A Set of Powerful Negative Selection Systems for Unmodified Enterobacteriaceae

Nucleic Acids Res. 2015 Jul 27;43(13):e83. doi: 10.1093/nar/gkv248. Epub 2015 Mar 23.

Abstract

Creation of defined genetic mutations is a powerful method for dissecting mechanisms of bacterial disease; however, many genetic tools are only developed for laboratory strains. We have designed a modular and general negative selection strategy based on inducible toxins that provides high selection stringency in clinical Escherichia coli and Salmonella isolates. No strain- or species-specific optimization is needed, yet this system achieves better selection stringency than all previously reported negative selection systems usable in unmodified E. coli strains. The high stringency enables use of negative instead of positive selection in phage-mediated generalized transduction and also allows transfer of alleles between arbitrary strains of E. coli without requiring phage. The modular design should also allow further extension to other bacteria. This negative selection system thus overcomes disadvantages of existing systems, enabling definitive genetic experiments in both lab and clinical isolates of E. coli and other Enterobacteriaceae.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Bacterial Toxins / biosynthesis
  • Bacterial Toxins / genetics
  • Cell Engineering
  • Chromosomes, Bacterial
  • Cloning, Molecular / methods
  • Enterobacteriaceae / genetics*
  • Escherichia coli / genetics*
  • Escherichia coli / isolation & purification
  • Genetic Loci
  • Humans
  • Mutation*
  • Salmonella enterica / genetics
  • Salmonella enterica / isolation & purification
  • Transduction, Genetic

Substances

  • Bacterial Toxins