This Article Full Text (PDF) Other Versions of this Article: AEM.02147-06v1 73/3/777    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Le Roux, F. Articles by Mazel, D. Search for Related Content PubMed PubMed Citation Articles by Le Roux, F. Articles by Mazel, D. Agricola Articles by Le Roux, F. Articles by Mazel, D.

 Previous Article  |  Next Article 

Appl. Environ. Microbiol. doi:10.1128/AEM.02147-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Construction of a Vibrio splendidus Vsm metalloprotease mutant using a novel counter-selectable suicide vector

Unité de plasticité du génome bactérien, Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France; Laboratoire de génétique et pathologie, Ifremer, BP33, 17390 La Tremblade, France

^* To whom correspondence should be addressed. Email: fleroux{at}pasteur.fr,

	Abstract

Vibrio splendidus is a dominant culturable Vibrio in seawater, but strains related to this group are also associated with mortalities in a variety of marine animals. The determinants encoding the pathogenic properties of these strains are still poorly known, however, the recent sequencing of the genome of V. splendidus LGP32, an oyster pathogen, offers the opportunity to decipher the basis of the virulence properties by the disruption of candidate genes. We have developed a novel suicide vector based on the pir-dependant R6K replicative origin, which can be transferred by RP4 based conjugation to, potentially, any Vibrio strain, and which also carries the plasmid F toxin ccdB gene under the control of the P_BAD promoter. We demonstrate that this genetic system allows the efficient counter-selection of integrated plasmids in presence of arabinose, both in V. splendidus and V. cholerae, and as such permits the efficient marker-less allelic replacement in these species. We used this technique to construct several mutants of V. splendidus LGP32, including a derivative deleted of a secreted metalloprotease gene, vsm. We show that this gene is essential for the LGP32 extracellular products toxicity when injected in oysters, but is not necessary for the virulence of bacteria in the model of oyster infection after bacterial injection.

This article has been cited by other articles:

• Lakhal, F., Bury-Mone, S., Nomane, Y., Le Goic, N., Paillard, C., Jacq, A. (2008). DjlA, a Membrane-Anchored DnaJ-Like Protein, Is Required for Cytotoxicity of Clam Pathogen Vibrio tapetis to Hemocytes. Appl. Environ. Microbiol. 74: 5750-5758 [Abstract] [Full Text]  
• Hasegawa, H., Lind, E. J., Boin, M. A., Hase, C. C. (2008). The Extracellular Metalloprotease of Vibrio tubiashii Is a Major Virulence Factor for Pacific Oyster (Crassostrea gigas) Larvae. Appl. Environ. Microbiol. 74: 4101-4110 [Abstract] [Full Text]  
• Picardeau, M. (2008). Conjugative Transfer between Escherichia coli and Leptospira spp. as a New Genetic Tool. Appl. Environ. Microbiol. 74: 319-322 [Abstract] [Full Text]