Insertion sequence IS26 is widely recognised as an essential player in the mobilisation and dissemination of antibiotic resistance genes in Gram-negative bacteria. Despite its pervasiveness, the mechanisms by which IS26 and its encoded transposase, Tnp26, is able to drive genetic diversity on bacteria is not well understood.
In this study, a putative helix-helix-turn-helix (H-HTH) motif was identified in the N-terminal region of Tnp26 by bioinformatic and computational means. This tri-helical bundle was proposed to be necessary for Tnp26 to recognise and bind to the terminal inverted repeats (IR) which form the boundaries of IS26.
Using in vitro mutagenesis, specific amino acid substitutions were introduced disrupting helix 2 (E30P), helix 3 (W50P) or the turn (G39W), and the resultant mutant IS26 were tested using a transposition assay. Replicative transposition was not detectable, over 150-fold lower than the wild-type. The mutant IS26 were also tested for targeted (conservative) transposition to second IS26. When both IS26 participating in conservative transposition were mutated, the transposition frequency of IS26 mutants (encoding Tnp26 E30P, G39W and W50P) was reduced over 100-fold, in comparison to the wild-type. However, there was a less than 5-fold reduction when only one of the two participating IS26 were mutated.
The residues computationally predicted to be oriented outward from helix 3, the ‘recognition helix’, were also altered. In the conservative reaction, transposition of mutant IS26, encoding Tnp26 R49A, Y54A and R63A, had a 30-50 fold reduction when one IS26 was mutated, indicating a more direct involvement of these residues in IR-recognition and protein-DNA binding. qRT-PCR provided evidence that Tnp26 is able to bind specifically to the IRs and auto-regulate its own expression.
Understanding the function of Tnp26 and interactions with its encoding insertion sequence, IS26, will allow us to better understand the transposition mechanism, and elucidate how other members of the IS6 family are able to mobilise antibiotic resistance genes in both Gram-negative and Gram-positive bacteria.