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The black-pigmented Gram-negative anaerobe Porphyromonas gingivalis has been implicated in human periodontal diseases and expresses a number of exoglycosidase activities that may be capable of degrading the oligosaccharides from host glycoconjugates. The first stage in characterising the role of the beta-N-Acetyl-hexosaminidase activity from this microorganism was the cloning of the nahA gene from P. gingivalis W83 which encodes for a protein with this enzyme activity. The nahA gene was cloned in E. coli by constructing a plasmid expression library of Sau3A-generated P. gingivalis W83 chromosomal DNA fragments. Expression of beta-N-Acetyl-hexosaminidase activity was detected by cleavage of the fluorogenic substrates 4-methylumbelliferyl-N-Acetyl-beta-D-glucosaminide and 4-methylumbelliferyl-N-Acetyl-beta-D-galactosaminide. Southern blot analysis suggested that the nahA gene was present as a single copy in all P. gingivalis strains tested. In contrast, sequences homologous to the nahA gene were not detectable in the closely related species P. endodontalis and P. asaccharolytica. The nahA gene was 2331 base pairs long and encoded a protein of 777 amino acids with a predicted molecular weight of 87 kDa. A characteristic signal peptide for an acylated lipoprotein was present at the amino terminus suggesting that the mature beta-N-Acetyl-hexosaminidase may be a lipoprotein attached to the outer membrane of P. gingivalis. Protein homology studies suggested that NahA and beta-N-Acetyl-hexosaminidases from eukaryotes and prokaryotes contain homologous active site domains with similar catalytic arginine residues. DNA sequence analysis 5' to the nahA gene identified another open reading frame, and a potential hairpin structure which may be involved in regulating gene expression. Lastly, a suicide plasmid that may allow the site specific inactivation of the nahA gene on the chromosome of P. gingivalis was constructed. The results presented in this thesis may contribute to future studies including the generation of an isogenic mutant of P. gingivalis lacking beta-N-Acetyl-hexosaminidase activity. |
