Two species of the mutants streptococci (MS), Streptococcus mutants and Streptococcus sobrinus, are the major etiologic agents of human dental caries. Among the properties of these microorganisms that contribute to their virulence (cariogenicity) are the ability 1) to adhere to enamel surfaces; 2) to produce copious amounts of organic acids, particularly lactic acid, from dietary carbohydrates; 3) to compete effectively with other members of the oral microbial flora, particularly during periods of carbohydrate limitation; and 4) to exhibit high tolerance for acid environments. The first three traits have been linked to the expression by the MS of multiple mechanisms for the metabolism of sucrose, a major dietary carbohydrate worldwide. The broad long-range goals of this project are to develop and utilize genetic and molecular tools to study the specifically, S. mutants NCTC10449 and S. sobrinus 6715. Several laboratories have reported the cloning and sequencing of a variety of sucrose metabolic genes, and molecular mechanisms for their manipulation have been devised. The tools for microbial physiology likewise have been applied to studies on the activates and regulation of sucrose metabolic gene products. There have been only a few published reports in which the tools of genetics molecular biology, and microbial physiology were applied to analyses of the sucrose metabolic capacity of the MS. The immediate goal of the proposed study is to study is to combined all of these tools in an assessment and comparison of the roles of the scrA and scrB gene products in the metabolism of sucrose by S. mutans and S. sobrinus by pursuit of the following specific aims. 1) Construct and test a mobilizable E. coli/Streptococcus shuttle vector suitable for mutagenesis by allelic replacement of sucrose catabolic genes of S. sobrinus. Recent results suggest the feasibility of this approach for this non-transformable MS species. 2) Utilize a Lactococcus lactis model system to assess the requirements for expression of scrA, encoding EII, from each MS strain. 3) Insertionally inactivate by allelic replacement, the scrA gene of each MS strain, and assess the effects in continuous culture on sucrose transport, types of transport, acid production, and metabolic endproducts. 4) Construct by allelic replacement, double mutants (scrA and scrB) of each strain, and assess effects as in aim 3, as well as the role of scrB in the hydrolysis of sucrose-phosphate and sucrose. 5) Examine transcriptional/transnational regulation of scrA via reporter genes, and assays for EII activity.
