Targeted Conservative Cointegrate Formation Mediated by IS Family Members Requires Sequence Identity at the Reacting End.

IS forms cointegrates using two distinct routes, a copy-in mechanism involving one insertion sequence (IS) and a target and a targeted conservative mechanism involving two ISs in different DNA molecules. In this study, the ability of IS and some close relatives, IS, IS, and a natural hybrid, IS, which are found predominantly in spp., to interact was examined. IS consists of 175 bp from IS at the left end, with the remainder from IS These ISs all have the same 14-bp terminal inverted repeats, and the Tnp26, Tnp1006, and Tnp1008 transposases, with pairwise identities of 83.7% to 93.1%, should be able to recognize each other's ends. In a -negative strain, IS, IS, and IS each formed cointegrates via the copy-in route and via the targeted conservative route, albeit at frequencies for the targeted reaction at least 10-fold lower than for IS However, using mixed pairs, targeted cointegration was detected only when IS was paired with the IS/ hybrid, which also encodes Tnp1008, and the targeted cointegrates formed all arose from a reaction occurring at the end where the DNA sequences are identical. The reaction also occurred at the end with extended DNA identity using IS paired with IS::, an artificially constructed IS derivative that includes the gene. Thus, both identical transposases and identical DNA sequences at the reacting end were required. These features indicate that the targeted conservative pathway proceeds via a single transposase-catalyzed strand transfer, followed by migration and resolution of the Holliday junction formed. The IS family includes the ISs that are commonly found associated with antibiotic resistance genes in multiply resistant Gram-negative and Gram-positive bacteria. IS is most prevalent in Gram-negative species and can generate the clusters of antibiotic resistance genes interspersed with directly oriented IS seen in multiply resistant pathogens. This ability relies on the novel dual mechanistic capabilities of IS family members. However, the mechanism underlying the recently discovered targeted conservative mode of cointegrate formation mediated by IS, ISIS, and IS, which is unlike any previously studied IS movement mechanism, is not well understood. An important question is what features of the IS and the transposase are key to allowing IS family members to undertake targeted conservative reaction. In this study, this question was addressed using mixed-partner crosses involving IS and naturally occurring close relatives of IS that are found near resistance genes in and are widespread in species.