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Related Experiment Videos

Integron cassette insertion: a recombination process involving a folded single strand substrate.

Marie Bouvier1, Gaëlle Demarre, Didier Mazel

  • 1Unité Postulante Plasticité du Génome Bactérien, CNRS URA 2171, Institut Pasteur, Paris, France.

The EMBO Journal
|December 13, 2005
PubMed
Summary

Integron integrases facilitate antibiotic resistance gene spread. A novel model shows the attC site folds into a double-stranded DNA for efficient cassette insertion by integrase IntI1.

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Area of Science:

  • Molecular Biology
  • Microbiology
  • Genetics

Background:

  • Integrons are key genetic elements driving antibiotic resistance gene dissemination in Gram-negative bacteria.
  • Integron integrases (IntI) catalyze the insertion of gene cassettes, containing attC sites, into specific platform sites (attI).

Purpose of the Study:

  • To elucidate the in vivo mechanism of integron gene cassette insertion.
  • To investigate the role of attC strand orientation and structure in the recombination process mediated by IntI1.

Main Methods:

  • Utilized a plasmid conjugation-based recombination assay to deliver single-stranded attC and attI substrates.
  • Analyzed recombination efficiency with varying single-stranded attC and attI orientations.
  • Investigated the impact of mutations in the putative attC stem-loop structure.

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Main Results:

  • Recombination efficiency was strand-specific, with one attC strand showing 1000-fold higher recombination than the other.
  • Recombination was highly unfavorable when the attI site was transferred as a single strand.
  • Mutations in the attC stem-loop disrupted the insertion process.

Conclusions:

  • A novel model for integron cassette insertion is proposed, where the single-stranded attC bottom strand folds into a double-stranded structure.
  • This folded attC site acts as the recombination substrate for integrase IntI1.
  • Cassette insertion occurs via a single-stranded intermediate requiring subsequent processing.