Oral Presentation Australian Society for Microbiology Annual Scientific Meeting 2021

The DNA-binding domain of Tnp26, the transposase of insertion sequence IS26 (#46)

Carol H Pong 1 , Christopher J Harmer 1 , Janine K Flores 1 , Sandro F Ataide 1 , Ruth M Hall 1
  1. School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW, Australia

Insertion sequence IS26 plays a significant role in disseminating antibiotic resistance genes and generating genetic diversity in Gram-negative bacteria. IS26 differs from other bacterial IS as it cannot move as a discreet unit. Rather, it mobilises by two mechanisms, a copy-in mode and a targeted conservative mode, both of which result in the formation of cointegrates. However, the way IS26 is able to interact with its transposase, Tnp26, to mobilise DNA is not well understood.

IS26 (820 bp) is bounded by identical 14 bp terminal inverted repeats (TIR) and encodes the DDE transposase Tnp26 (234 aa). Protein homology identified a tri-helical helix-turn-helix (HTH) motif near the N-terminus of Tnp26 (aa I13–R53) that was investigated as the putative DNA-binding domain of Tnp26. The functional characteristics of the HTH were examined in vivo via mating-out assays to measure cointegration activity, and in vitro using electrophoretic mobility shift assays and microscale thermophoresis to detect protein-DNA binding.

Residues conserved in the predicted HTH motif of 65 known IS26 family members were substituted in Tnp26, and these alterations significantly reduced cointegration activity. Tnp26 was purified as a His-MBP fusion, and this protein bound to dsDNA probes containing the IS26 TIR sequence. Tnp26 with aa substitutions in helix 3 did not bind to the TIR, consistent with its predicted role as the end-recognition domain. Deletion derivative Tnp26 M1–P56 was sufficient for sequence-specific binding but only at high concentrations, indicating that the catalytic core or other parts of Tnp26 enhance binding affinity. 

Tnp26 binding to probes containing TIRR (right TIR) with transversions of conserved guanine bases to thymine (1G>T 2G>T, 7G>T, and 10G>T) abolished cointegrate formation. Protein-DNA binding was detected with 1G>T 2G>T and 10G>T probes, but not with the 7G>T probe. This suggests that 7G forms a critical part of the Tnp26 binding site. Together, these findings indicate that the HTH motif serves as the IS26 end-recognition domain for Tnp26.