The Conjugative Transposon Tn 5397 Has a Strong Preference for Integration into Its Clostridium difficile Target Site

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Abstract
Tn 5397 is a conjugative transposon, originally isolated from Clostridium difficile . The Tn 5397 transposase TndX is related to the phage-encoded serine integrases and the Clostridium perfringens Tn 4451 transposase TnpX. TndX is required for the insertion and excision of the transposon. Tn 5397 inserts at one locus, attB Cd , 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 Tn 5397 , attB Cd was introduced into the B. subtilis genome. When Tn 5397 was transferred into this strain, 100% of the 50 independent transconjugants tested had Tn 5397 inserted into attB Cd . This experiment was repeated using a 50-bp attB Cd with no loss of target preference. The mutation of the 5′ GA to 5′ TC in the attB Cd target site caused a switch in the polarity of insertion of Tn 5397 , which is consistent with this dinucleotide being at the crossover site and in keeping with the mechanism of other serine recombinases. Tn 5397 could also transpose into 50-bp sequences encoding the end joints attL and attR but, surprisingly, could not recombine into the circular joint of Tn 5397 , attTn . Purified TndX was shown to bind specifically to 50-bp attB Cd , attL , attR , attTn , and attB Bs 3 with relative binding affinities attTn ≈ attR > attL > attB Cd > attB Bs 3 . We conclude that TndX has a strong preference for attB Cd over other potential recombination sites in the B. subtilis genome and therefore behaves as a site-specific recombinase.