We performed the first broad study aiming at the reconstruction of the evolutionary history of vibrios by means of multilocus sequence analysis of nine genes. Overall, 14 distinct clades were recognized using the SplitsTree decomposition method. Some of these clades may correspond to families, e.g., the clades Salinivibrio and Photobacteria, while other clades, e.g., Splendidus and Harveyi, correspond to genera. The common ancestor of all vibrios was estimated to have been present 600 million years ago. We can define species of vibrios as groups of strains that share >95% gene sequence similarity and >99.4% amino acid identity based on the eight protein-coding housekeeping genes. The gene sequence data were used to refine the standard online electronic taxonomic scheme for vibrios (http://www.taxvibrio.lncc.br).Vibrios are widespread in the aquatic environment, occupying a variety of ecological niches, such as the human and animal gut, the surface of chitinous organisms, most notably copepods, and the coral mucus layer. A better understanding of the ecology and the patterns of distribution of vibrios relies on the online electronic taxonomy. Polyphasic taxonomic studies of vibrios performed in recent years have underpinned this new paradigm in studies of the biodiversity and systematics of this group (16,17,19). Currently, we recognize 78 species of vibrios distributed into five phylogenetic robust clades corresponding to the genera Vibrio, Photobacterium, Salinivibrio, Enterovibrio, and Grimontia based on 16S rRNA gene sequences (16,17,19). Both genome content and architecture indicate that these genera share a common ancestor (12). In addition, the genera within vibrios are defined on the basis of their shared sequence similarities in different loci. Species within the genus Vibrio share at least 85% gene sequence similarity in recA, rpoA, and pyrH (18).Species of vibrios are defined as clusters of strains with high phenotypic and genotypic similarities. Clusters comprise strains with highly similar genomes as determined by multilocus sequence analysis (MLSA), amplified fragment length polymorphism analysis, and DNA-DNA hybridization (DDH) (16,17,19). Formal delineation of bacterial species still relies on DDH, with a cutoff level of Ͼ70% DDH similarity, but this technique is time-consuming and can be performed in relatively few laboratories and, more importantly, the DDH data are not cumulative in online databases. Clearly, a reliable and straightforward alternative is the use of MLSA. The usefulness of MLSA in the taxonomy of vibrios was described in previous papers (e.g., references 15 and 18). Overall, species form discrete clusters on the basis of recA, rpoA, and pyrH, with a species cutoff level of Ͼ94% gene sequence similarity (18). However, some groups of species, e.g., the Vibrio splendidus and Vibrio harveyi species groups, were somewhat fuzzy on the basis of recA, gyrB, and gapA (15, 18). Thus, it is very important to evaluate additional genetic markers that can distinguish closely related species o...

The prevalence of the two-chromosome configuration was investigated in 34 species of vibrios and closely related species. Pulsed-field gel electrophoresis of undigested genomic DNA suggested that vibrios commonly have two chromosomes. The size of the large chromosome is predominantly within a narrow range (3.0 to 3.3 Mb), whereas the size of the small chromosome varies considerably among the vibrios (0.8 to 2.4 Mb). This fact suggests that the structure of the small chromosome is more flexible than that of the large chromosome during the evolution of vibrios.Vibrios, which are gram-negative halophilic bacteria that include more than 60 species, comprise the major culturable bacteria in marine and estuarine environments (10, 40). Although many marine bacteria are known to be difficult to grow in artificial media containing rich nutrients, most vibrios grow well on ordinary peptone-containing media. Vibrios have high degradative ability (e.g., chitin digestion and production of various extracellular proteases) (1, 28). Several Vibrio species are devastating pathogens for fish, shellfish, coral, and mammals (10,30,40). The best documented example is Vibrio cholerae, the etiological agent of cholera. When exposed to certain conditions, some vibrios become nonculturable without losing their respiratory activity. This phenomenon is called the "viable but nonculturable" state (5, 9). Nonpathogenic vibrios are also known. For example, V. mediterranei plays a role as the first colonizer of the gut of turbot larvae and may prevent the colonization of the gut by opportunistic bacteria (17). V. fischeri plays an important role in the development of the light organ of juvenile squid of the species Euprymna scolopes (44). V. natriegens, which produces nitrogenase, plays an important role in providing fixed nitrogen de novo to marine ecosystems (6). The optimal temperatures for growth of V. salmonicida, V. logei, and V. wodanis are as low as those for the growth of psychrophilic bacteria (12,26). Thus, vibrios with various features and lifestyles exist.Previously, we reported that some species of the genus Vibrio, including V. parahaemolyticus, V. cholerae, V. anguillarum, V. fluvialis, and V. vulnificus, which cause illness in humans, fish, and other animals, possess two chromosomes (39, 46). Trucksis et al. also reported that V. cholerae and V. mimicus possess two chromosomes (42). Recently, the wholegenome sequences of V. cholerae, V. parahaemolyticus, and V. vulnificus were published; these studies confirmed that their genome structures consist of two circular chromosomes (2,16,24). In this study, we extensively investigated the genome structure of vibrios, including pathogenic and nonpathogenic strains, and of species closely related to the genus Vibrio to evaluate the prevalence of the two-chromosome structure among these bacteria.The strains we examined are listed in Table 1. The vibrios were grown in Luria-Bertani broth (34) or Difco marine broth 2216. Bacteria of the genera Plesiomonas and Aeromonas were grown in tryp...

