Construction of a Vibrio splendidus mutant lacking the metalloprotease gene vsm by use of a novel counterselectable suicide vector.
Vibrio splendidus is a dominant culturable Vibrio in seawater, and strains related to this species are also associated with mortality in a variety of marine animals. The determinants encoding the pathogenic properties of these strains are still poorly understood; however, the recent sequencing of the genome of V. splendidus LGP32, an oyster pathogen, provides an opportunity to decipher the basis of the virulence properties by disruption of candidate genes. We developed a novel suicide vector based on the pir-dependent R6K replicative origin, which potentially can be transferred by RP4-based conjugation to any Vibrio strain and which also carries the plasmid F toxin ccdB gene under control of the PBAD promoter. We demonstrated that this genetic system allows efficient counterselection of integrated plasmids in the presence of arabinose in both V. splendidus and Vibrio cholerae and thus permits efficient markerless allelic replacement in these species. We used this technique to construct several mutants of V. splendidus LGP32, including a derivative with a secreted metalloprotease gene, vsm, deleted. We found that this gene is essential for LGP32 extracellular product toxicity when the extracellular products are injected into oysters but is not necessary for virulence of bacteria in the oyster infection model when bacteria are injected.
Le Roux
Frédérique
F
Unité Postulante Plasticité du Génome Bactérien, CNRS URA 2171, Institut Pasteur, 25 rue du Dr Roux, 75015, Paris, France. fleroux@pasteur.fr
Binesse
Johan
J
Saulnier
Denis
D
Mazel
Didier
D
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001427:Bacterial Toxins / Q000235:genetics*; D003227:Conjugation, Genetic; D017353:Gene Deletion; D005821:Genetic Techniques; D005822:Genetic Vectors; D045726:Metalloproteases / Q000235:genetics*; D008969:Molecular Sequence Data; D010125:Ostreidae / Q000382:microbiology; D010957:Plasmids / Q000235:genetics; D017422:Sequence Analysis, DNA; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity
D001426:Bacterial Proteins; D001427:Bacterial Toxins; C068356:CcdB protein, Plasmid F; D045726:Metalloproteases