Isolation And Characterization Of Tetrodotoxin-producing Bacterial Stains RG-3B6 And RG-33B From The Puffer Fish Fugu Rubripes And Detection Of Tetrodotoxin In The Fermentation Production

Tetrodotoxin (TTX) is one of the most potent non-protein neurotoxins that blocks sodium channels and thus inhibits the propagation of action potentials. TTX was widely used as an important pharmacological reagent for neurobiological research. A recent study showed that TTX may be one of the most promising analgesic, detoxification agent and local anesthetic in the future which is currently under phase II clinic trials. All these will increase the market requirement for TTX. Conventionally, TTX is purified from ovaries and livers of puffer fish and the yield is limited. Isolation of TTX-producing bacteria will make it possible to produce large amount of TTX in vitro without destroying the puffer fish resource.The origin of TTX is one of the most controversial topics. Since TTX was originally discovered and isolated from certain species of fish belonging to the order of Tetraodontiforms, for a long time, puffer fish was thought to produce and accumulate TTX within their body. Lot of reserch was conducted but no gland structure was identified. Furthermore, TTX and its analogs were detected in several phyla of vertebrates and invertebrates. The wide distribution of TTX among genetically unrelated animals made the true origin of TTX one of the most interesting and controversial topics. TTX-producing bacteria have been reported to be isolated from different TTX-bearing animals but no bacterial production of TTX was achieved. It has been known for many years that the toxicity of the ovaries and livers of puffer fish is more potent than that of other organs, but whether the anatomical variation of toxicity is due to the different distribution of TTX-producing bacteria is not known.In the present study, three speciemen of the female puffer fish Fugu rubripes were collected from the Bohai Sea of China for the isolating and screening of the TTX-producing bacteria.Our results demonstrated that (1) the distribution of TTX-producing strains and the bacterial production of TTX is close related with the toxicity of each tissue in puffer fish; (2) based on morphological observations, physiological, biochemical characteristics and 16S rDNA alignment, the two TTX-producing strains were identified as Nocardiopsis dassonville and Bacillus Jusiformii, in our knowledge Nocardiopsis dassonville was the first time which was reported to produce TTX; (3) bacterial production of TTX was achieved and idendified by the chemical and biological methods. Our results supported the postulation of bacterial origin of TTX.In summary, the distribution and toxicity of TTX-producing bacteria in puffer fish Fugu rupries was investigated. Bacterial production of TTX was purified and identified by biological and instrument analysis. Our result will make it possible to produce TTX via fermentation and give solid ground on the postulation of bacterial origin of TTX. More research is needed to elucidate the mechanism of TTX synthesis and the role of TTX in bacteria.