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Applied and Environmental Microbiology, November 2004, p. 6887-6891, Vol. 70, No. 11
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.11.6887-6891.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

New Vector for Efficient Allelic Replacement in Naturally Nontransformable, Low-GC-Content, Gram-Positive Bacteria{dagger}

Maryvonne Arnaud, Arnaud Chastanet, and Michel Débarbouillé*

Unité de Biologie des Bactéries Pathogènes à Gram Positif, Institut Pasteur, Centre National de la Recherche Scientifique, Paris, France

Received 9 April 2004/ Accepted 28 June 2004

ABSTRACT

A shuttle vector designated pMAD was constructed for quickly generating gene inactivation mutants in naturally nontransformable gram-positive bacteria. This vector allows, on X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) plates, a quick colorimetric blue-white discrimination of bacteria which have lost the plasmid, greatly facilitating clone identification during mutagenesis. The plasmid was used in Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus to efficiently construct mutants with or without an associated antibiotic resistance gene.

* Corresponding author. Mailing address: Unité de Biologie des Bactéries Pathogènes à Gram Positif, Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France. Phone: (33 1) 44 38 94 87. Fax: (33 1) 45 68 89 38. E-mail: mdebarbo{at}pasteur.fr.

FOOTNOTES

{dagger} This paper is dedicated to the memory of Maryvonne Arnaud, who died on 1 February 2004.

Applied and Environmental Microbiology, November 2004, p. 6887-6891, Vol. 70, No. 11
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.11.6887-6891.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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