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.

In Gram-negative bacteria, IS 26 recruits antibiotic resistance genes into the mobile gene pool by forming transposons carrying many different resistance genes. In addition to replicative transposition, IS 26 was recently shown to use a novel conservative movement mechanism in which an incoming IS 26 targets a preexisting one. Here, we have demonstrated how IS 26 -bounded class I transposons can be produced from translocatable units (TUs) containing only an IS 26 and a resistance gene via the conservative reaction. TUs were incorporated next to an existing IS 26 , creating a class I transposon, and if the targeted IS 26 is in a transposon, the product resembles two transposons sharing a central IS 26 , a configuration observed in some resistance regions and when a transposon is tandemly duplicated. Though homologous recombination could also incorporate a TU, Tnp26 is far more efficient. This provides insight into how IS 26 builds transposons and brings additional transposons into resistance regions.

SUMMARY: In Gram-negative bacteria, the insertion sequence IS 26 is highly active in disseminating antibiotic resistance genes. IS 26 can recruit a gene or group of genes into the mobile gene pool and support their continued dissemination to new locations by creating pseudo-compound transposons (PCTs) that can be further mobilized by the insertion sequence (IS). IS 26 can also enhance expression of adjacent potential resistance genes. IS 26 encodes a DDE transposase but has unique properties. It forms cointegrates between two separate DNA molecules using two mechanisms. The well-known copy-in (replicative) route generates an additional IS copy and duplicates the target site. The recently discovered and more efficient and targeted conservative mechanism requires an IS in both participating molecules and does not generate any new sequence. The unit of movement for PCTs, known as a translocatable unit or TU, includes only one IS 26 . TU formed by homologous recombination between the bounding IS 26 s can be reincorporated via either cointegration route. However, the targeted conservative reaction is key to generation of arrays of overlapping PCTs seen in resistant pathogens. Using the copy-in route, IS 26 can also act on a site in the same DNA molecule, either inverting adjacent DNA or generating an adjacent deletion plus a circular molecule carrying the DNA segment lost and an IS copy. If reincorporated, these circular molecules create a new PCT. IS 26 is the best characterized IS in the IS 26 family, which includes IS 257 /IS 431 , ISSau10, IS 1216 , IS 1006 , and IS 1008 that are also implicated in spreading resistance genes in Gram-positive and Gram-negative pathogens.

Pseudomonas aeruginosaairway infection is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. Variousin vitromodels have been developed to studyP. aeruginosapathobiology in the CF lung. In this study we produced a modified artificial-sputum medium (ASMDM) more closely resembling CF sputum than previous models, and extended previous work by using strain PAO1 arrays to examine the global transcription profiles ofP. aeruginosastrain UCBPP-PA14 under early exponential-phase and stationary-phase growth. In early exponential phase, 38/39 nutrition-related genes were upregulated in line with data from previousin vitromodels using UCBPP-PA14. Additionally, 23 type III secretion system (T3SS) genes, several anaerobic respiration genes and 24 quorum-sensing (QS)-related genes were upregulated in ASMDM, suggesting enhanced virulence factor expression and priming for anaerobic growth and biofilm formation. Under stationary phase growth in ASMDM, macroscopic clumps resembling microcolonies were evident in UCBPP-PA14 and CF strains, and over 40 potentially important genes were differentially expressed relative to stationary-phase growth in Luria broth. Most notably, QS-related and T3SS genes were downregulated in ASMDM, and iron-acquisition and assimilatory nitrate reductase genes were upregulated, simulating the iron-depleted, microaerophilic/anaerobic environment of CF sputum. ASMDM thus appears to be highly suitable for gene expression studies ofP. aeruginosain CF.