The large resolvase TndX is required and sufficient for integration and excision of derivatives of the novel conjugative transposon Tn5397

Tn5397 is a novel conjugative transposon, originally isolated from Clostrid ium difficile. This element can transfer between C. difficile strains and t o and from Bacillus subtilis, It encodes a conjugation system that is very similar to that of Tn916. However, insertion and excision of Tn5397 appears to be dependent on the product of the element encoded gene tndX, a member of the large resolvase family of site-specific recombinases. To test the ro le of tndX, the gene was cloned and the protein was expressed in Escherichi a coli. The ability of TndX to catalyze the insertion and excision of deriv atives (minitransposons) of Tn5397 representing the putative circular and i ntegrated forms, respectively, was investigated. TndX was required for both insertion and excision. Mutagenesis studies showed that some of the highly conserved amino acids at the N-terminal resolvase domain and the C-termina l nonconserved region of TndX are essential for activity. Analysis of the t arget site choices showed that the cloned Tn5397 targets from C. difficile and B. subtilis were still hot spots for the minitransposon insertion in E. coli.