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Characterization of the transposase encoded by IS256, the prototype of a major family of bacterial insertion sequence elements.

ABSTRACT: IS256 is the founding member of the IS256 family of insertion sequence (IS) elements. These elements encode a poorly characterized transposase, which features a conserved DDE catalytic motif and produces circular IS intermediates. Here, we characterized the IS256 transposase as a DNA-binding protein and obtained insight into the subdomain organization and functional properties of this prototype enzyme of IS256 family transposases. Recombinant forms of the transposase were shown to bind specifically to inverted repeats present in the IS256 noncoding regions. A DNA-binding domain was identified in the N-terminal part of the transposase, and a mutagenesis study targeting conserved amino acid residues in this region revealed a putative helix-turn-helix structure as a key element involved in DNA binding. Furthermore, we obtained evidence to suggest that the terminal nucleotides of IS256 are critically involved in IS circularization. Although small deletions at both ends reduced the formation of IS circles, changes at the left-hand IS256 terminus proved to be significantly more detrimental to circle production. Taken together, the data lead us to suggest that the IS256 transposase-mediated circularization reaction preferentially starts with a sequence-specific first-strand cleavage at the left-hand IS terminus.

SUBMITTER: Hennig S

PROVIDER: S-EPMC2916423 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Characterization of the transposase encoded by IS256, the prototype of a major family of bacterial insertion sequence elements.

Hennig Susanne S Ziebuhr Wilma W

Journal of bacteriology 20100611 16

IS256 is the founding member of the IS256 family of insertion sequence (IS) elements. These elements encode a poorly characterized transposase, which features a conserved DDE catalytic motif and produces circular IS intermediates. Here, we characterized the IS256 transposase as a DNA-binding protein and obtained insight into the subdomain organization and functional properties of this prototype enzyme of IS256 family transposases. Recombinant forms of the transposase were shown to bind specifica ...[more]

PMID: 20543074

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Project description:BackgroundInsertion sequences (IS) are small transposable elements, commonly found in bacterial genomes. Identifying the location of IS in bacterial genomes can be useful for a variety of purposes including epidemiological tracking and predicting antibiotic resistance. However IS are commonly present in multiple copies in a single genome, which complicates genome assembly and the identification of IS insertion sites. Here we present ISMapper, a mapping-based tool for identification of the site and orientation of IS insertions in bacterial genomes, directly from paired-end short read data.ResultsISMapper was validated using three types of short read data: (i) simulated reads from a variety of species, (ii) Illumina reads from 5 isolates for which finished genome sequences were available for comparison, and (iii) Illumina reads from 7 Acinetobacter baumannii isolates for which predicted IS locations were tested using PCR. A total of 20 genomes, including 13 species and 32 distinct IS, were used for validation. ISMapper correctly identified 97 % of known IS insertions in the analysis of simulated reads, and 98 % in real Illumina reads. Subsampling of real Illumina reads to lower depths indicated ISMapper was able to correctly detect insertions for average genome-wide read depths >20x, although read depths >50x were required to obtain confident calls that were highly-supported by evidence from reads. All ISAba1 insertions identified by ISMapper in the A. baumannii genomes were confirmed by PCR. In each A. baumannii genome, ISMapper successfully identified an IS insertion upstream of the ampC beta-lactamase that could explain phenotypic resistance to third-generation cephalosporins. The utility of ISMapper was further demonstrated by profiling genome-wide IS6110 insertions in 138 publicly available Mycobacterium tuberculosis genomes, revealing lineage-specific insertions and multiple insertion hotspots.ConclusionsISMapper provides a rapid and robust method for identifying IS insertion sites directly from short read data, with a high degree of accuracy demonstrated across a wide range of bacteria.

Project description:BackgroundMobile elements are involved in genomic rearrangements and virulence acquisition, and hence, are important elements in bacterial genome evolution. The insertion of some specific Insertion Sequences had been associated with repetitive extragenic palindromic (REP) elements. Considering that there are a sufficient number of available genomes with described REPs, and exploiting the advantage of the traceability of transposition events in genomes, we decided to exhaustively analyze the relationship between REP sequences and mobile elements.ResultsThis global multigenome study highlights the importance of repetitive extragenic palindromic elements as target sequences for transposases. The study is based on the analysis of the DNA regions surrounding the 981 instances of Insertion Sequence elements with respect to the positioning of REP sequences in the 19 available annotated microbial genomes corresponding to species of bacteria with reported REP sequences. This analysis has allowed the detection of the specific insertion into REP sequences for ISPsy8 in Pseudomonas syringae DC3000, ISPa11 in P. aeruginosa PA01, ISPpu9 and ISPpu10 in P. putida KT2440, and ISRm22 and ISRm19 in Sinorhizobium meliloti 1021 genome. Preference for insertion in extragenic spaces with REP sequences has also been detected for ISPsy7 in P. syringae DC3000, ISRm5 in S. meliloti and ISNm1106 in Neisseria meningitidis MC58 and Z2491 genomes. Probably, the association with REP elements that we have detected analyzing genomes is only the tip of the iceberg, and this association could be even more frequent in natural isolates.ConclusionOur findings characterize REP elements as hot spots for transposition and reinforce the relationship between REP sequences and genomic plasticity mediated by mobile elements. In addition, this study defines a subset of REP-recognizer transposases with high target selectivity that can be useful in the development of new tools for genome manipulation.

Dataset Information

Characterization of the transposase encoded by IS256, the prototype of a major family of bacterial insertion sequence elements.

Publications

Characterization of the transposase encoded by IS256, the prototype of a major family of bacterial insertion sequence elements.

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