Streptococcus mutans is recognized as a prime etiologic agent in human dental caries (tooth decay). The long range objective of our research is to understand, at the molecular level, the genetic and biochemical base of S. mutans' oral colonization ability and virulence. As a primary apprach to this problem, we shall dissect the genome of S. mutans using a recobinant DNA system that employs transformable Streptococcus sanguis as a host. In this application, we propose to refine our newly-developed oral streptococcal recombinant DNA system by constructing more useful plasmid vectors and by isolating mutants of the S. sanguis host that will permit better utilization of our system. These include recombination deficient mutants as well as mutants that will permit the direct selection of genes specifying sucrase activity. Using standard recombinant DNA methods, we shall clone S. mutans genes involved in sucrose-glucan metabolism including glucosyl (GT) and fructosyl transferases, dextranase (dex), invertase, phosphosucrase and glucan binding protein. Strains of S. sanguis and S. mutans bearing chimeric plasmids will be used to study the role of the GT, dex and glucan binding determinants in sucrose-glucan-mediated adherence in vitro. Specific information relative to the total number and kinds of GT genes will be obtained including the physiologic interaction of their products with each other as well as with glucan binding protein and dextranase. The plasmid chimeras constructed in this work also will be used to determine the organization of genes specifying sucrase activity on the S. mutans genome. Strains of S. mutans and S. sanguis constructed in this work will be assayed for cariogenicity in germ-free rats in order to test the relative importance of the above genes in determining virulence.
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