Elsevier

Plasmid

Volume 55, Issue 1, January 2006, Pages 58-63
Plasmid

Short communication
Allelic replacement in Staphylococcus aureus with inducible counter-selection

https://doi.org/10.1016/j.plasmid.2005.05.005Get rights and content

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.

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Acknowledgments

We thank Melanie Marketon and Angelika Gründling for suggestions during the course of these studies and members of our laboratory for critical comments on the manuscript. Work in the laboratory of Olaf Schneewind is supported by United States Public Health Awards from the National Institutes of Allergy and Infectious Diseases, Infectious Diseases Branch AI38897 and AI52474.

References (23)

  • P. Gay et al.

    Cloning structural gene sacB, which codes for exoenzyme levansucrase of Bacillus subtilis: expression of the gene in Escherichia coli

    J. Bacteriol.

    (1983)

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