Bacterial colonization and invasion depend upon a microbe's ability to attach to a host and multiply. Oral bacteria, like other human commensals and pathogens, have evolved specific surface proteins for this purpose. Some of these molecules are associated with scaffolding structures, e.g., pili or fimbriae, and others are integral parts of the organism's outer membrane. Several adhesins on the surface of Prevotella loescheii have been the focus of studies in this laboratory. The gene for a 75 kDa lectin-like protein was cloned and its structure deduced from the nucleotide sequence. Translation of the adhesin mRNA occurs via a frameshifting hop in which 27 nucleotides are bypassed. Since bypasses are rarely seen among prokaryotes, it was essential to establish that the amino acid sequences prior to and immediately following the hop were the expected residues. Recent attempts to sequence this region yielded the anticipated amino acid sequence proving that a 27 nucleotide bypass occurs during translation. Elements of the """"""""hop"""""""", e.g., the shift region, stem loop and pseudoknot are being assessed for their effect on translation of the adhesin and heterologous proteins. Beta-galactosidase constructs with the 228 nucleotide hop positioned in frame at the 5' end of the gene indicated that synthesis of the enzyme in Escherichia coli was being retarded compared to wild type production. Current studies using site-specific mutagenesis have demonstrated that regions within the stem loop and pseudoknot are absolutely essential for read-through of the adhesin mRNA. Attempts to identify and clone the gene encoding a P. loescheii non-lectin adhesin using degenerated probes based on the first 10 amino acids of the N-terminal sequence are continuing. The gene of a P. loescheii surface component believed to be a support protein was cloned and sequenced. Comparisons indicated that the deduced sequence showed extensive identity to both eukaryotic and prokaryotic enolases. The partially purified protein from which the N-terminal amino acid sequence was obtained shows enolase activity. The role of this enzyme is being investigated.
