The degradation of fructans, which are synthesized by a variety of oral bacteria, is believed to contribute to the initiation and progression of dental caries. Fructans accumulate rapidly in plaque following a dietary sucrose intake, functioning as a temporary store of carbohydrate outside the cell. These polysaccharides can then be hydrolyzed by the fructanase of S. mutans. This process is believed to extend both the depth and duration of the acid challenge to the tooth surface. This proposal seeks to understand the molecular aspects of fructan metabolism by the oral streptococci and to elucidate the mechanisms underlying the complex regulation of the fruA (fructanase) gene of S. mutans. To accomplish this, the DNA sequence of the fruA gene, which has been cloned and characterized previously by the P.I., and DNA flanking fruA will be determined. Insertional inactivation of the gene will be done to construct fruA-defective S. mutans. These strains will be evaluated for their ability to initiate caries in the rat model, and for a variety of properties related to pathogenesis and fructan biology, in vitro. Recent evidence demonstrates the linkage of fruA to other important carbohydrate metabolism genes. Using the polymerase chain reaction, chromosome walking, DNA sequencing and, targeted mutagenesis, the genes adjacent to fruA will be isolated and characterized. Then, with a unique combination of chemostat culture and molecular biology, a study of the inducibility, glucose repressibility, and growth-rate dependence of fruA expression will be undertaken. These studies will define the level at which expression of fruA is controlled in response to each of these variables through the use of MRNA analysis coupled with biochemical and immunologic quantification of fruA protein. Dot hybridization will be used to quantitate transcription levels, and Northern Blotting and primer extension will be used to characterize MRNA size and transcriptional initiation sites. Culture supernates will be assayed for fructanase activity biochemically, and for fruA protein by ELISA with specific antiserum. Gene fusions will then be used to identify cis- acting factors controlling catabolite repression of fructanase synthesis. These studies will be extended to examine regulation and regulatory mechanisms involved in differential gene expression of bacteria adhering to solid surfaces in the chemostat, as well as the responses to intermittent carbohydrate pulses of bacteria growing at steady state. Through collaborative efforts, these studies will be extended to examine environmental effects on the expression of other virulence genes, specifically the glucosyltransferases.
