Production of Tetrodotoxin by the Intestinal Bacteria of a Puffer Fish Takifugu Niphobles*1

Nippon Suisan Gakkaishi 55(12), 2199-2203 (1989)

Production of Tetrodotoxin by the Intestinal Bacteria of a Puffer Fish Takifugu niphobles*1

Takashi Matsui,*2 Shuichi Taketsugu,*2 Kentaro Kodama, *3 Akira Ishii,*3 Kunio Yamamori,*4 and Chiaki Shimizu5(Received August 9, 1989)

We surveyed tetrodotoxin (TTX) production on 15 bactria strains isolated from the intestine of a puffer fish caught at Hakatajima, Ehime Prefecture, where the puffer fish is highly toxic. The mouse bioassay revealed that all culture broths were none toxic or very weakly toxic with the ex ception of one strain (code-named SA-4). The SA-4 strain was confirmed for its toxicity by the mouse bioassay, and mice died after showing the characteristic symptoms for TTX intoxication. The amount of toxin was 15 MU in 250ml of culture broth. After bioassays, all the remained solu tions were purified through a Toyopearl HW-40 column, an Amberlite CG-50 column (NH+ form), and a charcoal column. Chromatography of purified toxins demonstrated that 2 strains in addition to SA-4 strain contained TTX and its derivatives in their broths. The cells of SA-4 strain were proved to be lethal to mice, and the existence of TTX was confirmed. The purified toxins and authentic TTX were hydrolyzed to 2-amino-6-hydroxyl-methyl-8-hydroxyquinazoline (C9-base), and converted into its TMS-C9-bases to be analyzed by a GC-MS analyzer. Mass fragment ions m/z 407 and 392, which were specific for TMS-C9-base, appeared in the same retention time. The results from detail examinations done on SA-4 strain led us to the conclusion that SA-4 strain was Schewanella putrefaciens.

Despite high toxicity, puffer fish is one of the amounts of TTX and its derivatives in many biota. most valuable fish in Japan on account of its good In fact, Yasumoto et al.6) and Noguchi et al.7) taste. Puffer fish toxin, tetrodotoxin (TTX) has independently confirmed by using the TTX ana a unique chemical structure, shows the specific lyzer that Alteromonas sp. and Vibrio sp. produced action of blocking sodium channels of excitable TTX and anhydTTX, respectively. The situation membranes, and has extensively been studied led us to survey TTX production in bacteria isolat mainly by Japanese researchers. ed from the intestine of puffer fish Takifugu ni Nevertheless, its biogenesis remains obscure. phobles caught at Hakatajima, Ehime Prefecture, We first provided experimental evidence to sug where the puffer fish has highest toxicity as far as gest its exogenous origin in puffer fish, demonstrat we examined in Japan.3) We confirmed that ing the absence of the toxin in cultured puffer fish Shewanella putrefaciens definitely produced TTX and showing the remarkable regional as well as and anhydTTX and that another 2 strains pro individual variation of toxicity.1-3) On the other duced the toxins in small amounts. The present hand, Yasumoto et al. reported the presence of paper deals with these results. anhydrotetrodotoxin (anhydTTX), 4-epi-tetrodotoxin (4-epiTTX) and tetrodonic acid in addition Materials and Methods to TTX in two species of puffer fish4) and con structed a fluorometric HPLC analyzer (TTX Fish Specimen analyzer) for these compounds.5) The TTX Puffer fish Takifugu niphobles (female; body analyzer made possible the detection of small weight; 55g, toxicity; liver 5, 960 and intestine

*1 Studies on the toxification of puffer fish-I.*2 Laboratory of Marine Biochemistry, Faculty of Agriculture, The University of Tokyo, Bunkyo, To

kyo 113, Japan(松居隆,武次周一:東京大学農学部水産化学研究室). *3 Fermentation Research Laboratories, Sankyo Co., Shinagawa, Tokyo140, Japan(小玉健太郎,石井

晃:三共株式会社〓酵研究所). *4 School of Fisheries Science, Kitasato University, Sanriku, Iwate O22-O1, Japan(山森邦夫:北里大学水産学部). *5 Daishin Marine Products Co., Ltd., Showa, Sakaiminato, Tottori684, Japan(清水千秋:株式会社大伸水産). 2200 Matsui, Taketsugu, Kodama, Ishii, Yamamori, and Shimizu

Table 1. Composition of the medium for isolation purification was performed through a Toyopearl HW-40 column (2 •~ 100 cm; solvent 0.05N acetic Glucose 5 g Polypeptone (Daigo) 5 g acid). The TTX fractions from HW-40 column were •@ concentrated and put on an Amberlite CG-50 Beef ex. (Difco) 5 g Agar 15 g column (1 •~ 5cm, NH+ form), washed with 10ml Artificial sea water 1,000 ml of water and then eluted with 20ml of 0.5N acetic pH 7.2 acid. The eluate was concentrated and treated with second charcoal column chromatography.

The TTX fraction finally obtained was concentrat 3,890 MU/g) was caught by line fishing at Haka ed to 0.5ml and analyzed by a TTX analyzer. tajima in November 1985, fasted for 3 days and The bacteria, code-named SA-4 of which culture transported alive to our laboratory. broth was confirmed to contain TTX and its •@ de

rivatives, were harvested by centrifugation from Isolation of Bacteria 1l medium and the bacterial cells were suspended The intestine was removed from the fish, weigh in 0.1 % acetic acid and heated in a boiling bath ed, cut into pieces, and homogenized with several for 10 min. The suspension was centrifuged at volumes of sterilized artificial sea water (Jamarine 10,000 rpm for 20 min and the precipitates were S). After homogenization, the suspension of re-extracted in the same manner. The combined microorganisms was diluted, spreaded with glass supernatants were concentrated to 6ml and the rod on agar medium of which composition is test solutions were checked for TTX or its de shown in Table 1. After incubation for 3-7 days rivatives as mentioned above. at 23•Ž, colonies appearing on the agar plate were •@ divided into several types according to cultural Fluorometric HPLC Analysis and morphological characteristics. Each of them A TTX analyzer, which was developed by Yasu was picked up and streaked on the surface of agar moto et al.5) was used for detection of TTX or its medium for purification. Finally, 15 kinds of derivatives. The mobile phase was prepared by bacteria were isolated from the puffer fish in adding conc. NH4OH to an aqueous solution testine and code-named from SA-1 through SA-15. containing 3% MeCN, 0.005 N heptafluorobutyric acid and 0.05N acetic acid (pH5.0). The flow Fermentation of TTX rates of the mobile phase and of 4N NaOH were A loopful growth of isolates was incubated in a kept at 0.5ml/min. For detecting the fluorescent 500ml Sakaguchi flask containing 100ml of the products, the excitation and emission wavelengths medium which was composed of phytone (BBL) were set at 363 and 510nm, respectively. 10g, artificial sea water 500ml, and distilled water

500ml, pH7.2. The flasks were incubated without GC-MS Spectrography shaking at 26•Ž for 10 to 15 days. Authentic TTX or partially purified toxins from bacteria cells and culture broths, which were con Screening of Toxin from the Culture Borth or firmed to contain TTX by mouse bioassay and by Bacterial Cell the TTX analyzer, were converted into trimethy The culture broths of 15 strains were examined lsilyl(TMS)-2-amino-6-hydroxyl-methyl-8-hydro- for the presence of TTX and its derivatives. The xyquinazoline (C9-base) by the method of Ohtsuka culture broths (250ml each) were acidified to pH et al.9) as follows: TTX or the bacterial toxins were 5.0 with acetic acid and centrifuged at 8,000 rpm. hydrolyzed in 7.4% KOH for 30min in a boiling The supernatant was diluted to 750ml with water water both. The alkali hydrolyzates were neutraliz and then passed through a charcoal column (2 •~ ed with HC•¬ and passed •@ through a C18-SEP-PAC 30cm). After washing the column with 200ml cartridge column. The column was washed with of water, the adsorbate was eluted with 20% EtOH 2ml of water and then 2ml of 20% MeOH. The containing 1% acetic acid. The eluate was eva adsorbed C9-base was eluted with 5ml of MeOH

porated, made up to 6ml with water, and used as and concentrated to dryness. The residue was a test solution. One ml of the test solution was dissolved in 50ƒÊl of N, N-dimethylformamide employed for the determination of lethal toxicity (DMF), and then 200 ƒÊl of N,O-Bis(trimethylsily1) to mice according to the standard method de trifluoroacetamide and 10ƒÊl of trimethylchloro scribed in Shokuhin Eisei Kensa Shishin II. 8) silane were added. The reaction mixture was Using remained 5ml of the test solution, further heated at 100•Ž for 30 min in order to convert C9- - TTX Production by Bacteria from a Puffer Fish 2201

base into its TMS derivatives . The TMS-derivatives were subjected to GC-MS on a Shimadzu GC- MS QP-1,000 [column: 5% SE-52 (Uniport HP

60-80 mesh), 3 mm •~ 1m, temp. 220-260•Ž (5•Ž/

min); •@ ionization voltage 70V, carrier gas helium

30ml/min].

Identification of Bacteria The media for identification was prepared with artificial sea water. All of the phenotypic char acteristics and DNA base composion were de termined by the method of Kodama et a1.10) Ex traction and purification of quinones were carried out by the method described by Tamaoka et al. 11) Ubiquinone systems and menaquinone systems were determined by HPLC analysis and mass ana lysis, respectively.

Results and Discussion

Yasumoto et al.6) isolated TTX and anhydTTX

from the culture broth of Alteromonas sp. isolated

from a red calcareous alga, Jania sp. Therefore, we first conducted the screening for the toxins in

the culture broths. In the mouse bioassay of the

test solutions, except one sample code-named SA-4,

it was observed that some mice showed char

acteristic symptoms for TTX intoxication but Fig. 1. Chromatograms of partially purified toxins

completely recovered until next morning and that from culture broth (top) and cells (middle), and

other mice became dull and died without typical standard TTX, containing a trace of anhydTTX

TTX symptoms by the next morning after the (bottom) with the TTX analyzer. injection. From this result, we concluded that

they were none toxic or even if they had toxins, symptoms of TTX toxication. As shown in Fig.

the toxicities were very low. Only one strain, 1, the partially purified toxins from both bacterial cells and broth gave peaks, of which retention SA-4 was confirmed for its toxicity by the mouse times were in good agreement with those of TTX bioassay, and the amount of toxin totaled 15MU in 250 ml of culture broth. When mice were and anhydTTX. This showed confidently that the toxins were TTX and anhydTTX. injected with 1ml of the test solution, were ob For further confirmation that SA-4 strain pro served the typical signs of TTX intoxication such duced and secreted TTX and its derivatives, as dyspnea and convulsion, and then the animal died. authentic TTX and the purified toxins were hy

Partially purified TTX fractions were concen drolyzed in alkaline solution to C9-bases and the C9-bases were changed to TMS derivatives by the trated to 500ƒÊl and 100ƒÊ1 each out of these •@ solu tions was analyzed by the fluorescent TTX ana method of Ohtsuka et al.9) in order to be analyzed by the GC-MS analyzer. Mass fragment ions lyzer. It was revealed that 2 strains, code-named m/z 407 and 392, which were specific for tri SA-3 and SA-11 in addition to SA-4 contained methylsylil C9-base, appeared in the same reten TTX, 4-epiTTX and/or anhydTTX (data not tion time, irrespective of samples analyzed. (Fig shown). . 2) The results allowed us to conclude that the Since it became certain that the broth of SA-4 bacterial toxins are TTX and its derivatives. contained TTX by the mouse assay, the bacterial Strain SA-4 was nonsporeforming, gram-nega •@cells of this strain were surveyed for toxins by the tive, monotrichously flagellated rod-shaped orga method mentioned above. Due to the •@restriction nism measuring 1.0 by 3.0ƒÊm (Table 2) . It of amount of toxin, only one mouse was used for required Na+ for its growth. DNA base com the bioassay but the mouse was killed with typical 2202 Matsui, Taketsugu, Kodama, Ishii, Yamamori, and Shimizu

Table 2. Morphological, cultural, and biochemical characteristics of strain SA-4

Cell: Rod (1.0 x 3.0 µm) Na+ requirement: + Endospre: - DNase: + Flagellation: Monotrichous H2S production: + Gram stain: - Nitrate to Nitrite: + Pigmentation: Orange Urease: + OF test Hydrolysis of Oxidative: - Gelatin: + Fermentative: - Tween 80: + Catalase: + ONPG: - Oxidase: +

tified as Shewanella putrefaciens (Derby and Hammer) Macdonell and Colwell. Yasumoto et a1. 5, 6)reported the ability of pro ducing TTX in Pseudomonas sp. isolated from the skin of puffer fish Takifugu poecilonolus as well as Alteromonas from red alga. On the other hand, Noguchi et al. 7) also reported Vibrio sp. isolated from a xanthid crab to produce TTX and anhyd TTX. This study revealed for the first time that S. putrefaciens isolated from the intestine of highly toxic wild puffer fish Takifugu niphobles produced TTX and anhydTTX and secreted into the culture broth. This result strongly suggests that the toxification of the puffer fish is more likely caused by the bacteria. In order to verify the speculation, however, it is necessary to obtain more precise experimental data concerning the amount of toxin and its derivatives. Studies along these lines are now in progress.

Acknowledgments

Fig. 2. Selected ion-monitored chromatograms of This work was supported in part by a Grant-in- TMS C9-bases from TTX (bottom), and of the Aid for Scientific Research from the Ministry of corresponding derivatives of alkali-hydrolyzates of purified toxins from the bacterial cells (middle) Education, Science and Culture of Japan. The and culture broth (top). authors are indebted to Dr. Yukio Ohtsuka, Forensic Science Laboratory of Fukuoka Pre fectural Police H. Q. for GC-MS analysis. Thanks position of strain SA-4 was 44.2%. Therefore, are also due to Professor Takeshi Yasumoto, strain SA-4 belongs to Shewanella putrefaciens or Faculty of Agriculture, Tohoku University for his genus Alteromonas.12, 13) S. puterfaciens differed valuable suggestions. from the genus Alteromonas in quinone system. The quinone system of S. putrefaciens is ubiquinone Q-7 and Q-8, and menaquinone MK-7, and meth References ylmenaquinone MMK-7. On the other hand, the 1) T. Matsui, S. Hamada, and S. Konosu: Nippon quinone system of the genus Alteromonas is ubiq Suisan Gakkaishi, 47, 535-537 (1981). uinone Q-8. Strain SA-4 had ubiquinone Q-7 2) T. Matsui, H. Sato, S. Hamada, and C. Shimizu: and Q-8, menaquinone MK-7, and methylmenq Nippon Suisan Gakkaishi, 48, 253 (1982). uinone MMK-7. By its phenotypic and chemot 3) T. Matsui, S. Hamada, and K. Yamamori: axonomical characteristics, strain SA-4 was iden- Nippon Suisan Gakkaishi, 48, 1179 (1982). TTX Production by Bacteria from a Puffer Fish 2203

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Nippon Suisan Gakkaishi: Formerly Bull. Japan. Soc. Sci. Fish.