Allelic replacement in Staphylococcus aureus with inducible counter-selection

Taeok Bae, Olaf Schneewind

Research output: Contribution to journalArticle

374 Scopus citations

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°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.

Original languageEnglish (US)
Pages (from-to)58-63
Number of pages6
JournalPlasmid
Volume55
Issue number1
DOIs
StatePublished - Jan 1 2006
Externally publishedYes

Keywords

  • Allelic replacement
  • Antisense RNA
  • Phage lambda recombination
  • Staphylococcus aureus
  • rocA
  • secY

ASJC Scopus subject areas

  • Molecular Biology

Fingerprint Dive into the research topics of 'Allelic replacement in Staphylococcus aureus with inducible counter-selection'. Together they form a unique fingerprint.

  • Cite this