Friday, 31 January 2014

TTX biosynthesis remains a mystery!



In addition to the TTX studies already summarised here, I have been looking for a paper that tried to identify the metabolic pathways that potential TTX producing bacteria are utilising. Adam’s last entry already focused on the origin of TTX and the same authors have published this paper in order to investigate it further.     
Chau et al. isolated the bacteria in the tissues of the blue-ringed octopus Hapalochlaena sp. and the sea slug Pleurobranchaea maculata which have both been suggested to have associated bacteria that synthesise TTX. Microbial communities of the both specimens were examined by culturing samples from 4 different agars and colonies were subject to genomic DNA extraction. 16S rRNA genes were amplified so that bacteria could be identified and a phylogenetic tree encompassing all isolated bacteria was established for both animals.   
Most notably, the slug and the octopus showed a greater diversity of bacterial species than previously estimated with 22 and 27 unique bacterial strains, respectively and in general, over 60% belonged to Alteromonadales with Pseudoalteromonas and Alteromonas as most representative genera. High abundance of these genera suggested that they play a key role in the hosts’ physiology and perhaps are also involved in the TTX production which acts as a defence mechanism to the host and therefore increases its fitness.


Additionally, all isolates were screened for three gene types (PKS, NRPS and AMT genes) which were assumed to be involved in the biosynthesis of TTX. Some isolates screened positive for any of the targeted genes and in particular two strains of Pseudoalteromonas from the octopus contained all three genes. However, the PKS and NRPS genes identified coded for unique enzymes which have not been found in any characterised toxin biosynthesis pathways and thus the genes could not be confirmed to be involved in TTX production; yet further analyses are needed to reject or confirm these genes responsible for the toxin synthesis.         
AMT gene sequences from isolates of octopus tissues showed high similarity to AMT genes (e.g. cyrA) that are involved in the biosynthesis of cyclindrospermopsin, a toxin which is assumed have similar molecular components as TTX. Therefore, screening suspect microorganisms for AMT genes could be key for investigating the TTX origin.


In general, it is still unknown how exactly TTX is synthesised. However, this paper provides a basis for understanding the microbial diversity of TTX containing organisms. The authors have only examined two animals and analysing more animals of the same species is important so that it can be determined whether microbial diversity differs between individuals of the same species. It has to be determined whether only the associated bacteria are responsible for TTX, or if perhaps metabolic substances produced by the animals are utilised by the bacteria. Moreover, metagenomic analyses and culture independent identification would be more appropriate to get an insight of the whole microbiota since the majority of all bacteria are not culturable, as well as the targeting of more than just three genes suspected to be responsible for the TTX synthesis.



Chau, R., Kalaitzis, J.A., Wood, S.A., Neilan, B.A., 2013. Diversity and Biosynthetic Potential of Culturable Microbes Associated with Toxic Marine Animals. Marine Drugs, 11, pp. 2695 – 2712.
 

3 comments:

  1. Perhaps many of these bacteria are non-culturable because of their dependence on symbiosis. I would not be surprised if TTX could only be synthesised when host and bacterial gene cooperate, or through some complex quorum sensing similar to that in other symbioses such as the bobtail squid and its bio-luminescent bacteria.

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  2. I agree with you Dean, I think it highly likely that some kind of sybmbioses or quorum sensing (or combination) is necessary for TTX production. I think that if, or when, researchers find a way of culturing more TTX producing bacteria, potentially by trial and error of combinations of isolates, that atomic force microscopy (as seen in Malfatti & Azam 2009) may offer further insights of the relationships involved. Furthermore, as highlighted in the paper, the mechanics of determining biosynthesis pathways in bacteria are incredibly difficult and this, coupled with (to date) unculturable bacteria makes this area of research incredibly challenging! I believe that the jury will be out on this score for some time to come!

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