Vibrio tubiashii is a recently reemerging pathogen of larval bivalve mollusks, causing both toxigenic and invasive disease. Marine Vibrio spp. produce an array of extracellular products as potential pathogenicity factors. Culture supernatants of V. tubiashii have been shown to be toxic to oyster larvae and were reported to contain a metalloprotease and a cytolysin/hemolysin. However, the structural genes responsible for these proteins have yet to be identified, and it is uncertain which extracellular products play a role in pathogenicity. We investigated the effects of the metalloprotease and hemolysin secreted by V. tubiashii on its ability to kill Pacific oyster (Crassostrea gigas) larvae. While V. tubiashii supernatants treated with metalloprotease inhibitors severely reduced the toxicity to oyster larvae, inhibition of the hemolytic activity did not affect larval toxicity. We identified structural genes of V. tubiashii encoding a metalloprotease (vtpA) and a hemolysin (vthA). Sequence analyses revealed that VtpA shared high homology with metalloproteases from a variety of Vibrio species, while VthA showed high homology only to the cytolysin/hemolysin of Vibrio vulnificus. Compared to the wild-type strain, a VtpA mutant of V. tubiashii not only produced reduced amounts of protease but also showed decreased toxicity to C. gigas larvae. Vibrio cholerae strains carrying the vtpA or vthA gene successfully secreted the heterologous protein. Culture supernatants of V. cholerae carrying vtpA but not vthA were highly toxic to Pacific oyster larvae. Together, these results suggest that the V. tubiashii extracellular metalloprotease is important in its pathogenicity to C. gigas larvae.Vibriosis caused by marine Vibrio species is considered one of the most serious diseases of hatchery-reared oyster larvae (10,11,17,47,52). The disease is characterized by a rapid and dramatic reduction in larval motility, detached vela, and necrotic soft tissue, which lead to high mortality rates, exceeding 90% within 1 day of infection (45). Pathogenic agents that cause larval bivalve vibriosis have intermittently and severely curtailed shellfish hatchery production on the Atlantic and Pacific coasts of the United States, causing substantial losses in the industry (3, 10, 13). Vibrio tubiashii, a bacterial species first reported by Tubiash et al. (51), was identified as a causative agent of vibriosis (originally referred to as bacillary necrosis) in larval and juvenile bivalves of the hard clam (Mercenaria mercenaria) and Eastern oyster (Crassostrea virginica). Estes et al. (14) characterized a number of pathogenic and nonpathogenic bacterial strains from diseased Pacific oysters (Crassostrea gigas) at shellfish hatcheries on the Pacific coast of North America and described some of the highly pathogenic bacterial isolates as V. tubiashii.The genus Vibrio is the largest member of the family Vibrionaceae, which includes gram-negative and curved rod-shaped facultative anaerobes. The genus consists of at least 30 known species, which are w...

Vibrio cholerae has three sets of chemotaxis (Che) proteins, including three histidine kinases (CheA) and four response regulators (CheY) that are encoded by three che gene clusters. We deleted the cheY genes individually or in combination and found that only the cheY3 deletion impaired chemotaxis, reinforcing the previous conclusion that che cluster II is involved in chemotaxis. However, this does not exclude the involvement of the other clusters in chemotaxis. In other bacteria, phospho-CheY binds directly to the flagellar motor to modulate its rotation, and CheY overexpression, even without CheA, causes extremely biased swimming behavior. We reasoned that a V. cholerae CheY homolog, if it directly controls flagellar rotation, should also induce extreme swimming behavior when overproduced. This was the case for CheY3 (che cluster II). However, no other CheY homolog, including the putative CheY (CheY0) protein encoded outside the che clusters, affected swimming, demonstrating that these CheY homologs cannot act directly on the flagellar motor. CheY4 very slightly enhanced the spreading of an Escherichia coli cheZ mutant in semisolid agar, raising the possibility that it can affect chemotaxis by removing a phosphoryl group from CheY3. We also found that V. cholerae CheY3 and E. coli CheY are only partially exchangeable. Mutagenic analyses suggested that this may come from coevolution of the interacting pair of proteins, CheY and the motor protein FliM. Taken together, it is likely that the principal roles of che clusters I and III as well as cheY0 are to control functions other than chemotaxis.

