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Biosynthetic intermediate analysis and functional homology reveal a saxitoxin gene cluster in cyanobacteria.

ABSTRACT: Saxitoxin (STX) and its analogues cause the paralytic shellfish poisoning (PSP) syndrome, which afflicts human health and impacts coastal shellfish economies worldwide. PSP toxins are unique alkaloids, being produced by both prokaryotes and eukaryotes. Here we describe a candidate PSP toxin biosynthesis gene cluster (sxt) from Cylindrospermopsis raciborskii T3. The saxitoxin biosynthetic pathway is encoded by more than 35 kb, and comparative sequence analysis assigns 30 catalytic functions to 26 proteins. STX biosynthesis is initiated with arginine, S-adenosylmethionine, and acetate by a new type of polyketide synthase, which can putatively perform a methylation of acetate, and a Claisen condensation reaction between propionate and arginine. Further steps involve enzymes catalyzing three heterocyclizations and various tailoring reactions that result in the numerous isoforms of saxitoxin. In the absence of a gene transfer system in these microorganisms, we have revised the description of the known STX biosynthetic pathway, with in silico functional inferences based on sxt open reading frames combined with liquid chromatography-tandem mass spectrometry analysis of the biosynthetic intermediates. Our results indicate the evolutionary origin for the production of PSP toxins in an ancestral cyanobacterium with genetic contributions from diverse phylogenetic lineages of bacteria and provide a quantum addition to the catalytic collective available for future combinatorial biosyntheses. The distribution of these genes also supports the idea of the involvement of this gene cluster in STX production in various cyanobacteria.

SUBMITTER: Kellmann R

PROVIDER: S-EPMC2446512 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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Biosynthetic intermediate analysis and functional homology reveal a saxitoxin gene cluster in cyanobacteria.

Kellmann Ralf R Mihali Troco Kaan TK Jeon Young Jae YJ Pickford Russell R Pomati Francesco F Neilan Brett A BA

Applied and environmental microbiology 20080516 13

Saxitoxin (STX) and its analogues cause the paralytic shellfish poisoning (PSP) syndrome, which afflicts human health and impacts coastal shellfish economies worldwide. PSP toxins are unique alkaloids, being produced by both prokaryotes and eukaryotes. Here we describe a candidate PSP toxin biosynthesis gene cluster (sxt) from Cylindrospermopsis raciborskii T3. The saxitoxin biosynthetic pathway is encoded by more than 35 kb, and comparative sequence analysis assigns 30 catalytic functions to 26 ...[more]

PMID: 18487408

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Project description:BACKGROUND:Paralytic shellfish poisoning (PSP) is a potentially fatal syndrome associated with the consumption of shellfish that have accumulated saxitoxin (STX). STX is produced by microscopic marine dinoflagellate algae. Little is known about the origin and spread of saxitoxin genes in these under-studied eukaryotes. Fortuitously, some freshwater cyanobacteria also produce STX, providing an ideal model for studying its biosynthesis. Here we focus on saxitoxin-producing cyanobacteria and their non-toxic sisters to elucidate the origin of genes involved in the putative STX biosynthetic pathway. METHODOLOGY/PRINCIPAL FINDINGS:We generated a draft genome assembly of the saxitoxin-producing (STX+) cyanobacterium Anabaena circinalis ACBU02 and searched for 26 candidate saxitoxin-genes (named sxtA to sxtZ) that were recently identified in the toxic strain Cylindrospermopsis raciborskii T3. We also generated a draft assembly of the non-toxic (STX-) sister Anabaena circinalis ACFR02 to aid the identification of saxitoxin-specific genes. Comparative phylogenomic analyses revealed that nine putative STX genes were horizontally transferred from non-cyanobacterial sources, whereas one key gene (sxtA) originated in STX+ cyanobacteria via two independent horizontal transfers followed by fusion. In total, of the 26 candidate saxitoxin-genes, 13 are of cyanobacterial provenance and are monophyletic among the STX+ taxa, four are shared amongst STX+ and STX-cyanobacteria, and the remaining nine genes are specific to STX+ cyanobacteria. CONCLUSIONS/SIGNIFICANCE:Our results provide evidence that the assembly of STX genes in ACBU02 involved multiple HGT events from different sources followed presumably by coordination of the expression of foreign and native genes in the common ancestor of STX+ cyanobacteria. The ability to produce saxitoxin was subsequently lost multiple independent times resulting in a nested relationship of STX+ and STX- strains among Anabaena circinalis strains.

Project description:Dinoflagellates produce a variety of toxic secondary metabolites that have a significant impact on marine ecosystems and fisheries. Saxitoxin (STX), the cause of paralytic shellfish poisoning, is produced by three marine dinoflagellate genera and is also made by some freshwater cyanobacteria. Genes involved in STX synthesis have been identified in cyanobacteria but are yet to be reported in the massive genomes of dinoflagellates. We have assembled comprehensive transcriptome data sets for several STX-producing dinoflagellates and a related non-toxic species and have identified 265 putative homologs of 13 cyanobacterial STX synthesis genes, including all of the genes directly involved in toxin synthesis. Putative homologs of four proteins group closely in phylogenies with cyanobacteria and are likely the functional homologs of sxtA, sxtG, and sxtB in dinoflagellates. However, the phylogenies do not support the transfer of these genes directly between toxic cyanobacteria and dinoflagellates. SxtA is split into two proteins in the dinoflagellates corresponding to the N-terminal portion containing the methyltransferase and acyl carrier protein domains and a C-terminal portion with the aminotransferase domain. Homologs of sxtB and N-terminal sxtA are present in non-toxic strains, suggesting their functions may not be limited to saxitoxin production. Only homologs of the C-terminus of sxtA and sxtG were found exclusively in toxic strains. A more thorough survey of STX+ dinoflagellates will be needed to determine if these two genes may be specific to SXT production in dinoflagellates. The A. tamarense transcriptome does not contain homologs for the remaining STX genes. Nevertheless, we identified candidate genes with similar predicted biochemical activities that account for the missing functions. These results suggest that the STX synthesis pathway was likely assembled independently in the distantly related cyanobacteria and dinoflagellates, although using some evolutionarily related proteins. The biological role of STX is not well understood in either cyanobacteria or dinoflagellates. However, STX production in these two ecologically distinct groups of organisms suggests that this toxin confers a benefit to producers that we do not yet fully understand.

Dataset Information

Biosynthetic intermediate analysis and functional homology reveal a saxitoxin gene cluster in cyanobacteria.

Publications

Biosynthetic intermediate analysis and functional homology reveal a saxitoxin gene cluster in cyanobacteria.

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