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 beta-D- phosphogalactoside galactohydrolase gene of Lactobacillus casei was completed. It displayed high homology to the same gene isolated from Staphylococcus aureus and Streptococcus lactis. Homology was also found to the beta-phosphoglucoside glucohydrolase gene of Escherichia coli, but not to beta- galactosidases. Factor III of the lactose PEP:PTS was cloned from the L. casei plasmid, pLZ64, into E. coli and its nucleotide sequence determined by the Sanger dideoxy chain-termination method. The third gene of the gram-positive lac operon, encoding the Enzyme II lactose of the lactose PEP:PTS was cloned and its nucleotide sequence is being determined. The three genes were found to comprise an operon-like structure. High frequency transformation of whole, untreated, cells of Lactobacillus casei was obtained for the first time using high voltage electroporation. Broad host range vectors, capable of replication in gram-positive and gram-negative hosts, and suitable for gene cloning, were introduced into lactobacilli. Transfection and transformation of Lactobacillus bulgaricus was observed using these same vectors.
