IS26 (820 bp) is a major player in the mobilisation of antibiotic resistance genes in Gram-negative bacteria, and is able to perform both random replicative and targeted conservative transposition. However, how IS26 uses its transposase, Tnp26 (234 aa), to do this is not understood.
We proposed that a putative helix-helix-turn-helix (H-HTH) motif in the N-terminal region of Tnp26 (aa 13–53) is necessary for Tnp26 to recognise and bind to the 14 bp inverted repeats (IR) at the IS boundaries. Single aa substitutions were introduced disrupting Tnp26 helix 2 (E30P), helix 3 (W50P) or the turn (G39I/G39W). Resultant mutant IS26 were tested using a series of transposition assays. Replicative transposition was not detectable, >150-fold lower than the wild-type. For targeted transposition, there was >100-fold reduction when both IS26 were mutated, but <5-fold reduction when only one of the two participating IS26 were mutated. However, single aa substitutions in residues computationally predicted to be outwardly-oriented from Tnp26 helix 1 (K23A) or helix 3 (R49A or Y54A) reduced transposition frequency by 40–100-fold when only one IS26 was mutated. This is consistent with significant involvement of these residues in IR-recognition.
To gain further insight into the mechanism of targeted integration, cointegrates from transposition assays were screened to determine which IS26 copy carried the mutation. For mutants at the N-terminal end of IS26 (within bases 1–252, aa M1–R63), the mutation appeared on the left or right side in 14.7% and 57.9%, respectively (total of 95 cointegrates), which is consistent with recombination at either IS end. However, the mutation also appeared in both (20%) and neither (7.4%) IS26 of the cointegrate. This gene conversion is consistent with a single-strand crossover, followed by branch migration and repair of the mismatch, prior to replication to complete cointegration formation.