Construction of a plasmid vector for transformation of Porphyromonas gingivalis

A host-vector system for transformation of Porphyromonas gingivalis was constructed using a set (1) strains that can incorporate plasmid DNA by electroporation regardless of its source and (2) stable vector plasmids with a selectable marker. First, restriction-negative mutants were isolated, because P. gingivalis possesses restriction modification systems by which DNA introduced by transformation even from heterologous strains of the same species is excluded. For screening of the mutants, plasmid pE5-2 was employed since it could be transconjugated (mobilized) to P. gingivalis from Escherichia coli and is able to replicate in this species, albeit not stably. pE5-2 DNA prepared from E coli was introduced by electroporation into chemically mutagenized P. gingivalis cells. By this method, three putative restriction-negative clones were selected. These strains exhibited a capacity for electroporation with plasmid DNAs both from E. coli and from various P. gingivalis strains at a similar efficiency. Using one of the derivatives thus obtained, YH522, we then screened for plasmids that could replicate stably in P. gingivalis. Since no plasmids were found from P. gingivalis, cryptic plasmids from other species of black-pigmented oral anaerobic rods were examined for their ability to transform P. gingivalis. A series of plasmids constructed by ligation with pBR322 for replication in E. coli and the EcoRI-B fragment from pBF4 containing erythromycin resistance were prepared from E. coli and were used for electroporation of P. gingivalis. Among these, a recombinant plasmid containing the replicon of pYHBA1 from Porphyromonas asaccharolytica, designated pYH400, was found to be incorporated into the restriction-negative P. gingivalis strain and replicated stably. This set of recipient strains and stable plasmids with a selectable marker constitutes the first practical host-vector system for this species.