Characterisation of the paralytic shellfish toxin biosynthesis gene clusters in Anabaena circinalis AWQC131C and Aphanizomenon sp. NH-5

<p>Abstract</p> <p>Background</p> <p>Saxitoxin and its analogues collectively known as the paralytic shellfish toxins (PSTs) are neurotoxic alkaloids and are the cause of the syndrome named paralytic shellfish poisoning. PSTs are produced by a unique biosynthetic pathway, which involves reactions that are rare in microbial metabolic pathways. Nevertheless, distantly related organisms such as dinoflagellates and cyanobacteria appear to produce these toxins using the same pathway. Hypothesised explanations for such an unusual phylogenetic distribution of this shared uncommon metabolic pathway, include a polyphyletic origin, an involvement of symbiotic bacteria, and horizontal gene transfer.</p> <p>Results</p> <p>We describe the identification, annotation and bioinformatic characterisation of the putative paralytic shellfish toxin biosynthesis clusters in an Australian isolate of <it>Anabaena circinalis </it>and an American isolate of <it>Aphanizomenon sp</it>., both members of the <it>Nostocales</it>. These putative PST gene clusters span approximately 28 kb and contain genes coding for the biosynthesis and export of the toxin. A putative insertion/excision site in the Australian <it>Anabaena circinalis </it>AWQC131C was identified, and the organization and evolution of the gene clusters are discussed. A biosynthetic pathway leading to the formation of saxitoxin and its analogues in these organisms is proposed.</p> <p>Conclusion</p> <p>The PST biosynthesis gene cluster presents a mosaic structure, whereby genes have apparently transposed in segments of varying size, resulting in different gene arrangements in all three <it>sxt </it>clusters sequenced so far. The gene cluster organizational structure and sequence similarity seems to reflect the phylogeny of the producer organisms, indicating that the gene clusters have an ancient origin, or that their lateral transfer was also an ancient event. The knowledge we gain from the characterisation of the PST biosynthesis gene clusters, including the identity and sequence of the genes involved in the biosynthesis, may also afford the identification of these gene clusters in dinoflagellates, the cause of human mortalities and significant financial loss to the tourism and shellfish industries.</p

A Putative Gene Cluster from a Lyngbya wollei Bloom that Encodes Paralytic Shellfish Toxin Biosynthesis

Saxitoxin and its analogs cause the paralytic shellfish-poisoning syndrome, adversely affecting human health and coastal shellfish industries worldwide. Here we report the isolation, sequencing, annotation, and predicted pathway of the saxitoxin biosynthetic gene cluster in the cyanobacterium Lyngbya wollei. The gene cluster spans 36 kb and encodes enzymes for the biosynthesis and export of the toxins. The Lyngbya wollei saxitoxin gene cluster differs from previously identified saxitoxin clusters as it contains genes that are unique to this cluster, whereby the carbamoyltransferase is truncated and replaced by an acyltransferase, explaining the unique toxin profile presented by Lyngbya wollei. These findings will enable the creation of toxin probes, for water monitoring purposes, as well as proof-of-concept for the combinatorial biosynthesis of these natural occurring alkaloids for the production of novel, biologically active compounds

Neurotoxic Alkaloids: Saxitoxin and Its Analogs

Saxitoxin (STX) and its 57 analogs are a broad group of natural neurotoxic alkaloids, commonly known as the paralytic shellfish toxins (PSTs). PSTs are the causative agents of paralytic shellfish poisoning (PSP) and are mostly associated with marine dinoflagellates (eukaryotes) and freshwater cyanobacteria (prokaryotes), which form extensive blooms around the world. PST producing dinoflagellates belong to the genera Alexandrium, Gymnodinium and Pyrodinium whilst production has been identified in several cyanobacterial genera including Anabaena, Cylindrospermopsis, Aphanizomenon Planktothrix and Lyngbya. STX and its analogs can be structurally classified into several classes such as non-sulfated, mono-sulfated, di-sulfated, decarbamoylated and the recently discovered hydrophobic analogs—each with varying levels of toxicity. Biotransformation of the PSTs into other PST analogs has been identified within marine invertebrates, humans and bacteria. An improved understanding of PST transformation into less toxic analogs and degradation, both chemically or enzymatically, will be important for the development of methods for the detoxification of contaminated water supplies and of shellfish destined for consumption. Some PSTs also have demonstrated pharmaceutical potential as a long-term anesthetic in the treatment of anal fissures and for chronic tension-type headache. The recent elucidation of the saxitoxin biosynthetic gene cluster in cyanobacteria and the identification of new PST analogs will present opportunities to further explore the pharmaceutical potential of these intriguing alkaloids

Structure of the saxitoxin analogs identified in <i>Lyngbya wollei</i> (adapted from [7]).

<p>Structure of the saxitoxin analogs identified in <i>Lyngbya wollei</i> (adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0014657#pone.0014657-Onodera1" target="_blank">[7]</a>).</p

Organization of the PST biosynthesis cluster in <i>Lyngbya wollei</i>, scale indicates gene size in base pairs, direction of arrowed boxes indicates direction of transcription.

<p>Organization of the PST biosynthesis cluster in <i>Lyngbya wollei</i>, scale indicates gene size in base pairs, direction of arrowed boxes indicates direction of transcription.</p

Proposed biosynthetic pathway for the PSTs produced by <i>Lyngbya wollei</i>.

<p>The gray shaded area highlights the steps that are unique to <i>L. wollei</i>.</p

The saxitoxin biosynthesis genes in <i>Lyngbya wollei</i> and their homology-based functions identified using BlastX (percentage identity is reported).

<p>*Indicates a PsiBLAST search was used.</p