The ability of motile bacteria to swim toward or away from specific environmental stimuli, such as nutrients, oxygen, or light provides cells with a survival advantage, especially under nutrient-limiting conditions. This behavior, called chemotaxis, is mediated by the bacteria changing direction by briefly reversing the direction of rotation of the flagellar motors. A sophisticated signal transduction system, consisting of signal transducer proteins, a histidine kinase, a response regulator, a coupling protein, and enzymes that mediate sensory adaptation, relates the input signal to the flagellar motor. Chemotaxis has been extensively studied in bacteria such as Escherichia coli and Salmonella enterica serovar Typhimurium, and depends on the activity of single copies of proteins in a linear pathway. However, growing evidence suggests that chemotaxis in other bacteria is more complex with many bacterial species having multiple paralogues of the various chemotaxis genes found in E. coli and, in most cases, the detailed functions of these potentially redundant genes have not been elucidated. Although the completed genome of Vibrio cholerae, the causative agent of cholera, predicted a multitude of genes with homology to known chemotaxis-related genes, little is known about their relative contribution to chemotaxis or other cellular functions. Furthermore, the role of chemotaxis during the environmental or infectious phases of this organism is not yet fully understood. This review will focus on the complex relationship between chemotaxis and virulence in V. cholerae.

The mrp operon from Vibrio cholerae encoding a putative multisubunit Na⁺/H⁺ antiporter was cloned and functionally expressed in the antiporter-deficient strain of Escherichia coli EP432. Cells of EP432 expressing Vc-Mrp exhibited resistance to Na⁺ and Li⁺ as well as to natural bile salts such as sodium cholate and taurocholate. When assayed in everted membrane vesicles of the E. coli EP432 host, Vc-Mrp had sufficiently high antiport activity to facilitate the first extensive analysis of Mrp system from a Gram-negative bacterium encoded by a group 2 mrp operon. Vc-Mrp was found to exchange protons for Li⁺, Na⁺, and K⁺ ions in pH-dependent manner with maximal activity at pH 9.0–9.5. Exchange was electrogenic (more than one H⁺ translocated per cation moved in opposite direction). The apparent K_m at pH 9.0 was 1.08, 1.30, and 68.5 mM for Li⁺, Na⁺, and K⁺, respectively. Kinetic analyses suggested that Vc-Mrp operates in a binding exchange mode with all cations and protons competing for binding to the antiporter. The robust ion antiport activity of Vc-Mrp in sub-bacterial vesicles and its effect on bile resistance of the heterologous host make Vc-Mrp an attractive experimental model for the further studies of biochemistry and physiology of Mrp systems.

The ability to move toward favorable environmental conditions, called chemotaxis, is common among motile bacteria. In particular, aerotaxis has been extensively studied in Escherichia coli and was shown to be dependent on the aer and tsr genes. Three putative aer gene homologs were identified in the Vibrio cholerae genome, designated aer-1 (VC0512), aer-2 (VCA0658), and aer-3 (VCA0988). Deletion analyses indicated that only one of them, aer-2, actively mediates an aerotaxis response, as assayed in succinate soft agar plates as well as a capillary assay. Complementation studies confirmed that Aer-2 is involved in aerotaxis in V. cholerae. In addition, overexpression of aer-2 resulted in a marked increase of the aerotactic response in soft agar plates. No observable phenotypes in V. cholerae mutants deleted in the aer-1 or aer-3 genes were detected under standard aerotaxis testing conditions. Furthermore, the V. cholerae aer-1 and aer-3 genes, even when expressed from a strong independent promoter, did not produce any observable phenotypes. As found in other bacterial species, the results presented in this study indicate the presence of a secondary aerotaxis transducer in V. cholerae.