The competence regulon in Streptococcus mutans biofilms

描述：变形链球菌存在于牙菌斑的生物膜中，它从饮食中的碳水化合物产生酸，引起龋齿。S.变形杆菌在生物膜中使用群体感应信号系统来激活遗传能力、耐酸性并影响生物膜结构。本申请解决了细胞-细胞和环境信号激活这种生物膜表型的机制。S.变形杆菌群体感应系统由comCDE基因编码，comCDE基因编码感受态刺激肽（CSP）前体、组氨酸激酶和反应调节剂。我们期待回答：1）CSP激活的哪些基因参与了生物膜的形成？2)这条途径是如何调节的？3)我们能否通过抑制该途径来减弱链球菌的生物膜表型？变种人？4)其他环境信号激活生物膜表型吗？将使用差异显示（ddPCR）和2D凝胶电泳来鉴定由CSP激活的基因和蛋白质。一个同基因comC（CSP缺陷）突变体将被用来破译这个调节子。将比较有和没有外源合成CSP的ddPCR和2D表达谱。将使用新的等位基因交换技术克隆和突变表达改变的编码产物的基因;将测试突变体的遗传能力。将通过SEM和共聚焦扫描激光显微镜（CSLM）评估突变生物膜的细胞分离和3D结构。在这些基因中构建的报告基因融合体（gfp、lacZ和luc）将用于使用荧光和发光分析以及CSLM来测量响应于CSP的时间上的基因表达，以深入了解CSP信号传导级联如何起作用以真实的时间影响生物膜表型。将测试CSP类似物对信号转导过程的抑制。S.变形菌还具有两个成分信号传导系统（TCSTS）来感知环境信号。生物信息学分析在S.突变体和它们的单个基因有缺陷的突变体。使用机器人生长监测器，将检查突变体在存在环境应力（酸、盐、过氧化物、SDS和高糖浓度）的情况下生长的能力，以阐明TCSTS的功能。这些实验将有助于深入了解S.变形菌和潜在的其他生物膜形成细菌。

DESCRIPTION: Streptococcus mutans resides in the biofilm of Dental plaque where it produces acid from dietary carbohydrate to cause caries. S. mutans uses a quorum-sensing signaling system in biofilms to activate genetic competence, acid tolerance and influence biofilm architecture. This application addresses mechanisms by which cell-cell and environmental signals activate this biofilm phenotype. S. mutans quorum-sensing system is encoded by the comCDE genes that encode a competence-stimulating peptide (CSP) precursor, a histidine kinase and a response regulator. We expect to answer: 1) Which of the genes activated by CSP are involved in biofilm formation? 2) How is this pathway regulated? 3) Can we inhibit this pathway to attenuate the biofilm phenotype of S. mutans? 4) Do other environmental signals activate the biofilm phenotype? Differential display (ddPCR) and 2D gel electrophoresis will be used to identify genes and proteins that are activated by the CSP. An isogenic comC (CSP-deficient) mutant will be used to decipher this regulon. Its ddPCR and 2D expression profiles with and without exogenous synthetic CSP will be compared. Genes encoding products with altered expression will be cloned and mutated using a novel allelic exchange technique; the mutants genetic competence will be tested. Cell segregation and the 3D architecture of the mutant biofilms will be assessed by SEM and Confocal Scanning Laser Microscopy (CSLM). Reporter gene fusions (gfp, lacZ and luc) constructed in these genes will be used to measure gene expression temporally in response to CSP using fluorometric and luminetric analysis and CSLM to give insight into how the CSP signaling cascade functions to influence biofilm phenotype in real time. CSP analogs will be tested for their inhibition of the signal transduction process. S. mutans also has two component signal transduction systems (TCSTS) to sense environmental signals. Bioinformatic analyses have identified 13 TCSTS in S. mutans, and mutants defective in their individual genes were constructed. Using a robotic growth monitor, the mutants will be examined for the ability to grow in the presence of environmental stresses: acid, salts, peroxide, SDS, and high sugar concentration to elucidate the function of the TCSTS. These experiments will give insight into the signaling mechanisms used by S. mutans and potentially other biofilm-forming bacteria.