Allelic Replacement in Staphylococcus Aureus With Inducible Counter-Selection

Plasmid. 2006 Jan;55(1):58-63. doi: 10.1016/j.plasmid.2005.05.005. Epub 2005 Jul 26.

Abstract

A method for rapid selection of allelic replacement mutations in the chromosome of Staphylococcus aureus is described. Plasmid pKOR1, an Escherichia coli/S. aureus shuttle vector, permits rapid cloning via lambda recombination and ccdB selection. Plasmid transformation of staphylococci and growth at 43 degrees C, a non-permissive condition for pKOR1 replication, selects for homologous recombination and pKOR1 integration into the bacterial chromosome. Anhydrotetracycline-mediated induction of pKOR1-encoded secY antisense transcripts via the Pxyl/tetO promoter, a condition that is not compatible with staphylococcal growth, selects for chromosomal excision and loss of plasmid. Using this strategy, allelic replacements in S. aureus rocA were generated at frequencies that obviated the need for antibiotic marker selection.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • 1-Pyrroline-5-Carboxylate Dehydrogenase / genetics*
  • 1-Pyrroline-5-Carboxylate Dehydrogenase / metabolism
  • Alleles*
  • Bacteriophage lambda / genetics
  • Chromosomes, Bacterial / genetics*
  • Cloning, Molecular
  • DNA, Bacterial
  • DNA, Recombinant
  • Escherichia coli / genetics
  • Genes, Bacterial
  • Genetic Vectors / genetics*
  • Humans
  • Mutation / genetics*
  • Plasmids / genetics*
  • RNA, Antisense / genetics
  • RNA, Antisense / metabolism
  • Recombination, Genetic
  • Staphylococcus aureus / genetics*
  • Tetracycline Resistance
  • Tetracyclines / pharmacology
  • Transformation, Genetic

Substances

  • DNA, Bacterial
  • DNA, Recombinant
  • RNA, Antisense
  • Tetracyclines
  • 4-epianhydrotetracycline
  • 1-Pyrroline-5-Carboxylate Dehydrogenase