Characterisation of the IS26-encoded transposase, Tnp26

Abstract

Insertion sequence IS26 plays a major role in the dissemination of antibiotic resistance genes in Gram-negative bacteria. Recently, IS26 has been shown to have unique abilities which are not shared by other IS families. Two domains predicted using in silico modelling were examined: the extreme N-terminus of Tnp26 (aa 1–9): a possible multimerisation domain, and the adjacent H-HTH fold (aa 10–56): a possible DNA-binding domain. The effect of amino acid substitutions targeting highly conserved residues in these two domains was investigated using in vivo cointegration assays, and electrophoretic mobility shift assays (EMSA) to test binding of purified Tnp26 protein to DNA substrates containing the terminal inverted repeats (TIR) of IS26. Two phenylalanine residues in the extreme N-terminus, F4 and F9, were established to be required for cointegration. EMSAs showed that changes to Tnp26 F4 and F9 impacted DNA-binding. Substitutions which altered the secondary structure of the H-HTH fold or replaced positively-charged/planar residues with alanine also affected cointegration. Fusion proteins carrying Tnp26 aa 1–56 demonstrated sequence-specific DNA recognition in EMSAs, but at high protein concentrations (compared to full-length Tnp26). A natural IS26 variant (encoding Tnp26 G184N) associated with enhanced activity was investigated in vivo to quantify the impact of the aa change on cointegration. Additional Tnp26 variants with different aa at the same position were also investigated to determine the residue properties required to establish enhanced activity. To further understand the role of IS26 movement in the real world, the evolution of an IS26-flanked tet(C) transposon (Tn6184) was analysed. Using bioinformatics analysis, comparisons of structures similar to Tn6184 revealed several transposons which could be generated by IS26-mediated activity, demonstrating the versatility of IS26 in driving evolution. The properties and conjugative ability of the plasmid carrying Tn6184, pSRC22-2, were also investigated. Understanding Tnp26 activity may improve understanding of IS26 movement, and elucidate how other members of its family are able to mobilise genetic material and drive diversification of Gram-negative and Gram-positive bacteria.