PREVOTELLA LOESCHEII ADHESIN GENE EXPRESSION

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The overall objective of the proposed research is to elucidate the mechanism(s) of gene expression used to synthesize the plaA adhesin gene product (SO34 protein) by the oral bacterium, Prevotella loescheii. The fimbrial-associated SO34 protein recognizes galactoside-containing receptors on Streptococcus oralis 34. The plaA gene contains short and long open reading frames (ORFs) consisting of 150 and 2310 base pairs (bp) respectively. Expression of the adhesin gene requires bypassing a 29 nucleotide (nt) gap in its coding sequence on the plaA messenger RNA (mRNA). The proposed mechanism of plaA expression is programmed frameshifting and ribosomal bypass. Features of the gap region consistent with this mechanism include: 1) the flanking of the bypass region with two identical (UAA) termination codons; 2) two runs of four or more identical bases (slippery sequences), 3) the ability to form a stem loop at the beginning of the large ORF, and 4) the ability of bases in the loop to form a pseudoknot. The proposed experiments will test whether the above features of the plaA mRNA structure are essential for efficient gap bypass. Site-directed mutagenesis will be used to after specific plaA nucleotides which may play a role in gap bypass. An assay which uses a beta-galactosidase reporter gene will be used to measure ribosomal gap bypass efficiency. In addition, these studies will lay the foundation for future functional studies of adhesin action. Engineering efficient and stable expression of the SO34 adhesin protein will facilitate studies of adhesion binding and possibly, the design of peptides which may be engineered to prevent adhesin-receptor binding.