Gene Cloning in Clostridium difficile Using Tn 916 as a Shuttle Conjugative Transposon

Abstract

A pBR322-based vector, pCI195, containing a 4.2-kb region of the conjugative transposon Tn 919 was used as a vector for gene cloning in Clostridium difficile. The plasmid was found to integrate into the chromosome of a Bacillus subtilis strain that contained Tn 916ΔE. Southern blot analysis of the recombinant demonstrated that pCI195 had inserted into Tn 916ΔE by a recombination event. The transposon::plasmid structure could be transferred, by filter mating, from B. subtilis to C. difficile (at a frequency of 10 -8 per donor), where it entered the chromosome at a specific site. Segregation of plasmid and transposon markers was observed on transfer, although the Tn 916ΔE::pCI195 was stably maintained in C. difficile. To demonstrate that pCI195 could be used for gene cloning in C. diffficile, a 1.1-kb fragment of the C. difficile toxin B gene was cloned into pCI195 to generate pPPM100. Tn 916ΔE::pPPM100 was transferred into a nontoxigenic C. difficile strain by filter mating, where it entered the genome at a specific site. pCI195 should be useful as a general cloning vector for C. difficile, as the transposon::plasmid structure could be transferred to different C. difficile strains. This is the first report of gene cloning in C. difficile.