Characterization of the ends and target sites of the novel conjugative transposon Tn5397 from Clostridium difficile: Excision and circularization is mediated by the large resolvase, TndX

Tn5397 is a conjugative transposon that was originally isolated from Clostr idium digicile, Previous analysis had shown that the central region of Tn53 97 was closely related to the conjugative transposon Tn916, However, in thi s work we obtained the DNA sequence of the ends of Tn5397 and showed that t hey are completely different to those of Tn916, Tn5397 did not contain the int and xis genes, which are required for the excision and integration of T n916. Instead, the right end of Tn5397 contained a gene, tndX; that appears to encode a member of the large resolvase family of site-specific recombin ases. TndX is closely related to the TnpX resolvase from the mobilizable bu t nonconjugative chloramphenicol resistance transposons, Tn4451 from Clostr idium perfringens and Tn4453 from C. difficile. Like the latter elements, i nserted copies of Tn5397 were flanked by a direct repeat of a CA dinucleoti de, The Tn5397 target sites were also shown to contain a central GA dinucle otide, Excision of the element ill C, difficile completely regenerated the original target sequence. A circular form of the transposon, in which the l eft and right ends of the element were separated by a GA dinucleotide, was detected by PCR in both Bacillus subtilis and C. difficile, A Tn5397 mutant in which part of tndX was deleted was constructed in B. subtilis, This mut ant was nonconjugative and did not produce the circular form of Tn5397, ind icating that the TndX resolvase has an essential role in the excision and t ransposition of Tn5397 and is thus the first example of a member of the lar ge resolvase family of recombinases being involved in conjugative transposo n mobility. Finally, we showed that introduction of Tn916 into a strain con taining Tn5397 induced the loss of the latter element in 95.6% of recipient s.