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