Four strains of tetrodotoxin-producing bacteria isolated from a red alga and from pufferfish were characterized. Two of these strains are members of the genus Listonella MacDonell and Colwell. The phenotypic characteristics, guanine-plus-cytosine contents, and base sequences of the 16s rRNAs of these organisms indicated that they are members of ListoneUa pelagia (Vibrio pelagius) biovar II. The other two strains are members of the genus Alteromonas Baumann et al. and the genus Shewanella MacDonell and ColweU. These two strains are mutually distinct and distinct from the previously described Alteromonas and Shewanella species and therefore are placed in new species. The names Shewanella alga and Alteromonas tetraodonis are proposed for these organisms; the type strains are strains OK-1 and GFC, respectively.Tetrodotoxin, which is one of the strongest neurotoxins known (9, has been isolated from various species of animals, mostly animals which live in marine habitats (11,17). Although these animals themselves have long been considered the producers of tetrodotoxin, the recent isolation of tetrodotoxin-producing marine bacteria from various sources (12,17,18) suggests that the toxin is in fact produced by bacteria that are associated with the animals and their food. A presumptive Pseudomonas strain that was isolated from a red clacareous alga (Jania sp.) was reported to produce tetrodotoxin by Yasumoto et al. (18). Yasumoto et al. also reported the isolation of three other toxin-producing strains, one from the red alga and the other two from the surface slime of a pufferfish (Fugu poecilonotus) (19). In this paper we characterize and classify these four strains. Kotaki et al. (8) isolated strains OK-lT (T = type strain) and OK-2 from the surface of a red alga (Jania sp.) by using a medium containing 0.5% peptone (Nissui Seiyaku, Ltd.), 0.25% yeast extract (Nissui Seiyaku, Ltd.), 0.1% glucose, 3.0% NaC1, and 1.5% agar. The other two strains (strains GFB and GFCT) were isolated by Yasumoto and Yotsu from the skin slime of a pufferfish, using a similar medium (19). Along with strains OK-lT and GFCT, Alteromonas haloplanktis ATCC 14393T, Alteromonas nigrificans IAM 13010T, Pseudornonas nautica IAM 12929T, Deleya marina IAM 12928=, and Shewanella putrefaciens IAM 12079T were used for the analysis of 16s rRNA sequences. MATERIALS AND METHODS Strains.The strains were maintained in a semisolid agar medium (ORI medium) containing 0.1% Proteose Peptone no. 3 (Difco Laboratories, Detroit, Mich.), 0.1% yeast extract (Difco), 0.05% Phytone (BBL Microbiology Systems, Cockeysville, Md.), 0.02% sodium thiosulfate, 0.005% sodium sulfite, 0.04% ferric citrate, and 0.3% agar (Difco) in a mixture of aged seawater and distilled water (3: 1). The pH of the medium was adjusted to pH 7.6. Phenotypic characterization. A preliminary morphological and biochemical survey showed that two of the strains, strains OK-lT and GFCT, might belong to either the genus

The phylogenetic relationships of 50 reference strains, mostly marine bacteria which require Na+ for growth, were determined on the basis of 600 16s rRNA nucleotides by using reverse transcriptase sequencing. Strains belonging to 10 genera were included (four genera of the family Vibrionaceae, the genus Aeromonas of the family Aeromonaduceae, and the genera Alteromonas, Marinomonas, Shewanella, Pseudomonas, and Deleya). The sequences were aligned, the similarity values and evolutionary distance values were determined, and a phylogenetic tree was constructed by using the neighbor-joining method. On the basis of our results, the family Vibrionaceae was separated into at least seven groups (genera and families). Kbrio murinus clearly was on a line of descent that was remote from other vibrios. As determined by the similarity and evolutionary distance values, V. marinus is more distantly related to the family Vibrionaceae than the members of the Aeromonadaceae are. Also, Vibrio cholerae strains formed a separate group with Vibrio mimicus at the genus level. Of 30 species of the Vibrionaceae, 17 formed a large phylogenetic cluster. The genus Listonella was found to be a heterogeneous group, and the species were distributed in various subgroups of the Vibrionaceae. The separation of the family Aeromonadaceae from the family Vibrionaceae and the separation of the genera Marinomonas and Shewanella from the genus Alteromonas were confirmed in this phylogenetic study. However, a marine Pseudomonas species, Pseudomonas rtuutica, was clearly separated from two terrestrial Pseudomonas species. Each group that was separated by the phylogenetic analysis had characteristic 16s rRNA sequence patterns that were common only to species in that group. Therefore, the characteristic sequences described in this paper may be useful for identification purposes.Heterotrophic bacteria which are gram negative and motile by means of flagella are commonly isolated from marine environments and apparently are a major component of the bacterial flora of the sea. On the basis of their ability to ferment carbohydrates, these organisms can be divided into two groups. The fermentative strains have been assigned to the genera Vibrio, Listonella, Photobacterium, Colwellia (lo), and Aeromonas, and the nonfermentative strains have been included in the genera Alteromonas, Pseudomonas, Alcaligenes, Deleya, Marinomonas, Shewanella , and Flavobacterium (1).The family Hbrionaceae is one of the most important bacterial groups in marine environments. Members of this family often predominate in the bacterial flora of seawater, plankton, and fish. In a survey carried out in the West Pacific Ocean, vibrios accounted for nearly 80% of the bacterial population in surface seawater (31). Members of the Vibrionaceae are also the main organisms present in the intestinal flora of marine fish (30). In addition, some members of the Vibrionaceae are important pathogens for humans and animals.The marine members of the Kbrionaceae have been the subject of many taxonomic stud...

On the basis of the 16S rRNA sequence data analysis among the closely related species, the specific primers for Cytophaga psychrophila were constructed.The specificity in amplifying the 16S rRNA of C.psychrophila was confirmed by using some selected strains of the related species. In addition, it was revealed that these specific primers distinguished C. psychrophila from other principal fish pathogens.The present PCR technique is expected to be a powerful tool for the diagnosis of cold-water disease.