GENE REGULATION IN S MUTANS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。变形链球菌被认为是人类龋齿的主要病原，是一个全球性的口腔健康问题。变形链球菌也是感染性心内膜炎的主要原因。变形链球菌产生广泛的毒力因子，这是生物膜形成所需要的，通常被称为牙菌斑。这些毒力因子由几个双组分系统（TCS）调节。典型的TCS由膜相关的传感器激酶蛋白组成，该蛋白对特定的环境信号作出反应，并在保守的组氨酸残基上自动磷酸化。磷酸化的残基随后被转移到细胞质反应调节剂（RR）进行激活。这种激活的RR调节某些靶基因的表达，例如那些生物膜形成所需的基因。在变形链球菌中，已经确定了13种假定的TCS。其中，CovR是一种重要的RR，在A群链球菌中抑制15%的基因，在B群链球菌中抑制6%的基因。突变链球菌的covR失活导致生物膜形成改变，相应的突变体是低龋型的。利用DNA微阵列技术，我们的实验室已经发现约有100个基因受变异链球菌CovR的调控。例如，CovR抑制编码葡聚糖结合蛋白的gbpC基因和编码葡萄糖基转移酶的gtfB基因。与其他链球菌不同，变形链球菌CovR的同源组氨酸激酶（HK）不在CovR位点附近。为了更好地了解牙菌斑形成所必需的环境线索，本研究的重点是确定CovR的同源HK（从14个假定的HK中）。为此，构建了14个质粒，通过单次交叉整合使所有14个推测的HK基因失活。由于CovR调节PgbpC和PgtfB，因此也产生了转录报告基因融合，其中将PgbpC- gusa或PgtfB- gusa的单个拷贝插入到异位染色体位置。本项目的目的是灭活PgbpC-gusA或Pgtf- gusA报告菌株中的全部14个HK基因，并研究gusA在这些启动子中的表达。我们期望找到一个或几个HK基因可以调节这些启动子，因此可能是CovR的同源HK。一旦我们确定同源HK，我们未来的目标将是使用纯化蛋白研究CovR和HK的体外相互作用。

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.