The role of tandem IS dimers in IS911 transposition.
Using a combined in vivo and in vitro approach, we demonstrated that the transposition products generated by IS911 from a dimeric donor plasmid are different from those generated from a plasmid monomer. When carried by a monomeric plasmid donor, free IS911 transposon circles are generated by intra-IS recombination in which one IS end undergoes attack by the other. These represent transposition intermediates that undergo integration using the abutted left (IRL) and right (IRR) ends of the element, the active IRR-IRL junction, to generate simple insertions. In contrast, the two IS911 copies carried by a dimeric donor plasmid not only underwent intra-IS recombination to generate transposon circles but additionally participated in inter-IS recombination. This also creates an active IRR-IRL junction by generating a head-to-tail IS tandem dimer ([IS]2) in which one of the original plasmid backbone copies is eliminated in the formation of the junction. Both transposon circles and IS tandem dimers are generated from an intermediate in which two transposon ends are retained by a single strand joint to generate a figure 8 molecule. Inter-IS figure 8 molecules generated in vitro could be resolved into the [IS]2 form following introduction into a host strain by transformation. Resolution did not require IS911 transposase. The [IS]2 structure was stable in the absence of transposase but was highly unstable in its presence both in vivo and in vitro. Previous studies had demonstrated that the IRR-IRL junction promotes efficient intermolecular integration and intramolecular deletions both in vivo and in vitro. Integration of the [IS]2 derivative would result in a product that resembles a co-integrate structure. It is also shown here that the IRR-IRL junction of the [IS]2 form and derivative structures can specifically target one of the other ends in an intramolecular transposition reaction to generate transposon circles in vitro. These results not only demonstrate that IS911 (and presumably other members of the IS3 family) is capable of generating a range of transposition products, it also provides a mechanistic framework which explains the formation and activity of such structures previously observed for several other unrelated IS elements. This behaviour is probably characteristic of a large number of IS elements.
Turlan
C
C
Laboratoire de Microbiologie et Génétique Moléculaires, CNRS UPR9007, 118 Rte de Narbonne, F31062 Toulouse Cedex, France.
Ton-Hoang
B
B
Chandler
M
M
D001426:Bacterial Proteins / Q000235:genetics; D004251:DNA Transposable Elements; D004270:DNA, Circular; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D009154:Mutation; D010957:Plasmids / Q000235:genetics*; D011995:Recombination, Genetic; D019895:Transposases / Q000378:metabolism
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004270:DNA, Circular; D029968:Escherichia coli Proteins; C085896:OrfB protein, E coli; D019895:Transposases