ABSTRACT The insertion sequence IS 26 plays a key role in disseminating antibiotic resistance genes in Gram-negative bacteria, forming regions containing more than one antibiotic resistance gene that are flanked by and interspersed with copies of IS 26 . A model presented for a second mode of IS 26 movement that explains the structure of these regions involves a translocatable unit consisting of a unique DNA segment carrying an antibiotic resistance (or other) gene and a single IS copy. Structures resembling class I transposons are generated via RecA-independent incorporation of a translocatable unit next to a second IS 26 such that the ISs are in direct orientation. Repeating this process would lead to arrays of resistance genes with directly oriented copies of IS 26 at each end and between each unique segment. This model requires that IS 26 recognizes another IS 26 as a target, and in transposition experiments, the frequency of cointegrate formation was 60-fold higher when the target plasmid contained IS 26 . This reaction was conservative, with no additional IS 26 or target site duplication generated, and orientation specific as the IS 26 s in the cointegrates were always in the same orientation. Consequently, the cointegrates were identical to those formed via the known mode of IS 26 movement when a target IS 26 was not present. Intact transposase genes in both IS 26 s were required for high-frequency cointegrate formation as inactivation of either one reduced the frequency 30-fold. However, the IS 26 target specificity was retained. Conversion of each residue in the DDE motif of the Tnp26 transposase also reduced the cointegration frequency. IMPORTANCE Resistance to antibiotics belonging to several of the different classes used to treat infections is a critical problem. Multiply antibiotic-resistant bacteria usually carry large regions containing several antibiotic resistance genes, and in Gram-negative bacteria, IS 26 is often seen in these clusters. A model to explain the unusual structure of regions containing multiple IS 26 copies, each associated with a resistance gene, was not available, and the mechanism of their formation was unexplored. IS 26 -flanked structures deceptively resemble class I transposons, but this work reveals that the features of IS 26 movement do not resemble those of the IS and class I transposons studied to date. IS 26 uses a novel movement mechanism that defines a new family of mobile genetic elements that we have called “translocatable units.” The IS 26 mechanism also explains the properties of IS 257 (IS 431 ) and IS 1216 , which belong to the same IS family and mobilize resistance genes in Gram-positive staphylococci and enterococci.

Movement of IS 26 -Associated Antibiotic Resistance Genes Occurs via a Translocatable Unit That Includes a Single IS 26 and Preferentially Inserts Adjacent to Another IS 26