This project seeks to use genetic, biochemical and physiological approaches to investigate the pathogenicity of oral bacteria. The two specific areas of investigation are: 1) characterization of plasmid-coded and chromosomal metabolic genes from oral bacteria and 2) development of systems for genetic exchange in oral bacteria. The nucleotide sequence of the EnzymeIIlac gene of Lactobacillus casei was completed. It displayed high homology to the same genes isolated from staphylococcus aureus and Streptococcus lactis. Little homology was observed in comparison with other EnzymeII found in Escherichia coli, Salmonella typhimurium or Bacillus subtilis. The genes encoding the EnzymeIIlac, P-beta-gal and Factor IIlac were found to comprise an operon-like structure. Site-directed mutagenesis has been used to prepare mutant EnzymeII proteins with altered catalytic activity. The sequence of the plasmid-coded beta-galactosidase of Lactobacillus casei has been completed: the adjacent lac permease gene has been cloned and its sequence is being determined. L. casei has been utilized for the cloning and expression of heterologous genes such as alpha-amylase, subtilisin and beta-gal. Expression and secretion of foreign proteins has been observed; improved vectors and promoters have been identified. Using electroporation, L. bulgaricus and L. acidophilus have been transformed. The feasibility of use of these food related bacteria that colonize the gastrointestinal system as vectors for gene delivery to humans is being examined.
