INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, OCt. 1990, p. 331-336 0020-7713/9O/O403 3 1-06$02.

00/0
Copyright 8 1990, International Union of Microbiological Societies

Vol. 40, No.4

Taxonomy of Four Marine Bacterial Strains That Produce Tetrodotoxin

US10 SIMIDU,'* KUMIKO KITA-TSUKAMOTO,l TAKESHI YASUMOT0,2 A N D MAR1 YOTSU2 Ocean Research Institute, University of Tokyo, Minamidai 1-15-1, Nakano-ku, Tokyo 164,' and Faculty o f Agriculture, Tohoku University, Tsutsumi-dori, Sendai, Miyagi 980,2Japan
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 1 6 s 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 (Fugupoecilonotus) (19). In this paper we characterize and classify these four strains.
MATERIALS AND METHODS 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 12079Twere used for the analysis of 1 6 s rRNA sequences. 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 Pseudomonas or the genus Alteromonas. The other two

* Corresponding author.
331

strains, strains OK-2 and GFB, appeared to be members of the family Vibrionaceae. Hence, detailed experiments to determine morphological, physiological, and biochemical characteristics were carried out by using primarily the methods of Baumann et al. (2,4) and West and Colwell(15). The major characteristics which we examined are shown in Table 1. A total of 107 tests were performed for the Vibrio strains, and 101 tests were performed for the Pseudornonas and Alteromonas strains. Determination of G+C content. Bacterial DNAs were purified by using the method of Marmur (lo), and guanineplus-cytosine (G+C) contents were determined by using a modification of the high-performance liquid chromatography method of Tamaoka and Komagata (13). Purified DNA was dissolved in 10 mM phosphate buffer (pH 7.0) at a concentration of 1mg/ml of buffer. The solution was heated at 100C for 15 min and then cooled rapidly in an ice bath. Then 10 p1 of the denatured DNA solution was mixed with 10 pl of a nuclease P1 (Yamasa Shoyu Co., Ltd., Chiba, Japan) solution (0.1 mg in 1 ml of 40 mM sodium acetate buffer containing 2 mM ZnSO,, pH 5.3), and the preparation was incubated at 50C for 1 h. The hydrolysate was stored at -25C prior to analysis. Reverse-phase high-performance liquid chromatography was carried out by using a Senshu Pak ODs-1251-N column (Senshu Kagaku, Ltd., Tokyo, Japan) packed with 5-pm octyldecyl silane (ODS) (M. Nagel, City, Federal Republic of Germany). The nucleotides were eluted with 10 mM phosphate buffer (PH 7.0) at a flow rate of 1 mumin at 30C. Nucleotides were detected with a Jasco model Uvidec 100-VI variable-wavelength detector (Japan Spectroscopic Co., Ltd., Tokyo, Japan) at 260 nm. The peak areas were measured with a Jasco model DP-L220LC data processor. A standard mixture of the four deoxyribonucleotides was purchased from Yamasa Shoyu Co., Ltd. Determination of 16s rRNA sequences. Sequencing of 16s rRNA was carried out by using a reverse transcriptase reaction and the method of Lane et al. (9). We used three different oligonucleotide primers that were specific to the 16s rRNA, and about 600 nucleotide bases were determined for each of the strains tested. The details of the sequencing method which we used are described elsewhere (K. KitaTsukamoto, H. Oyaizu, N. Nanba, and U. Simidu, submitted for publication).

332

SIMIDU ET AL.

INT.J. SYST.BACTERIOL.

TABLE 1. Characteristics of four tetrodotoxin-producingstrains and L. pelagia biovar 11"

Characteristic
L . pelagia biovar I1

Strain OK-2

Strain GFB

Strain OK-lT

Strain GFC*

Polar flagellum Lateral flagella when grown on solid media Swarming on solid complex media Polyhydroxybutyrate accumulation Pigmentation Arginine dihydrolase Oxidase Reduction of NO3- to NO2Luminescence Gas produced from: D-Glucose Mannose Production of acetoin and/or diacetyl Na+ required for growth Requirement for organic growth factor Growth at: 4C 35C 40C Production of Amylase Gelatinase Lipase Alginase Chitinase DNase Utilization of D-Xylose D-Arabinose D-Mannose D-Galactose Sucrose Trehalose Cellobiose Melibiose Lactose D-Fructose Maltose D-Ribose L-Rhamnose D-Glucose Salicin Gluconate Glucuronate D-Galacturonate Propionate Valerate Glutarate DL-Malate P-Hydroxybutyrate DL-Lactate Citrate a-Ketoglutarate Pyruvate Succinate Fumarate DL-Glycerate Acetate Butyrate Isobut yrate Caproate Caprate L-Tartrate Aconitate L- Aspartate D-Mannitol D-Sorbitol

