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Allelic replacement in Staphylococcus aureus with inducible counter-selection.

Authors
  • Bae, Taeok
  • Schneewind, Olaf
Type
Published Article
Journal
Plasmid
Publication Date
Jan 01, 2006
Volume
55
Issue
1
Pages
58–63
Identifiers
PMID: 16051359
Source
Medline
License
Unknown

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.

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