Alharbi, Sarah; (2021) Analysis of the Target Site Preference of the Conjugative Transposon Tn5397. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Integrative conjugative elements (ICEs) also called conjugative transposons, are discrete DNA segments that can excise and re-insert into a DNA molecule. They can also transfer between bacterial cells by conjugation and often carry antibiotic resistance genes (ARGs). ICEs have a diverse preference for the DNA sequence of their target sites. Some insert preferentially into a specific nucleotide sequence, while others have many integration sites. The reasons for this difference in the evolution of target site selectivity are not fully understood but likely represent alternative evolutionary strategies to maximise the likelihood of finding a suitable target in a DNA molecule. The multi-drug resistant strain Clostridioides difficile 630 harbours the tetracycline resistance-encoding element Tn5397, which has considerable target-site specificity. The transposition of Tn5397 is catalysed by the large serine recombinase, TndX. An important structural feature of the Tn5397 target-site is imperfect inverted repeats flanking a central GA dinucleotide (crossover site). This study aimed to investigate the Tn5397 insertion site preference. For this purpose, in vivo transposition assays (consisting of a mini-Tn5397 element and the tndX gene, encoding the recombinase, plus several mutant target sites (attCD) on different replicons) were established in Escherichia coli to reconstitute the excision and insertion reactions of Tn5397. The insertion of mini-Tn5397 into the target sites was detected by PCR across the left and the right mini-transposon::target site junctions. Current data demonstrate successful transposition of the mini-transposon into the wild-type target-site and that this target can tolerate specific mutations around, but not including, the central GA dinucleotide. The data presented here advance our knowledge of Tn5397 target-site selection and revealed specific positions at the target site that contribute to the target site preference. Analysis of mutant attCD sites and Tn5397 target sites identified in several hosts determined conserved nucleotides, which I inferred as the key bases by which TndX contacts attCD.

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