This study seeks to define, at the genetic and structural level, the Prevotella loescheii fimbrial-associated adhesins and the fimbriae bearing them. After cloning and sequencing the galactoside-specific fimbrial-associated adhesin gene, the interrupted translation region within the mature protein beginning at the codon for amino acid 29 (actually an ochre terminator) was characterized. Existence of a stretch of 138 nucleotides containing the two ochre terminators, a polyA slip region, stem loop and pseudoknot was demonstrated in genomic DNA and adhesin mRNA establishing that ribosomal translation requires a bypass or """"""""hop"""""""" of at least 29 nucleotides located between two ochre codons. The entire region was replicated by PCR and fused in frame into the 5' terminus of lacZ to demonstrate that E. coli is capable of reading through the bypass region efficiently; expression of the gene occurred at levels varying between 4 and 25% of the control. A DNA probe based on the sequence of an internal 10 amino acid peptide isolated from a digest of pure P. loescheii actinomyces-specific adhesin reacted with restricted genomic DNA from this oral bacterium. The probe was subsequently used to screen a GT-11 lambda phage genomic P. loescheii library. Currently, DNA containing the phage-positive insert is being isolated for cloning. Similarly, the N-terminal sequence of P. loescheii fimbriae was determined from a purified preparation of the structures. A radioactive oligonucleotide probe was prepared following reverse translation and was found to react with digested genomic DNA. The above mentioned P. loescheii lambda GT-11 library is currently being screened with the oligonucleotide. The ribitol-5-P dehydrogenase gene found in the ribitol operon has been cloned and is roughly 50% sequenced. The ribitol PTS transport is currently being sought in the flanking regions of the dehydrogenase gene.
