The Conjugative Transposon Tn5397 Has a Strong Preference for Integration into Its Clostridium difficile Target Site

Tn5397 is a conjugative transposon, originally isolated from Clostridium difficile. The Tn5397 transposase TndX is related to the phage-encoded serine integrases and the Clostridium perfringens Tn4451 transposase TnpX. TndX is required for the insertion and excision of the transposon. Tn5397 inserts at one locus, attBCd, in C. difficile but at multiple sites in Bacillus subtilis. Apart from a conserved 5′ GA dinucleotide at the recombination site, there appears to be little sequence conservation between the known target sites. To test the target site preference of Tn5397, attBCd was introduced into the B. subtilis genome. When Tn5397 was transferred into this strain, 100% of the 50 independent transconjugants tested had Tn5397 inserted into attBCd. This experiment was repeated using a 50-bp attBCd with no loss of target preference. The mutation of the 5′ GA to 5′ TC in the attBCd target site caused a switch in the polarity of insertion of Tn5397, which is consistent with this dinucleotide being at the crossover site and in keeping with the mechanism of other serine recombinases. Tn5397 could also transpose into 50-bp sequences encoding the end joints attL and attR but, surprisingly, could not recombine into the circular joint of Tn5397, attTn. Purified TndX was shown to bind specifically to 50-bp attBCd, attL, attR, attTn, and attBBs3 with relative binding affinities attTn ≈ attR > attL > attBCd > attBBs3. We conclude that TndX has a strong preference for attBCd over other potential recombination sites in the B. subtilis genome and therefore behaves as a site-specific recombinase.