Development of molecular genetics in oral bacteroides

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(1)Construction of a suicide plasmid (pKDCMZ) for Porphyromonas (Bacteroides) gingivalis.A suicide plasmid was constructed from a shuttle plasmid vector between E.coli and Bacteroides (pVAL-1). By use of pKDCMZ plasmid, a superoxide dismutase (SOD) -deficient mutant strain was constructed via gene replacement by a mutated sod. The SOD mutant strain showed a rapid viability loss on exposure to air, indicating that SOD is essential for aerotolerance of this organism.(2)Isolation of colorless mutant strains of P.gingivalis by use of Tn4351 transposon mutagenesis.Colorless mutant strains of P.gingivalis were isolated by trasconjugation between E.coli harboring R751 : : Tn4351OMEGA4 and P.gingivalis. A gene disrupted by Tn4351 was cloned from one of the colorless mutants and partially sequenced. The amino acid sequence of putative product of the gene (porR) has relatively high similarity with the amino acid sequences of proteins coded by Bacillus stearothermophilus degt, Saccharopolyspora erythraea eryC1, and Streptomyces peucetius dnrJ.These proteins are located at cytoplasmic membrane, functioning as a sensor in signal transduction.