Density-dependent surface growth may switch sessile phenotypes to planktonic. During years 08-13, sessile and planktonic S. sanguis and S. gordonii cells will be compared to test the hypothesis that expression of adhesin proteins is regulated by the process of adhesion. Specifically, we will (1) compare the adhesion specificities of PAAP+ S. sanguis and PAAP- S. gordonii in a biofilm model that facilitates adhesion, protein biosynthesis and growth at 37 C for 6 hours in chemically-defined synthetic medium. In this model, the two wild-type strains and selected mutants will tagged with different antibiotic resistance markers. The strains will be incubated alone or together to determine if adhesion is competitive when binding to saliva-coated hydroxyapatite in the presence or absence of specific antibodies against salivary antigens, or simulated pellicles formed from purified salivary macromolecules. Since preferred binding sites show discrete distributions on coated enamel chips, the two strains will be compared microscopically for topological distribution of binding. To learn if they express different adhesin phenotypes, (2) sessile and planktonic cells will be recovered periodically from the biofilm model and altered expression of streptococcal surface macromolecules will be analyzed by pulse-labeling, autoradiography and Western immunoblotting with antibodies against known proteins. Novel surface proteins regulated during adhesion will be analyzed by amino acid microsequencing, PCR synthesis of the target gene, insertional inactivation, analysis of the mutant for defects in adhesion and finally cloning and sequencing of the complete gene. Selected experiments will be simulated on enamel chips to determine the ultrastructural morphology of resultant biofilms. The specificity of adhesin proteins requires protection against oxidative stress. Preliminary data show partial cloning of the S. gordonii msrA gene, which encodes methionine sulfoxide reductase, a purported adhesin maintenance factor. To analyze post-translational modification and functional maintenance of adhesin proteins, (3) an msrA-negative mutant will be constructed and the methionine-rich diversity region of the adhesin, antigen I/II (SspA/SspB) will be analyzed for altered structure and function. These studies will serve as a definitive test of the hypothesis that the process of adhesion regulates expression of required proteins in a biofilm model.
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