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An analysis of the IS6/IS26 family of insertion sequences: is it a single family?

ABSTRACT: The relationships within a curated set of 112 insertion sequences (ISs) currently assigned to the IS6 family, here re-named the IS6/IS26 family, in the ISFinder database were examined. The encoded DDE transposases include a helix-helix-turn-helix (H-HTH) potential DNA binding domain N-terminal to the catalytic (DDE) domain, but 10 from Clostridia include one or two additional N-terminal domains. The transposase phylogeny clearly separated 75 derived from bacteria from 37 from archaea. The longer bacterial transposases also clustered separately. The 65 shorter bacterial transposases, including Tnp26 from IS26, formed six clades but share significant conservation in the H-HTH domain and in a short extension at the N-terminus, and several amino acids in the catalytic domain are completely or highly conserved. At the outer ends of these ISs, 14?bp were strongly conserved as terminal inverted repeats (TIRs) with the first two bases (GG) and the seventh base (G) present in all except one IS. The longer bacterial transposases are only distantly related to the short bacterial transposases, with only some amino acids conserved. The TIR consensus was longer and only one IS started with GG. The 37 archaeal transposases are only distantly related to either the short or the long bacterial transposases and different residues were conserved. Their TIRs are loosely related to the bacterial TIR consensus but are longer and many do not begin with GG. As they do not fit well with most bacterial ISs, the inclusion of the archaeal ISs and the longer bacterial ISs in the IS6/IS26 family is not appropriate.

SUBMITTER: Harmer CJ

PROVIDER: S-EPMC6807381 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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An analysis of the IS6/IS26 family of insertion sequences: is it a single family?

Harmer Christopher J CJ Harmer Christopher J CJ Hall Ruth M RM

Microbial genomics 20190905 9

The relationships within a curated set of 112 insertion sequences (ISs) currently assigned to the IS6 family, here re-named the IS6/IS26 family, in the ISFinder database were examined. The encoded DDE transposases include a helix-helix-turn-helix (H-HTH) potential DNA binding domain N-terminal to the catalytic (DDE) domain, but 10 from Clostridia include one or two additional N-terminal domains. The transposase phylogeny clearly separated 75 derived from bacteria from ...[more]

PMID: 31486766

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Project description:The insertion sequence IS26 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 IS26. A model presented for a second mode of IS26 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 IS26 such that the ISs are in direct orientation. Repeating this process would lead to arrays of resistance genes with directly oriented copies of IS26 at each end and between each unique segment. This model requires that IS26 recognizes another IS26 as a target, and in transposition experiments, the frequency of cointegrate formation was 60-fold higher when the target plasmid contained IS26. This reaction was conservative, with no additional IS26 or target site duplication generated, and orientation specific as the IS26s in the cointegrates were always in the same orientation. Consequently, the cointegrates were identical to those formed via the known mode of IS26 movement when a target IS26 was not present. Intact transposase genes in both IS26s were required for high-frequency cointegrate formation as inactivation of either one reduced the frequency 30-fold. However, the IS26 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, IS26 is often seen in these clusters. A model to explain the unusual structure of regions containing multiple IS26 copies, each associated with a resistance gene, was not available, and the mechanism of their formation was unexplored. IS26-flanked structures deceptively resemble class I transposons, but this work reveals that the features of IS26 movement do not resemble those of the IS and class I transposons studied to date. IS26 uses a novel movement mechanism that defines a new family of mobile genetic elements that we have called "translocatable units." The IS26 mechanism also explains the properties of IS257 (IS431) and IS1216, which belong to the same IS family and mobilize resistance genes in Gram-positive staphylococci and enterococci.

