GENE CLONING IN CLOSTRIDIUM-DIFFICILE USING TN916 AS A SHUTTLE CONJUGATIVE TRANSPOSON

A pBR322-based vector, pCI195, containing a 4.2-kb region of the conju gative transposon Tn919 was used as a vector for gene cloning in Clost ridium difficile. The plasmid was found to integrate into the chromoso me of a Bacillus subtilis strain that contained Tn916 Delta E. Souther n blot analysis of the recombinant demonstrated that pCI195 had insert ed into Tn916 Delta E by a recombination event. The transposon::plasmi d structure could be transferred, by filter mating, from B. subtilis t o C. difficile (at a frequency of 10(-8) per donor), where it entered the chromosome at a specific site. Segregation of plasmid and transpos on markers was observed on transfer, although the Tn916 Delta E::pCI19 5 was stably maintained in C. difficile. To demonstrate that pCI195 co uld be used for gene cloning in C. difficile, a 1.1-kb fragment of the C. difficile toxin B gene was cloned into pCI195 to generate pPPM 100 . Tn916 Delta E::pPPM100 was transferred into a nontoxigenic C. diffic ile strain by filter mating, where it entered the genome at a specific site. pCI195 should be useful as a general cloning vector for C. diff icile, as the transposon::plasmid structure could be transferred to di fferent C. difficile strains. This is the first report of gene