Fast and efficient three-step target-specific curing of a virulence plasmid in Salmonella enterica

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Abstract

Virulence plasmids borne by serovars of Salmonella enterica carry genes involved in its pathogenicity, as well as other functions. Characterization of phenotypes associated with virulence plasmids requires a system for efficiently curing strains of their virulence plasmids. Here, we developed a 3-step protocol for targeted curing of virulence plasmids. The protocol involves insertion of an I-SecI restriction site linked to an antibiotic resistance gene into the target plasmid using λ-Red mutagenesis, followed by the transformation with a temperature-sensitive auxiliary plasmid which carries I-SecI nuclease expressed from a tetracycline-inducible promoter. Finally, the auxiliary plasmid is removed by incubation at 42 °C and the plasmid-less strains are verified on antibiotic-containing media. This method is fast and very efficient: over 90 % of recovered colonies lacked their virulence plasmid.

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de Moraes, M. H., & Teplitski, M. (2015). Fast and efficient three-step target-specific curing of a virulence plasmid in Salmonella enterica. AMB Express, 5(1). https://doi.org/10.1186/s13568-015-0139-y

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