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 genes isolated from oral bacteria; and 2) development of systems for genetic exchange in oral bacteria. The amino acid residue that participates in transphosphorylation at the active center EnzymeII(lac) of Lactobacillus casei was determined. Replacement of each of the histidine and cysteine residues in EII with other amino acid by site-directed mutagenesis, followed by an analysis of their ability to phosphorylate lactose using an in vitro assay system, revealed that only cysteine-483 is required for enzymatic activity. Using the HOST-VECTOR system previously developed for the genetic manipulation of lactobacilli, the expression of a number of heterologous genes has been examined. Of particular interest, the genes encoding the EnzymeII(lac), p-beta-gal and Factor III(lac) of L. casei were transcriptionally fused to the strong constitutive beta- galactosidase promoted isolated from L. bulgaricus to create an operon- like structure which was inserted into the shuttle vector pBS19 forming pBS914. This construction was transformed into lactose negative L. casei strains; transformants were selected by their ability to grow on lactose. Efforts are now directed at construction of food grade vectors with lactobacilli used in food fermentations, or isolated from the gastrointestinal tract, for gene delivery and antigen presentation to humans.
