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Movement of IS26-associated antibiotic resistance genes occurs via a translocatable unit that includes a single IS26 and preferentially inserts adjacent to another IS26.

Harmer CJ, Moran RA, Hall RM - MBio (2014)

Bottom Line: Intact transposase genes in both IS26s were required for high-frequency cointegrate formation as inactivation of either one reduced the frequency 30-fold.Conversion of each residue in the DDE motif of the Tnp26 transposase also reduced the cointegration frequency.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.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular Bioscience, The University of Sydney, Sydney, Australia.

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Related in: MedlinePlus

Cointegrate formation between pRMH762 and R388. The R388 backbone is drawn to scale from GenBank accession no. BR000038 with key resistance genes, genes involved in replication (repA), and genes involved in conjugative transfer (tra) shown as arrows inside the circular backbone. Arrows pointing toward the circular backbone indicate the location of 20 mapped R388::pRMH762 cointegrates. The precise location and orientation of nine cointegrates were determined by sequencing. An arrow labeled 1 indicates a cointegrate with tnp26 facing clockwise, while an arrow labeled 2 indicates a cointegrate with tnp26 facing counterclockwise. An asterisk denotes the cointegrate used in the construction of R388::IS26.
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fig4: Cointegrate formation between pRMH762 and R388. The R388 backbone is drawn to scale from GenBank accession no. BR000038 with key resistance genes, genes involved in replication (repA), and genes involved in conjugative transfer (tra) shown as arrows inside the circular backbone. Arrows pointing toward the circular backbone indicate the location of 20 mapped R388::pRMH762 cointegrates. The precise location and orientation of nine cointegrates were determined by sequencing. An arrow labeled 1 indicates a cointegrate with tnp26 facing clockwise, while an arrow labeled 2 indicates a cointegrate with tnp26 facing counterclockwise. An asterisk denotes the cointegrate used in the construction of R388::IS26.

Mentions: All experiments were conducted in a recA mutant Escherichia coli background to ensure that the events detected had been catalyzed by the IS26-encoded transposase Tnp26. First, the ability of a single copy of IS26 to form cointegrates as described previously was confirmed using a mating-out assay to detect the cointegrates formed between the plasmid pRMH762 (which confers ampicillin resistance [Apr]), which contains a single IS26, and the conjugative plasmid R388 (which confers sulfonamide and trimethoprim resistance [Sur Tpr]) residing together in the E. coli recA mutant background. The Apr Tpr transconjugants carrying cointegrates were detected at an average of 2.9 × 10−6 cointegrates per Tpr transconjugant (Table 1). Digestion of several independent cointegrates with BglII and BsiWI mapped the location of the junctions between the two plasmids and showed that pRMH762 had incorporated randomly throughout the R388 backbone (Fig. 4). The precise boundaries between the two plasmids relative to one another were determined for 10 cointegrates using primers in the R388 backbone adjacent to the predicted cointegrate boundaries with primers internal to pRMH762 (RH1451 and RH1452) to amplify each boundary. Both orientations of the plasmids relative to one another were found. The sequence spanning each IS26 revealed that a copy of IS26 was found in direct orientation at each boundary, and a duplication of 8 bp of R388 had been generated.


Movement of IS26-associated antibiotic resistance genes occurs via a translocatable unit that includes a single IS26 and preferentially inserts adjacent to another IS26.

Harmer CJ, Moran RA, Hall RM - MBio (2014)

Cointegrate formation between pRMH762 and R388. The R388 backbone is drawn to scale from GenBank accession no. BR000038 with key resistance genes, genes involved in replication (repA), and genes involved in conjugative transfer (tra) shown as arrows inside the circular backbone. Arrows pointing toward the circular backbone indicate the location of 20 mapped R388::pRMH762 cointegrates. The precise location and orientation of nine cointegrates were determined by sequencing. An arrow labeled 1 indicates a cointegrate with tnp26 facing clockwise, while an arrow labeled 2 indicates a cointegrate with tnp26 facing counterclockwise. An asterisk denotes the cointegrate used in the construction of R388::IS26.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196232&req=5

fig4: Cointegrate formation between pRMH762 and R388. The R388 backbone is drawn to scale from GenBank accession no. BR000038 with key resistance genes, genes involved in replication (repA), and genes involved in conjugative transfer (tra) shown as arrows inside the circular backbone. Arrows pointing toward the circular backbone indicate the location of 20 mapped R388::pRMH762 cointegrates. The precise location and orientation of nine cointegrates were determined by sequencing. An arrow labeled 1 indicates a cointegrate with tnp26 facing clockwise, while an arrow labeled 2 indicates a cointegrate with tnp26 facing counterclockwise. An asterisk denotes the cointegrate used in the construction of R388::IS26.
Mentions: All experiments were conducted in a recA mutant Escherichia coli background to ensure that the events detected had been catalyzed by the IS26-encoded transposase Tnp26. First, the ability of a single copy of IS26 to form cointegrates as described previously was confirmed using a mating-out assay to detect the cointegrates formed between the plasmid pRMH762 (which confers ampicillin resistance [Apr]), which contains a single IS26, and the conjugative plasmid R388 (which confers sulfonamide and trimethoprim resistance [Sur Tpr]) residing together in the E. coli recA mutant background. The Apr Tpr transconjugants carrying cointegrates were detected at an average of 2.9 × 10−6 cointegrates per Tpr transconjugant (Table 1). Digestion of several independent cointegrates with BglII and BsiWI mapped the location of the junctions between the two plasmids and showed that pRMH762 had incorporated randomly throughout the R388 backbone (Fig. 4). The precise boundaries between the two plasmids relative to one another were determined for 10 cointegrates using primers in the R388 backbone adjacent to the predicted cointegrate boundaries with primers internal to pRMH762 (RH1451 and RH1452) to amplify each boundary. Both orientations of the plasmids relative to one another were found. The sequence spanning each IS26 revealed that a copy of IS26 was found in direct orientation at each boundary, and a duplication of 8 bp of R388 had been generated.

Bottom Line: Intact transposase genes in both IS26s were required for high-frequency cointegrate formation as inactivation of either one reduced the frequency 30-fold.Conversion of each residue in the DDE motif of the Tnp26 transposase also reduced the cointegration frequency.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.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular Bioscience, The University of Sydney, Sydney, Australia.

Show MeSH
Related in: MedlinePlus