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 PBAD 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.