Invited Speaker Australian Society for Microbiology Annual Scientific Meeting 2018

Transposon maker IS26, flagship of the versatile IS6 family of insertion sequences (#70)

Christopher J Harmer , Carol Pong , Ruth M Hall

In Gram-negative bacteria, the insertion sequence IS26 plays a major role in disseminating antibiotic resistance genes via the formation of compound transposons. Despite this, and despite being discovered over three decades ago, until recently IS26 was relatively neglected. Over the past five years we have shown that in addition to the well-known replicative cointegration mode, IS26 can use a conservative, self-targeted reaction to form a cointegrate between two DNA molecules that each contain a copy of the IS. This occurs in preference to the replicative mode when two IS26 are present. During the conservative reaction, the strand exchange(s) occur at the same end of the IS, but can occur at equal efficiency at either IS end. In the products of the conservative reaction, the two IS26 are always in direct orientation, resembling a traditional class 1 compound transposon, and this mechanism has been used to introduce new genes in vitro.


To determine the functional characteristics of the IS26 transposase (Tnp26) and to better understand the interactions between IS26 and Tnp26, we computationally modelled the protein. We have subdivided and investigated several putative domains, including a helix-helix-turn-helix DNA binding domain, the DDE catalytic core, and a potential dimerization domain at the extreme N-terminus. Site-directed mutagenesis was used to introduce amino acid substitutions into conserved residues, and their importance in transposase activity was determined. A natural variant of IS26 that carries an amino acid substitution in the DDE catalytic core is over-represented in intramolecular inversion and deletion events was also examined. This substitution was shown to significantly increase transposase activity.


The ability of Tnp26 to function in either a replicative or conservative mode is likely to explain the prominence of IS26-bounded transposons in the resistance regions found in Gram-negative bacteria. IS26 relatives that play important roles in the mobilization of resistance genes in Gram-positive bacteria will need to be re-examined to determine if they too can utilise this new mechanism.