Two-component signal transduction systems (TCSTS) are present across divergent bacterial species and function importantly in both environmental sensing and cell-cell communication. Each system is composed of a histidine kinase (HK) sensor protein coupled to a cognate response regulator (RR) element. Objectives: The present study was carried out to identify the complete complement of TCSTS in the oral pathogen Streptococcus mutans, and to determine the functional role of each component gene product in these systems. Methods: Published TCSTS seqeunce data from the related organism Streptococcus pneumoniae were compared with the S. mutans genome database available from the University of Oklahoma to identify putative TCSTS. A rapid PCR-based gene deletion strategy involving restriction-ligation and allelic replacement was employed for the systematic creation of knockout mutant strains, each with a different HK or RR component deleted. An automated, high throughput Bioscreen C Microbiology Analyzer was employed to assay the deletion mutants derived from wild-type NG8 or UA159 strains for growth in different stress conditions, including sodium chloride, sodium dodecyl sulphate, ethanol, hydrogen peroxide, acid, etc. Results: Thirteen HK/RR pairs, believed to represent the complete complement of TCSTS, were identified and located in the S. mutans genome. Specific primers were designed to individually delete 24 genes of 12 TCSTS by PCR ligation mutagenesis (one system consisted of comD and comE, genes that were previously deleted via insertion-duplication mutagenesis). Comparison of growth data of the 26 mutants with the wild-type strains indicated the physiological role of the TCSTS genes in cellular function. Conclusions: Using a combination of bioinformatics tools, molecular biology techniques and high-throughput screening, the insight into functional significance of all 13 TCSTS in S. mutans can be elucidated. Supported by PHS grant DE 013230 from the NIDCR.

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