Bacterial plasmids may show the origin of TTX

Tetrodotoxin (TTX) is a highly toxic neurotoxin found in a diverse range of genetically distant species. It has been proposed that bacteria are the true source of TTX within animals, such as pufferfish, gastropod, octopus etc.; however it is difficult to truly isolate this toxin’s source.

A study by Liu et al. (2015) wanted to identify the origin of TTX within cells of Aeromonas sp. Ne-1 isolated from the pufferfish, Takifugu obscurus. This was completed using the plasmid involved in TTX production, pNe-1. It was found that this plasmid’s copy number was associated with the bacteria’s ability to produce TTX; when the copy number decreased, so did the concentration of TTX. Unexpectedly, a degradation enzyme was discovered within the cells, which may explain why the energetically costly production of TTX reduced over time.

The plasmid of Aeromonas sp. Ne-1 was extracted and sequenced in order to determine the open reading frames (ORFs) present. At least 60 ORFs were recorded, with one known to be associated with E. coli insertion elements, and another associated with the incorporation of DNA into a larger DNA molecule. These potential genes could facilitate the transfer of the TTX pathway horizontally between organisms, which may explain the presence of TTX in over 14 different phyla.

A time-course bacterial cell culture was grown and at 12, 18, 21, 24, 31 and 42 hours samples were taken for plasmid extraction. The plasmid DNA was used in real-time qPCR to determine the average plasmid copy number. From 12 to 18 hours, there was an increase in the plasmid copy number, as expected. What is interesting is that after the 18^th hour, the copy numbers of this plasmid decreased sharply from 2.51 to 0.27, and eventually the plasmid was undetectable at 42 hours.

Another time-course cell culture was set up in order to measure the TTX concentrations at 24, 42, 66 and 96 hours. The results were similar to above, as after 42 hours (1.602 ng for each 10¹⁰ clone), the concentration dropped sharply; at 66 hours the concentration fell to 0.292 ng, and then down to 0.220 ng for each 10¹⁰ clone at 96 hours. This decrease in TTX concentration suggests an unknown TTX-degrading enzyme in the cell. A follow up experiment was conducted to determine the presence of such an enzyme, whereby a set amount of TTX was added to cultures. Compared to the control, at 66 and 96 hours there was 26-55% and 53-69% degradation of TTX, respectively.

This paper has shown how the plasmid copy number of pNE-1 from Aeromonas sp. Ne-1 is related to TTX production, and that there is a high possibility that the genes involved in this pathway are passed from each organism via horizontal gene transfer (HGT). What was unexpected was the presence of a TTX-degrading enzyme, which suggests that there is a homeostatic control mechanism within the bacteria that maintains stable levels of the energetically costly toxin.  

I believe that this work has furthered our understanding of the potential origins of this prevalent toxin, despite the widespread distribution across many phyla. For the first time, it has been seen that the bacteria Aeromonas sp. Ne-1 can produce and degrade the levels of TTX in order to control their internal environment. This along with the possible origins of this toxin being spread via HGT is a massive breakthrough in understanding how this pathway may have arrived in many animals today and what may regulate it.

Liu, J., Wei, F., Lu, Y., Ma, T., Zhao, J., Gong, X., & Bao, B. (2015). Production level of tetrodotoxin in Aeromonas is associated with the copy number of a plasmid. Toxicon, 101, 27-34.

http://www.sciencedirect.com/science/article/pii/S0041010115001063

A helpful website explaining some plasmid characteristics:

http://blog.addgene.org/plasmid-101-origin-of-replication