| dc.description | Streptococcus mutans adheres to enamel and may subsequently cause caries. A previously characterized surface protein releasing enzyme (SPRE), capable of detaching cells from model biofilms may be a means to detach cells from enamel prior to the development of caries. This work was undertaken to characterize the SPRE activity, and identify genes) responsible for, or affecting the activity or production of SPRE. Characterization of SPRE required development of a model system to assay detachment. In detachment experiments biofilms were formed on epon-hydroxyapatite (EHA) rods, which mimicked enamel. To mirror conditions in the oral cavity and to provide a specific mechanism for adherence EHA rods were conditioned with saliva or salivary agglutinin (SA). Stable biofilms were formed after 1 hour. Detachment was determined to be temperature dependent, and had a pH optimum of 5.5--6.0. In comparison to the controls, crude SPRE preparations were able to induce detachment of significantly greater percentages of cells from model biofilm. The susceptibility of SPRE to inactivation by pronase, suggested detachment was an active enzyme mediated process. A pool of transposon mutants was produced and screened. A putative SPRE defective mutant was isolated. In contrast to the wild type, SPRE prepared from the mutant lacked the ability to detach cells from biofilms. Genetic characterization revealed a single transposon insertion adjacent to the -35 region of an operon encoding a P-type ATPase similar to CopA and CopB of Enterocoecus hirae. The ATPase conferred high level (8 mM) copper resistance to S. mutans. The transposon insertion was postulated to allow increased expression of the operon by decreasing the affinity of a negative regulator, Copy, for its binding site overlapping the promoter. Increased expression of the ATPase in the mutant was believed to lower intracellular copper concentrations which may negatively effect SPRE. | en_US |
