The proposed research addresses 1) the unique mechanisms used by the oral bacterium Prevotella loescheii 1295 to synthesize the SO34 adhesin protein, and 2) the mechanisms of adhesin action. The SO34 adhesin, which is encoded by the plaA gene, is a lectin- like protein that recognizes galactoside-containing receptors on Streptococcus oralis 34 cells. PlaA expression requires bypassing a 29-nucleotide (nt) gap in its coding sequence on the PlaA messenger RNA (mRN). The proposed mechanism of PlaA expression is a programmed frameshifting hop. Features of the gap region consistent with this mechanism include: 1) the presence of two (UAA) termination condons flanking the bypass region, 2) two runs of four or more identical bases (slippery sequences), 3) the ability the plaA mRNA to form a stem-loop at the beginning of the large ORF, and 4) the potential of bases in the loop to form a pseudoknot.
Specific Aims of the proposed research are to: A0 Use site-directed and random mutagenesis to test whether the above features of the plaA mRNA structure are essential for efficient gap bypass. An assay that uses a beta-galactosidase reporter gence will be used to measure coding gap bypass efficiency. B) Express the SO34 adhesin and map its active site. C) Transfer and express the plaA adhesin in Prevotella and/or Bacteroides for studies of gene regulation and adhesin function. These experiments will significantly expand our understanding of genetic mechanisms in oral bacteria associated with dental plaque. Specifically, the proposed experiments will elucidate the unusual translation mechanisms used to by-pass the plaA coding gap. Expression of the plaA gene in related bacteria will facilitate testing the effect of the coding gap on plaA expression. The proposed studies will provide a solid foundation for understanding mechanisms of adhesin binding and the subsequent engineering of peptides that may prevent adhesin action. Understanding adhesin action and how to prevent adhesin binding to receptors will lay a foundation for the future development of agent that prevent plaque accumulation.
|Manch-Citron, J N; Shahani, P J; Schneider, R (2001) Cloning, characterization, and possible origin of the Prevotella loescheii dnaK homolog. Curr Microbiol 42:82-8|
|Manch-Citron, J N; Dey, A; Schneider, R et al. (1999) The translational hop junction and the 5' transcriptional start site for the Prevotella loescheii adhesin encoded by plaA. Curr Microbiol 38:22-6|