Project description:UnlabelledCarbapenemase-producing Enterobacteriaceae (CPE), which are resistant to most or all known antibiotics, constitute a global threat to public health. Transposable elements are often associated with antibiotic resistance determinants, suggesting a role in the emergence of resistance. One insertion sequence, IS26, is frequently associated with resistance determinants, but its role remains unclear. We have analyzed the genomic contexts of 70 IS26 copies in several clinical and surveillance CPE isolates from the National Institutes of Health Clinical Center. We used target site duplications and their patterns as guides and found that a large fraction of plasmid reorganizations result from IS26 replicative transpositions, including replicon fusions, DNA inversions, and deletions. Replicative transposition could also be inferred for transposon Tn4401, which harbors the carbapenemase blaKPC gene. Thus, replicative transposition is important in the ongoing reorganization of plasmids carrying multidrug-resistant determinants, an observation that carries substantial clinical and epidemiological implications for understanding how such extreme drug resistance phenotypes evolve.ImportanceAlthough IS26 is frequently reported to reside in resistance plasmids of clinical isolates, the characteristic hallmark of transposition, target site duplication (TSD), is generally not observed, raising questions about the mode of transposition for IS26. The previous observation of cointegrate formation during transposition implies that IS26 transposes via a replicative mechanism. The other possible outcome of replicative transposition is DNA inversion or deletion, when transposition occurs intramolecularly, and this would also generate a specific TSD pattern that might also serve as supporting evidence for the transposition mechanism. The numerous examples we present here demonstrate that replicative transposition, used by many mobile elements (including IS26 and Tn4401), is prevalent in the plasmids of clinical isolates and results in significant plasmid reorganization. This study also provides a method to trace the evolution of resistance plasmids based on TSD patterns.

Project description:UNLABELLED:We recently showed that, in the absence of RecA-dependent homologous recombination, the Tnp26 transposase catalyzes cointegrate formation via a conservative reaction between two preexisting IS26, and this is strongly preferred over replicative transposition to a new site. Here, the reverse reaction was investigated by assaying for precise excision of the central region together with a single IS26 from a compound transposon bounded by IS26. In a recA mutant strain, Tn4352, a kanamycin resistance transposon carrying the aphA1a gene, was stable. However, loss of kanamycin resistance due to precise excision of the translocatable unit (TU) from the closely related Tn4352B, leaving behind the second IS26, occurred at high frequency. Excision occurred when Tn4352B was in either a high- or low-copy-number plasmid. The excised circular segment, known as a TU, was detected by PCR. Excision required the IS26 transposase Tnp26. However, the Tnp26 of only one IS26 in Tn4352B was required, specifically the IS26 downstream of the aphA1a gene, and the excised TU included the active IS26. The frequency of Tn4352B TU loss was influenced by the context of the transposon, but the critical determinant of high-frequency excision was the presence of three G residues in Tn4352B replacing a single G in Tn4352. These G residues are located immediately adjacent to the two G residues at the left end of the IS26 that is upstream of the aphA1a gene. Transcription of tnp26 was not affected by the additional G residues, which appear to enhance Tnp26 cleavage at this end. IMPORTANCE:Resistance to antibiotics limits treatment options. In Gram-negative bacteria, IS26 plays a major role in the acquisition and dissemination of antibiotic resistance. IS257 (IS431) and IS1216, which belong to the same insertion sequence (IS) family, mobilize resistance genes in staphylococci and enterococci, respectively. Many different resistance genes are found in compound transposons bounded by IS26, and multiply and extensively antibiotic-resistant Gram-negative bacteria often include regions containing several antibiotic resistance genes and multiple copies of IS26. We recently showed that in addition to replicative transposition, IS26 can use a conservative movement mechanism in which an incoming IS26 targets a preexisting one, and this reaction can create these regions. This mechanism differs from that of all the ISs examined in detail thus far. Here, we have continued to extend understanding of the reactions carried out by IS26 by examining whether the reverse precise excision reaction is also catalyzed by the IS26 transposase.

Dataset Information

An analysis of the IS6/IS26 family of insertion sequences: is it a single family?

Publications

An analysis of the IS<i>6</i>/IS<i>26</i> family of insertion sequences: is it a single family?

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