Transposon Mutagenesis in Clostridium difficile

Genetic manipulation of Clostridium difficile is notoriously difficult, currently there is only one reliable method for generating random mutations in the organism and that is to use the conjugative transposon Tn916 . Tn916 enters the genome of most strains of C. difficile with no obvious target site preference. In order to use the genome strain C. difficile 630 for transposon mutagenesis a erythromycin-sensitive derivative C. difficile 630Δerm was constructed and the Tn916 derivative, Tn916 ΔE, was shown to enter the genome at multiple sites enabling the construction of a Tn916 insertion library.