Recombinant DNA technology and transposon mutagenesis will be used to examine, directly in oral streptococci, the pathways of sucrose metabolism by Streptococcus mutans. A thorough investigation of these pathways ins imperative because of the role of dietary sucrose in the development of dental caries. Problems inherent in the transformation of naturally competent strains of S. mutans and Streptococcus sanquis have prevented the direct cloning of many genetic determinants in these species. Genes associated with the metabolism of sucrose by S. mutans have been cloned in E. coli. However, the products of some of these genes are expressed differently by E. coli. Furthermore, the fermentation of sucrose to lactic acid, a key product in the induction of caries by S. mutans, is initiated by a sugar-specific phosphoenolpyruvate-dependent phospho-transferase system (suc-PTS). The genes for this system in Gram-negative bacteria are not complemented by their counterparts from Gram-positive bacteria. The projects to be pursued in this study will be aimed at developing the necessary tools for the analysis of sucrose metabolism directly in the oral streptococci, and will include the following. 1) A transformation system will be developed for S. mutans and S. sanquis that will permit the introduction of plasmid DNA by single-hit kinetics, rather than the two- hit kinetics associated with natural competence in these species. Such a system, which has been optimized for Streptococcus faecalis and functions with noncompetent strains of S. sanquis, will rely on electroporation, the utilization of the effects of high-voltage field pulses on the cell membrane to introduce DNA into a host cell. 2) Transposon delivery vehicles will be constructed from streptococcal plasmid derivatives which are unable to replicate at temperatures above 37 degrees C. These vehicles will contain a selectable antibiotic resistance determinant to permit assays for the loss of plasmid DNA, but retention, by chromosomal insertion of resistance to either tetracycline (Tn916) or erythromycin (Tn917) encoded by the transposon. 3) The transposon delivery vehicles will be used to obtain mutants of oral streptococci defective in homologous recombination (rec-), to prevent the chromosomal integration of cloned sequences, and in the isolation of strains with single mutations in genes associated with the metabolism of sucrose. 4) The entire system will be tested by attempts to clone, with vectors already constructed, and characterize, components of the suc-PTS system from chromosomal DNA isolated from sucrose-positive strains of S. mutans.
