Characterization of the Ends and Target Sites of the Novel Conjugative Transposon Tn 5397 from Clostridium difficile : Excision and Circularization Is Mediated by the Large Resolvase, TndX

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Abstract
Tn 5397 is a conjugative transposon that was originally isolated from Clostridium difficile . Previous analysis had shown that the central region of Tn 5397 was closely related to the conjugative transposon Tn 916 . However, in this work we obtained the DNA sequence of the ends of Tn 5397 and showed that they are completely different to those of Tn 916 . Tn 5397 did not contain the int and xis genes, which are required for the excision and integration of Tn 916 . Instead, the right end of Tn 5397 contained a gene, tndX , that appears to encode a member of the large resolvase family of site-specific recombinases. TndX is closely related to the TnpX resolvase from the mobilizable but nonconjugative chloramphenicol resistance transposons, Tn 4451 from Clostridium perfringens and Tn 4453 from C. difficile . Like the latter elements, inserted copies of Tn 5397 were flanked by a direct repeat of a GA dinucleotide. The Tn 5397 target sites were also shown to contain a central GA dinucleotide. Excision of the element in C. difficile completely regenerated the original target sequence. A circular form of the transposon, in which the left and right ends of the element were separated by a GA dinucleotide, was detected by PCR in both Bacillus subtilis and C. difficile . A Tn 5397 mutant in which part of tndX was deleted was constructed in B. subtilis . This mutant was nonconjugative and did not produce the circular form of Tn 5397 , indicating that the TndX resolvase has an essential role in the excision and transposition of Tn 5397 and is thus the first example of a member of the large resolvase family of recombinases being involved in conjugative transposon mobility. Finally, we showed that introduction of Tn 916 into a strain containing Tn 5397 induced the loss of the latter element in 95.6% of recipients.