Dental caries is recognized as the most prevalent infectious disease on earth and its treatment continues to have a major economic impact on developed countries. Streptococcus mutans is the principal etiologic agent of this disease and the unique ability of this organism to metabolize dietary sucrose is known to be crucial to virulence. Especially important in this regard is the production of extracellular glucans from sucrose by multiple glucosyltransferase enzymes. In the present application we propose to extend our studies utilizing molecular genetics to gain a more complete understanding of the biochemical genetic basis of glucan synthesis and other sucrose-related virulence traits. We shall complete our construction of S. mutans genomic libraries using lambda phage cloning and employ a variety of immunological and physiological screening techniques to identify the genetic determinants specifying glucan synthesis, dextranase and sucrase activity. Two such genes have already been identified, one of which seems involved in water-insoluble glucan synthesis. Using all of these cloned genes as probes in various types of hybridization strategies, we shall test the hypothesis that certain virulence related genes governing sucrose metabolism are linked in S. mutans. We shall construct defective copies of all of these genes in E. coli and introduce them back into S. mutans by transformation. Strains carrying single, double and multiple mutations constructed by this methodology will be studied to define the individual and collective roles of these determinants in sucrose-related glucan synthesis, adherence, cell aggregation, acid production and virulence in animals. Finally, we shall take advantage of our cloned glucosyltransferase genes as DNA probes to study the possibility that genetic expression of these loci is controlled. Specifically, because glucan synthesis appears to shut down spontaneously in cells cultured in vitro, it is our hypothesis that these genes are regulated by a control mechanism involving genomic rearrangement reminiscent of phase variation seen in other pathogenic bacteria. We shall examine the local genomic environment of the glucosyltransferase genes using appropriate DNA probes in order to test this hypothesis.
