Development of cloning vectors and estabiishment of transformation system for the periodontitis- pathogenic anaerobes

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Genetic transformation, by electroporation, of Porphyromonas gingivalis was accomplished with DNA of plasmid pE5-2, or its derivative pYT7. Prior to transformation, pE5-2 was transferred from E. coli to P. gingivalis strains by conjugation (mobilization with R751), and plasmid DNA was purified from the transconjugants thus obtained. Transformation was possible when the recipient strain and the donor from which the plasmid DNA was purified were homologous. If they were heterologous each other, transformation did not occur at all, or only occured at a very low frequency. This evidence suggests presence of the restriction-modification system in most of P. gingivalis strains. pYT7, a derivative of pE5-2, was obtained by self-ligation of the 8.0 kb fragment of AvaI-digested pE5-2 from P. gingivalis, which has several single-cutting restriction sites such as EcoRI, AvaI, ClaI and BstEII. However, it was not stable enough, though is much more stable than its parent, pE5-2, in P. gingivalis cells probably because its rep gene was originated in a relatively distant species, Bacteroides eggerthii.