+
ND -

+
+

ND ND -

+ + + + -

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + -

+ + + + + +

Continued on following page

VOL.40, 1990

BACTERIA THAT PRODUCE TETRODOTOXIN TABLE 1-Continued

Characteristic

333

L. pelagia biovar I1

Strain OK-2

Strain GFB

Strain OK-lT

Strain GFCT

meso-Inositol Ethanol Erythritol Glycerol Adonitol p-H ydrox ybenzoate L-a- Alanine D-a- Manine L-Leucine L-Glutamate L-Histidine L-Proline L-Tyrosine L-Threonine L-Lysine L- Arginine L-Ornithine L-Phenylalanine N-Acetylglucosamhe Metabolism of Maltose Galactose Lactose D-Mannose D-Cellobiose Sucrose Glucose Trehalose Melibiose D-Ribose L- Arabinose Xylose Rhamnose Mannitol Inositol Glycerol D-Sorbitol Dulcitol Inulin Salicin D-Fructose G+C content (mol%)

ND -

+ -

+ + + d + + + + + ND +
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND
4547

+ + + + + + -

+ + + + + + + -

+
F F F F F F F F F 0 0 F
-

+
F F F F F F F F F F 0 0 F
-

+ + + + + + + + +
0
0

+ + + + + + + + + + + + + + + + + +
0 0 0 0 0 0
0 -

0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 54

0 F 0
-

0 F 46

0 F 0 0 F
45

0 0 0 0 0 0 0 0 0 0 41

' + , Positive; -, negative; ND, not determined; d, variable; F, fermentative; 0, oxidative.

RESULTS AND DISCUSSION Phenotypic characteristics of the tetrodot oxin-producing strains, as well as their G+C contents, are shown in Table 1. Strains OK-2and GFB. Strains OK-2 and GFB exhibited quite similar characteristics despite their different sources. Strain OK-2 was isolated from an alga in Okinawa, Japan, and strain GFB was isolated from a pufferfish in northern Honshu Island, Japan. Of the 108 characteristics compared, only 7 were different (94% matching characteristics). The following characteristics were different: arginine dihydrolase activity, fermentation of melibiose, and utilization of Dribose, glutarate, or-ketoglutarate, L-histidine, and caprate. Both of these organisms hydrolyzed alginic acid, which is one of the rare characteristics found in members of the genera Vibrio and Listonella. Most of the characteristics of both strains were identical to those of Listonella pelagia (Vibrio pelagius) biovar 11. The main differences were requirements for organic growth factors, utilization of cellobiose, propionate, and aconitate, and (for strain GFB) the presence of arginine dihydrolase activity. Tison and Seidler (14) described a new species of the genus Vibrio, Vibrio
aestuarianus, which has arginine dihydrolase activity. Other characteristics of strain GFB were also identical to those of V . aestuarianus, except that strain GFB grew in the presence of 7% NaCl, whereas V . aestuarianus did not. However, the number of phenotypic characteristics reported for V . aestuarianus is too small (13 characteristics, including dependence on NaCl for growth) to draw a conclusion concerning whether this species is the same organism as strain GFB (and also L . pelagia). Although further examination of these organisms by using molecular genetic techniques might be needed for exact identification of strain GFB, the clear similarity between strain GFB and L . pelagia seems to indicate that strains OK-2 and GFB are L. pelagia biovar I1 strains. Strain OK-lT. The G+C content of strain OK-lT was 54 mol%. The high-performance liquid chromatography method for determining G+C contents of DNAs has been well established by a number of workers (6, 7, 13) and gives results similar to those obtained by melting point methods (possible differences are within 2%) (6, 13). Considering the possible 2% differences, the G+C content of strain OK-IT

334

SIMIDU ET AL.

