Genetic studies on Porphyromonas gingivalis W83.
thesisposted on 19.11.2015, 09:10 by Janette. Maley
Until recently molecular biological approaches to the study of putative virulence factors of the oral pathogen Porphyromonas gingivalis were limited. This was due to the absence of a genetic system which could facilitate the production of isogenic mutant derivatives. Among the many components produced, the capsule is believed to be of particular importance in the pathogenicity of P. gingivalis. In order to identify mutants lacking capsular polysaccharide, purified capsular material from P. gingivalis W83 was used to generate capsule-specific antisera. However, these antisera contained antibodies which recognised lipopolysaccharide. The use of polymyxin B-agarose was found to be useful in the removal of LPS-reactive material. Capsular polysaccharide was purified further and shown to be free of detectable LPS. A system for conjugal transfer of plasmid DNA from Escherichia coli to P. gingivalis was developed. This method was then used for the introduction of the suicide-vector R751::Tn4351?4 into P. gingivalis. Examination of a number of P. gingivalis Tn4351 mutants demonstrated the presence of multiple copies of Tn4351 in the chromosome of the mutants. Recovery of plasmid pNJR12 from P. gingivalis transconjugants led to the isolation of IS 1126. This insertion sequence was present in the chromosome of all strains of P. gingivalis examined but absent from other members of the genus. The amino acid sequence of the major open reading frame (ORF1) was derived from the nucleotide sequence of IS 1126. ORF1 contained the conserved motifs displayed by the transposase proteins of IS-elements belonging to the IS4 family.