Yeast extract (0.025%) and nalidixic acid (0.002%) were added to seawater samples and the samples were incubated for 6 h at 20 degrees C in the dark. Under these conditions, bacterial cells did not divide but grew to form elongated cells that are easily recognized by a direct microscopic method and epifluorescent microscopic technique. The number of cells thus obtained is proposed as a direct cound of viable bacterial cells (DVC). With open ocean samples, DVC was higher than 'viable' plate counts by up to three orders of magnitude and lower than the direct counts by about one order.

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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...

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Sixteen strains of aerobic bacteria which contain bacteriochlorophyll a were isolated from the samples collected in aerobic marine environments: thalli of Enteromorpha linza, Porphyra sp., Sargussum horneri; beach sand; and the surface seawater from Aburatsubo Inlet. When they occurred, their proportions among the aerobic heterotrophic populations ranged from 0.9 to 1.1% in the seaweed samples and from 1.2 to 6.3% in the beach sand samples and were 0.9% in the seawater sample. The results suggested that the aerobic photopigmented bacteria widely inhabit aerobic marine environments.