INT. J. SYST.BACTERIOL.
1210 1220 1230 1240 1250 CUUACGACCA GGGCUACACA CGUGCUACAA UGGCGCAUAC AAAGAGAAGC

E. coli

GFC Al.haloplanktis Al.nigrifaciens

OK- 1 S.putrifaciens
Ps.nautica Deleya marina

El
G U GGU G AAG G GCU G UU
1260 1270 1280 1290 1300 GACCUCGCGA GAGCAAGCGG ACCUCAUAAA GUGCGUCGUA GUCCGGAUUG

E . coli

GFC Al.haloplanktis Al.nigrifaciens

OK- 1 S.putrifaciens

IGA 1
UG CGUGG

U A U

Ps.nautica Deleya marina

C A A UACG

CA CA

U C U U C

AC

cuu c c

E.coli GFC Al.haloplanktis Al.nigrifaciens

OK- 1 S.putrifaciens

1310 1320 1330 1340 1350 GAGUCUGCAA CUCGACUCCA UGAAGUCGGA AUCGCUAGUA AUCGUGGAUC

Ps.nautica Deleya marina

C A

E.coli GFC Al.haloplanktis Al.nigrifaciens

OK-1 S.putrifaciens

1360 1370 1380 AGAAUGCCAC GGUGAAUACG

Ps.nautica Deleya marina

U G

FIG. 1. Partial sequences of the 16s rRNAs of the strains tested. The Escherichia coli sequence was obtained from reference 16; only nucleotide bases that were different from those of E . coli are indicated. The boxes indicate where the specific base pattern for the genera Listonella and Shewanella was observed.

could be on the low end of the values reported for the genus Pseudomonas and on the high end of the values reported for the genus Alteromonas (3). The G+C content of Shewanella putrifaciens (Alteromonasputrifaciens)has been reported to be 48 to 56 mol%, although this range is rather broad for a single species. A study of the base sequences of marine bacteria (KitaTsukamoto et al., submitted) showed that each bacterial genus has its own characteristic pattern that is common to species in the genus. The partial 1 6 s rRNA base sequence of strain OK-lT (Fig. 1)clearly shows that the sequence of this strain differs from the sequences of the genus Pseudomonas and the genus Alteromonas. The sequence of strain OK-lT is rather similar to that of S. putrifaciens. A phylogenetic tree constructed by using 600 1 6 s rRNA bases (Fig. 2) confirmed the close relationship between strain OK-lT and S. putrifaciens. However, the number of differences in the base sequences of strain OK-lT and S. putrifaciens is rather high (at least 21 base substitutions in a 600-base sequence). These

dG
Al.haloplanktis Al.nigrifaciens
OK- 1

S.putrifaciens

E. coli
Ps.nautica

D .marina

FIG. 2. Phylogenetic tree for the strains tested, based on a comparison of 600 nucleotide bases of 16s rRNA partial sequences in which three different primers were used. The tree was constructed by using the unweighted pair group (UPG) method.

VOL. 40, 1990

BACTERIA THAT PRODUCE TETRODOTOXIN

335

FIG. 3. Electron micrograph of strain OK-lT.

differences might even be enough to warrant placing the two organisms in separate genera. However, at present, the number of species in the genus Shewanella is limited, and we prefer to place strain OK-lT in this genus. We propose that

a new species, Shewanella alga, should be created for strain OK-lT; a description is given below. Shewanella alga sp. nov. Shewanella alga (al'ga. L. n., alga, alga) cells are gram-negative, straight rods (0.8 by 1.6

FIG. 4. Electron micrograph of strain GFCT.

336

SIMIDU ET AL.

INT. J. SYST. BACTEFUOL.

