Development of an integrative vector for the expression of antisense RNA in Clostridium difficile.

A method was developed to use the conjugative transposon Tn916 as a vector for introducing recombinant DNA into Clostridium difficile. This was used to introduce antisense RNA for the adhesin encoding gene cwp66 into C. difficile 79-685. RT-PCR demonstrated that cwp66 specific antisense RNA was produced. However, there was no statistically significant difference in the protein expression or in the adherence of recombinant C. difficile strains. This may be due to the amount of transcripts of the wild-type (sense) cwp66 outnumbering the antisense transcripts or secondary structures present within the cwp66 mRNA. Unlike in other strains of C. difficile, where Tn916 inserts into the genome at highly preferred sites, in C. difficile 79-685, it integrates into multiple sites opening up the possibility of using Tn916 as a mutagen in this strain.