds to specific environmental cues and is auto phosphorylated at a conserved histidine residue. This phosphorylated residue is then transferred to a cytoplasmic response regulator (RR) for activation. This activated RR modulates the expression of certain target genes, such as those needed for biofilm formation. In S. mutans, 13 putative TCS have been identified. Among them, CovR is an important RR that represses 15% of genes in Group A Streptococcus and 6% in Group B Streptococcus. The inactivation of the covR in S. mutans leads to to altered biofilm formation, and corresponding mutants are hypocariogenic. Using DNA microarray, our laboratory has shown that ~100 genes are regulated by CovR in S. mutans. For instance, CovR represses gbpC gene encoding a glucan-binding protein and gtfB gene encoding a glucosyl-transferase. Unlike other streptococcal species, the cognate histidine kinase (HK) for CovR in S. mutans is not located near the CovR locus. In order to better understand the environmental cues that are necessary for dental plaque formation, the focus of this research is to identify the cognate HK of CovR (out of 14 putative HK). Towards this end, 14 plasmids were constructed to inactivate all 14 putative HK genes by single-crossover integration. Since CovR regulates PgbpC and PgtfB, transcriptional reporter fusions were also generated in which a single copy of PgbpC-gusA or PgtfB-gusA was inserted in an ectopic chromosomal location. The goal of this project is to inactivate all 14 HK genes in the PgbpC-gusA or Pgtf- gusA reporter strains and study gusA expression from these promoters. We expect to find one or several HK genes will modulate these promoters and therefore may be the cognate HK for CovR. Once we identify the cognate HK, our future goal will be to study in vitro interaction of CovR and HK using purified proteins.