4. Baumann, P., and R. H. W. Schubert. 1984. Family 11. Vibrionaceae Veron 1965, p. 516-550. In N. R. Krieg and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore. 5. Fuhrman, F. A. 1986. Tetrodotoxin, tarichatoxin, and chiriquitoxin: historical perspectives. Ann. N. Y. Acad. Sci. 479:l-14. 6. Katayama-Fujimura, Y., Y. Komatsu, H. Kuraishi, and K. Kaneko. 1984. Estimation of DNA base composition by high performance liquid chromatography of its nuclease P1 hydrolysate. Agric. Biol. Chem. 48:3169-3172. 7. Katoh, A., T. Suzuki-Onozaki,T. Ohta, H. Ebine, M. Kumagai, M. Fujimoto, and A. Kuninaka. 1983. Microbiological identification of single cell proteins based on DNA-GC contents. Rep. Natl. Food Res. Inst. 43:79-89. 8. Kotaki, Y., Y. Oshima, and T. Yasumoto. 1985. Bacterial transformation of paralytic shellfish toxins in coral reef crabs and a marine snail. Nippon Suisan Gakkaishi 51:1009-1013. 9. Lane, D. J., B. Pace, G. J. Olsen, D. A. Stahl, M. L. Sogin, and N. R. Pace. 1985. Rapid determination of 16s ribosomal RNA sequences for phylogenetic analyses. Proc. Natl. Acad. Sci. USA 82:6955-6959. 10. Marmur, J. 1961. A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J. Mol. Biol. 3:208-218. 11. Maruyama, J., and T. Noguche. 1984. Tetrodotoxin-a review with special reference to its distribution in nature. Mer (Tokyo) 22: 299-304. 12. Simidu, U., T. Noguchi, T. Kou, Y. Shida, and K. Hashimoto. 1987. Marine bacteria that produce tetrodotoxin. Appl. Environ. Microbiol. 53:1714-1715. 13. Tamaoka, J., and K. Komagata. 1984. Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol. Lett. 25:125-128. 14. Tison, D. L., and R. J. Seidler. 1983. Vibrio aestuarianus: a new species from estuarine waters and shellfish. Int. J. Syst. Bacterial. 33:699-702. 15. West, P. A., and R. R. Colwell. 1984. Identification and classification of Vibrionaceae-an overview, p. 285-363. In R. R. Colwell (ed.), Vibrios in the environment. John Wiley & Sons, Inc., New York. 16. Woese, C. R., R. Gutell, R. Gupta, and H. F. Noller. 1983. Detailed analysis of the higher-order structure of 16s-like ribosomal ribonucleic acids. Microbiol. Rev. 47:621-669. 17. Yasumoto, T., H. Nagai, D. Yasumura, T. Michishita, A. Endo, M. Yotsu, and Y. Kotaki. 1986. Interspecies distribution and possible origin of tetrodotoxin. Ann. N.Y. Acad. Sci. 479: 4 4 51. 18. Yasumoto, T., D. Yasumura, M. Yotsu, T. Michishita, A. Endo, and Y. Kotaki. 1986. Bacterial production of tetrodotoxin and anhydrotetrodotoxin. Agric. Biol. Chem. 50:793-795. 19. Yotsu, M., T. Yamazaki, Y. Meguro, A. Endo, M. Murata, H. Naoki, and T. Yasumoto. 1987. Production of tetrodotoxin and its derivatives by Pseudomonas sp. isolated from the skin of a pufferfish. Toxicon 25:225-228.

pm) that are motile by means of single polar flagella (Fig. 3). Halophilic. Grows at 10 to 40C but not at 4C. Produces gelatinase, lipase, and DNase but not amylase, chitinase, or alginase. Utilizes 30 of the 59 organic compounds which we tested. Type strain OK-1 produces tetrodotoxin. The G+C content of the DNA is 54 mol%. Strain GFCT. The G+C content of strain GFCT is 41 mol%. The G+C content and other characteristics of this organism were in accord with the characteristics of the genus Alteromonas. The partial base sequence of strain GFCT clearly showed that this strain is a member of the genus Alteromonas (Fig. 1 and 2). However, strain GFCT is quite distinct from the previously described Alteromonas species. The levels of similarity (simple matching coefficients) between strain GFCT and the previously described Alteromonus species were very low; the highest level of similarity was with Alteromonas vaga (60.9%). Strain GFCT has higher metabolic activity than A . vaga. Unlike A . vaga, it produces gelatinase and lipase. Also, it utilizes many carbohydrates, including cellobiose, melibiose, lactose, and salicin, which A . vaga does not utilize. We propose the name Alteromonas tetraodonis sp. nov. for strain GFCT. A description of the species is given below. Alteromonas tetraodonis sp. nov. Alteromonas tetraodonis (tet'rao.do.nis. M . L. n. tetraodonis, of Tetraodontidae, a family of plectognathic fishes) cells are gram-negative, straight rods (1.0 by 2.4 pm) that are motile by means of single polar flagella (Fig. 4). Halophilic. Grows at 4 to 35"C, but not at 40C. Produces gelatinase, lipase and DNase but not amylase, chitinase, or alginase. Utilizes 54 of the 59 organic compounds which we tested. The type strain, strain GFC, produces tetrodotoxin. The G+C content of the DNA is 41 mol%. Strains OK-lT and GFCT have been deposited in the Culture Collection of the Institute of Applied Microbiology, University of Tokyo, Tokyo, Japan.
LITERATURE CITED Baumann, P., L. Baumann, R. D. Bowditch, and B. Beman. 1984. Taxonomy of Alteromonas: A . nigrifaciens sp. nov., nom. rev.; A. macleodii; and A. haloplanktis. Int. J. Syst. Bacteriol. 34:145-149. Baumann, P., L. Baumann, and M. Mandel. 1971. Taxonomy of marine bacteria: the genus Beneckea. J. Bacteriol. 107:268-294. Baumann, P., M. J. Gauthier, and L. Baumann. 1984. Genus Alteromonas Baumann, Baumann, Mandel and Allen 1972, p. 343-352. In N. R. Krieg and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore.