Isolation and Identification of a Tetrodotoxin-Producing Bacterium from the Wild Puffer Fish of Poyang Lake

MA Chun-yan，LIU Song，LU Yu，YU Bo

Jiangxi-OAI Joint Research Institute，Nanchang University，Nanchang 330047，China

Introduction

Tetrodotoxin(TTX，C11H17N3O8)is one of the most potential nonprotein neurotoxins.As a voltage-gated sodium channel blocker［1］，it can provide effective tool for the research in neurobiology and neurophysiology.New clinical studies suggest that low-dose TTX can safely relieve severe，treatment-resistant cancer pain［2］.At present，TTX is under investigation and development as a potential analgesic，detoxification agent and anesthetic.And the medicine experiment is currently under phase II clinic trials(International Wex Technologies，Inc.)［3］.All of this will increase the market requirement for TTX.

At present，there are three methods to produce TTX.In TTX traditional producing method，TTX is purified from the ovaries and livers of wild puffer fish［4］.This method will destroying puffer fish resources which are rare and precious.The yield of TTX produced by this method are limited due to the exhaustion of puffer fish resource.Nowadays，attempts at chemical synthesis of TTX have been successful，but these generally involve complex，time consuming，multi-step reactions，and result in low yields and expensive［5，6］.

Since 1984，a postulation that all TTX-bearing animals were infected by TTX-producing microorganisms living symbiotically within their bodies was brought forward［7］，and until 1986 when the first TTX-producing microorganisms were eventually isolated and identified from the TTX-bearing animals［8］.After that，TTX-producing bacteria have been isolated from many organ-isms［9］.Isolation and culture of TTX-producing bacteria could be the most reliable method for TTX producing，and which will made it possible to produce TTX without destroying the puffer fish resources.

Poyang Lake is the largest freshwater lake of China.It locates in Jiangxi province and reaches the middle and lower of the Yangtze River.The Takifugu obscurus is a kind of migratory puffer fish.Every spring when sexual maturation，they migrate for spawning from East sea of China to the Poyang Lake.So the wild Takifugu obscurus is one of the characteristic species of Poyang Lake in Springtime.

There is a prolific resource of puffer fish in Poyang Lake.However，few studies have investigated TTX-producing bacteria in local puffer fish.In this study，a TTX-producing bacteria was isolated from the ovary of a wild puffer fish(Takifugu obscurus)from Poyang Lake，and it was identified as Bacillus vallismortis by physiological-biochemical characteristics and genetic analysis based on 16S rRNA analysis.

Materials and Methods

Materials

Collection of samples

In April 2011，a wild Takifugu obscurus was caught from Poyang Lake and transported alive to the laboratory of Jiangxi-OAI Joint Research Institute(Nanchang，China).

Culture medium and agar plates

The composition of the agar medium and liquid medium used for bacterial isolation、batch culture，and morphological observations、physiological and biochemical analyze were as follows.Modified Emerson agar medium:0.3%peptone，0.3%yeast extract，0.3%beef extract，1%glucose，1%glucose，2%agar，pH of the medium was adjusted to 7.0，and it was sterilized for 30 min at 121 ℃.The liquid medium:0.5%peptone，0.2%of yeast extract，0.2%beef extract，2%glucose，pH of the medium was adjusted to 7.0，and it was sterilized for 30 min at 121℃.

Mouse bioassay

The toxicity mouse bioassay use the kunming male mouse purchased from the Department of Experimental Animal Science，Nanchang University，China，each weighing 20±2 g.

Apparatus and reagent

TTX standard was purchased from Sigma-aldrich Corporation(USA).

Active charcoal、polyacrylamide and all of the other reagent purchased from Shanghai yuanye Biotechnology Company.

The main apparatus as follows:Eectro-heating standingtemperature cultivator(Botai，XMT 1052-b-B);Centrifuge(Sigma，4-16K)，Laminar Flow Cabinets(Opti-Mair.ABC-4A1);Sterilizer(SANYO，MLS-3780);Constant temperature oscillator(China，SHZ-82);Mass Spectrograph(Waters，ZQ400);Spectrofluorophotometer(Hitachi，F-4500);Ultraviolet Lamp(China，ZF-2).

Methods of bioassay and chemical analyses

Isolation of bacteria

The ovary of the puffer fish was homogenized in a sterile mortar，and was suspended in sterile seawater，then serially diluted to three concentrations of 10-1，10-2and 10-3.A 0.5 mL of each diluted sample was aseptically spread on agar plates，which were incubated at 24 ℃for 7 days.A mixture of colonies was formed on each plate，and each discrete colony was transferred to new plate until pure culture was obtained on the basis of colony morphology.Each distinct colony was further subcultured for several times to ensure that it was a pure culture before bacterial identification.Eventually，four representative colonies were picked up and transferred to the liquid medium for mouse toxicity assay.The four purified strains were stored at-70℃in the presence of glycerol.

Isolation and extraction of toxin from bacterial cultures To screen the TTX-producing isolation，isolated strains were inoculated into the liquid medium and incubated at 28℃with shaking at 180 rpm for 3 days.The cultures were centrifuged at 8，000×g at 4℃ for 30 min.The supernatant was evaporated under reduced pressure at 40-45℃，and then was boiled at 100℃ for 10 min after the pH had been adjusted to 4.5，cooled and centrifuged to remove debris.Then the supernatant was loaded into an activated charcoal column(1×20 cm)after the pH was adjusted to 5.5 and the toxin absorbed in the charcoal was eluted with 1%acetic acid in 20%methanol.The eluate was evaporated under reduced pressure at 40℃，and lyophilized.The lyophilized toxin extract was dissolved in 0.03 M acetic acid and applied to a Bio-Gel P-2 column(1×80 cm)and eluted with 0.03 M acetic acid as the eluant.Toxic fractions were determined by mouse bioassay，which were then combined and concentrated in reduced pressure.Lyophilized again and subjected to mouse toxicity assay，thin-layer chromatography(TLC)，fluorimetric spectrophotometry，and electrospray ionization mass spectrometry(ESI-MS)for detection and identification of TTX，or TTX-related toxin.The medium materials used for the cultivation was examined as background.Mouse bioassay

The mouse bioassay employed in this study followed the standard guidelines［10］.The toxic extracts from the bacterial cultures(0.5 mL)were injected into Kunming male mouse each in groups of three animals.The toxicity of the extract was determined by the average lethal time of each injected mouse.which was correlated to the amount of toxin in the extract.The death time was recorded at the last grasping breath of the mouse.Lethal potency of TTX was expressed in mouse units(MU，mean+SD)，according to the standard dose-lethal time prepared by using the standard TTX.1 MU was defined as the amount of TTX which kills a mouse in 30 min after injection.Death of mice after 30 min was considered to be negative.

Thin-layer chromatography(TLC)

One of the four isolated strains was found to produce toxin by mouse bioassay，which named as TL-1.The isolated toxin from TL-1 cultures was checked by thinlayer chromatography(TLC)as Yu have described previously with a slightly modification［3］.TLC assay was carried out on TLC aluminium precoated plates(without fluorescent indicator).The solvent system used was pyridine-ethyl acetate-glacial acetic acid-water(10∶5∶3∶2，v/v).Both the standard TTX and purified bacterial samples were applied on TLC plates 1 cm apart from one another by capillary tubes.Toxins were visualized as a fluorescent spots under UV light(365 nm)after the plate was sprayed with 10%KOH solution and heated at 110℃for 10 min.

Fluorimetric assay of toxin with spectrophotometer

A fluorimetric method was used for detection of TTX and its derivatives.A small amount of toxin extracts from TL-1 or authentic toxin were dissolved in 1 ml of 4 N NaOH and heated in a boiling water bath for 45 min.After cooling，the intensity of fluorescence of the 2 amino-6-hydroxymethyl-8-hydroxyquinazoline(C9 base)，which should have been derived from TTX and related substances if present，was determined with a fluorimetric spectrophotometry(Hitachi F850)，with excitation and emission wavelengths set at 370 and 500 nm，respectively.

Electrospray ionization mass spectrometry(ESI-MS)

For mass spectrometry，about 1 ml of the toxin extracts from the bacterial cultures were dissolved in a small amount of 0.1%acetic acid，and the sample solution was applied to electrospray ionization-mass spectrometer(Waters，ZQ400)by flow injection at a flow rate of 0.2 mL/min.

Identification of the strain

The toxin-producing bacteria，strain TL-1，was characterized according to the standard procedures as previous described［11］.The morphology of strain TL-1 was observed and photographed with the light microscope and the transmission electronmicroscope(Microscopy Core Facility，Nanchang University，China).

For physiological and biochemical characteristics analysis，The range of pH，temperature and NaCl concentrations for the growth of the strain were investigated in agar medium，and nitrate reduction、Catalase activity were investigated in liquid medium.The ability of the strain to utilize substrates as sole carbon source was tested by supplementing the agar medium with 1%of the filter-sterilized carbon compound.

For identification of the strain，the 16S rDNA was amplified from genomic DNA by PCR using the primers F:5’-AGAGTTTGATCCTGGCTCAG-3’，and R:5’-ACGGCTACCTTGTTACGACT-3’.Then amplified fragments were subjected to 1.0%agarose gel electrophoresis，and recovered using the DNA Extraction kit(Takara，Japan).The purified and recovered DNA was cloned into the pGEM T-Vector according to manufacturer’s instruction(Takara，Japan).This DNA fragment was sequenced by Shanghai Invitrogen Biotechnology Co.，Ltd.The partial 16S rRNA sequence was compared with sequences in the GenBank database using Blast.Multiple alignments were carried out with the ClustalX and the Mega 5.0 program，and phylogenetic analysis was conducted with the neighbor-joining plot program.

