Adherence, carbohydrate polymer production, acidogenesis, and acidurance are virulence properties associated with the cariogenic bacterium Streptococcus mutans. Each of these properties contributes to some degree to the virulence of this organism. Little if anything is known about how this organism regulates the transcription and translation of its genes. This proposal seeks to elucidate the molecular control mechanisms of gene regulation in Streptococcus mutans Gs-5. In order to achieve this goal, fusion vectors will be generated that will facilitate the rapid identification, cloning and sequencing of regulatable promoters. Alternate approaches will be taken to the construction of these gene fusion vectors. First, the Streptococcus transposon Tn917 and variants of this element containing either a promoterless beta-galactosidase gene or a promoterless amylase gene will be used to search for regulatable promoters. A second approach will involve the use of an integration vector carrying a promoterless amylase gene. In both cases, once regulatable promoters have been identified, they can be cloned and sequenced. In addition, a recombination deficient strain of GS-5 will be constructed in order to aid in the analysis of the cloned promoters. Finally, mutants of GS-5 will be generated that are altered in their ability to regulate the cloned promoters. The techniques to be used involve genetic engineering, chemical and transposon mutagenesis, gene fusions, protein identification and genetic analysis.
