The metabolism of urea to produce ammonia by urease-producing bacteria is believed to play multiple roles in microbial ecology and pH homeostasis In dental plaque. Ureolysis may be a principal determinant impeding the initiation and progression of dental caries directly through neutralization of acids produced by glycolytic processes, as well as through fostering a plaque environment which is less conducive to highly acidogenic and acid-tolerant species. This proposal focuses on dissecting molecular genetic and biochemical aspects of ureases from bacteria isolated from the human oral cavity. A Staphylococcus species has been recovered from the plaque of a chronic renal failure patient demonstrating extremely high salivary urea levels. A preliminary characterization of this strain and a partial genetic characterization of the urease genes of this species has been accomplished. A genetic library of this organism has been constructed. The genes which encode for the urease enzyme will be isolated and characterized. Concomitant with this study, the cloning and characterization of the urease genes from Streptococcus salivarius 57.1, which has a stable urease phenotype, will be undertaken. The urease genes will be mapped using subcloning, transposon mutagenesis, and in vitro transcription/translation systems to define essential proteins for urease activity and regulation. Biochemical analysis, including Km, Vmax, subunit composition, and inhibitor profiles, will be derived for the Staph. and S. salivarius enzymes. Then, using recently developed vector systems for integration of foreign genes into the chromosome of S. mutans, the urease genes will be stably established in this organism. The S. mutans strains with the urease phenotype will be subjected to a discrete number in vitro tests to establish the stability and characterize the expression of the ure genes, and the ability of the bacterium to modulate environmental pH through ammonia production. The recombinant S. mutans expressing the urease genes will then be tested in two sets of experiments using the rat-caries model to assess the role of urea and urease in plaque ecology, and the function of urea and ureolytic species in the caries process. The proposal seeks to apply recombinant DNA technology and basic biochemical physiological techniques to better define the role of urease and ureolytic organisms in dental plaque and caries inhibition. Ultimately, the goal is to provide new Insight into the caries process with the intention of developing novel strategies for caries control, particularly in caries-prone and aging populations, and to define molecular characteristics of ureases of oral bacteria for future investigation of the role of this enzyme in calculus formation and periodontal diseases.
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