Results and discussion

The strain TL-1 with capability of TTX producing

Four strains isolated from the ovary of Takifugu obscurus were screened for toxicity by mouse bioassay.The strain TL-1 was found to produce toxin，mouse injected with the toxin extracts showed the typical symptoms of TTX intoxication:dyspnea、convulsion and were killed in 3-10 min［12］.The toxicity determined by the mouse bioassay was 46.5 MU in 1L of broth medium.

In TLC analysis，the fluorescent spots of the toxic extracts(observed under 365nm)had the similar Rf value(0.7-0.75)with standard TTX，which indicate the presence of TTX or related substance.The result of fluorimetric assay of toxin was shown in Fig.1.As indicated，a florescence peak with a maximal emission wavelength at 500 nm appeared.Meanwhile each of the alkaline hydrolyzates exhibited a maximum absorption at about 370 nm，which is indistinguishable from the authentic C9 base(data not show).This suggested the presence of C9 base which derived from the extracted toxin.

Fig.1 Fluorescence emission spectra of the alkaline degradation product of toxin extracts from the strain TL-1．

Fig.2 Electrospray ionization mass spectrum of toxin extracts from strain TL-1，TL-2，TL-3，TL-4．

The chemical identity of TTX extracted from the cultured strain TL-1，TL-2，TL-3，TL-4 were shown in Fig.2.The mass spectrogram of TL-1 shows the peak(100%relative intensity)from the bacterial isolates occurred at m/z 319.9，corresponding to the protonated molecular ion(M+H+)as TTX has a molecular mass of 319，but which was not appear in other spectrograms of another three strains.There were also many high peaks at m/z less than 320，which may be due to the sample applied for ESI-MS assay was not purified enough through the process described above in extraction of toxin from bacterial cultures.So more works were needed in the extraction of toxin from bacterial cultures to purify TTX from bacteria cultures.

In the morphological and biochemical analyses of the characteristics of the strain TL-1 were listed in Table 1.The colonies of strain TL-1 were round，white，opaque，regular edge，Convex，moist and had a diameter between 1 and 3 mm.The basic microscopic observations of the bacteria showed long，straight rods，and it was non-sporing and Gram-negative.The strain grows between 10 and 40℃growth was observed from pH 4.0 to pH 8.0，it can grow in the presence of NaCl at the rang of 0-6%，and the optimum condition was about pH 7.0，30℃，2%NaCl.The strain can hydrolyze starch，gelatin and reduce nitrate to nitrite.Their nutrient requirements in the culture media and the positive catalase test results demonstrated that all of them were either strict aerobic or facultative anaerobic chemoorganotrophs.

The amplified region of the 16S rRNA in strain TL-1 was about 1，595 bp，and its Genbank accession number was determined to be JQ690755.Using the online program BLAST，the related sequence was searched from the databank.The 16SrRNA similarity showed that the strain TL-1 was most closely related to genus Bacillus(Fig.3).It was found that TL-1 was closely related to Bacillus vallismortis DSM 11031 and Bacillus subtilis subsp．Subtilis DSM 10，both of the sequence similarity were 100%.And based on the phylogenetic analysis，the strain TL-1 was identified as Bacillus vallismortis．

Table 1 Physiological and biochemical characteristics of the TTX-producing strain TL-1

Discussion

The theory that TTX is originated from the symbiotic microorganisms found inside the TTX bearing organisms is gaining more general acceptance worldwide.And most of the TTX-producing bacteria were isolated from puffer fish habitats mainly located in sea or coastal waters.To the best of the authors’knowledge，this study is the first report about the TTX-producing bacteria isolated from puffer fishes collected in freshwater lake.

Most of the TTX-producing bacteria were classified into the genera of Vibrio，Pseudomonsa，Alterononas，Flavobacterium and Micrococcus［13］.In a recent study，Lu＆ Yi［14］(2009)isolated a Bacillus horikoshii from the liver of puffer fish which can produce tetrodotoxin.And Wang ＆ Fan［15］(2010)described characterization of a Bacillus species capable of producing tetrodotoxin from the puffer fish Fugu obscurus.These，along with our results，suggested that Bacillus species may be one of the parasitic or symbiotic bacteria which produce TTX during their growth.

In fact，the cultivation conditions for the bacteria in the laboratory are different from their natural habitat.It is hoped that further studies can be conducted to isolate the more productive strains of TTX-producing bacteria and to develop the optimum medium composition and fermentation conditions.Thus great production of TTX would be obtained from TTX-producing bacteria.Though the strain TL-1 in the Takifugu obscurus collected from freshwater identified capable of TTX-producing，currently，we cannot formally exclude the possibility that other Bacillus vallismortis from Takifugu obscurus in the marine or freshwater may also produce TTX.

Acknowledgements This study was financed by the Research Project of Jiangxi education department(Project No.GJJ09447)and was supported by Nanchang Key Laboratory of Biochemical Engineering(Nanchang，China).The authors are grateful to the Institute of Agricultural Sciences of Jiangxi(Nanchang，China)for providing technical assistance in identifying the puffer fish.

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