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.
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