The formation of dental plaque is initiated with the deposition of a limited number of gram-positive bacteria including Streptococcus spp. Colonization by these organisms provides a mechanism for the adherence of other plaque bacteria and thus contributes significantly to bacterial succession during the later stages of plaque formation. The expression of cell surface polysaccharides and/or proteins by these primary colonizers is correlated with their ability to attach to host tissues and interact with other plaque bacteria. The cell wall polysaccharides of several members of the oral streptococci including S. gordonii, S. mitis and S. oral is have been determined. Results show that the polysaccharides of these bacteria are complex polymers consisting of hexasaccharide or heptasaccharide repeating units linked by phosphodiester bonds and that each unit contains distinct regions for receptor binding activities and antigenic specificities. Whereas the structural characteristics of these polysaccharide receptors are well defined, the genes responsible for the expression of these molecules are unknown. A molecular study of the S. mitis polysaccharide receptor has been initiated and several polysaccharide-deficient mutants have been obtained by transposon insertional mutagenesis. The objective of the present proposal is to map and characterize the region(s) of the S. mitis chromosome involved in the synthesis of the polysaccharide.
The specific aims are: (l) to isolate S. mitis specific DNA fragments flanking the inserted transposon from polysaccharide-deficient mutants generated by transposon mutagenesis, (2) to use the flanking DNA sequences as DNA probes to map a region of the S. mitis chromosome involved in polysaccharide synthesis, assembly and export, and (3) to begin to characterize DNA fragments which contain the putative genes associated with polysaccharide synthesis. The long term goal of this study will be to delineate the functions of the various genes and the regulatory mechanisms which control expression of the polysaccharide receptor. The results will provide insights into the contributions of the polysaccharide in modulating bacterial ecology of the microenvironment. Since multiple genes located at various loci of the chromosome will be involved in the regulation and expression of the complex polysaccharide, the objectives of this proposal, while highly feasible, incur a fair amount of risk. Thus, this proposal is considered appropriate for an R03 application. The results generated from analyses of the existing mutants will provide preliminary data essential for an R0l application.
