Polyphyletic origin of saxitoxin biosynthesis genes in the marine dinoflagellate Alexandrium revealed by comparative transcriptomics.

The marine dinoflagellate Alexandrium is known to form harmful algal blooms, and at least 14 species within the genus can produce saxitoxins (STXs). STX biosynthesis genes (sxt) are individually revealed in toxic dinoflagellates; however, the evolutionary history remains controversial. Herein, we determined the transcriptome sequences of toxic Alexandrium (A. catenella and A. pacificum) and non-toxic Alexandrium (A. fraterculus and A. fragae) and characterized their sxt by focusing on evolutionary events and STX production. Comparative transcriptome analysis revealed higher homology of the sxt in toxic Alexandrium than in non-toxic species. Notably, non-toxic Alexandrium spp. were found to have lost two sxt core genes, namely sxtA4 and sxtG. Expression levels of 28 transcripts related to eight sxt core genes showed that sxtA, sxtG, and sxtI were relatively high (>1.5) in the toxic group compared to the non-toxic group. In contrast, the non-toxic group showed high expression levels in sxtU (1.9) and sxtD (1.7). Phylogenetic tree comparisons revealed distinct evolutionary patterns between 28S rDNA and sxtA, sxtB, sxtI, sxtD, and sxtU. However, similar topology was observed between 28S rDNA, sxtS, and sxtH/T. In the sxtB and sxtI phylogeny trees, toxic Alexandrium and cyanobacteria were clustered together, separating from non-toxic species. These suggest that Alexandrium may acquire sxt genes independently via horizontal gene transfer from toxic cyanobacteria and other multiple sources, demonstrating monocistronic transcripts of sxt in dinoflagellates.

Chemistry of a Unique Polyketide-like Synthase.

Like many complex natural products, the intricate architecture of saxitoxin (STX) has hindered full exploration of this scaffold's utility as a tool for studying voltage-gated sodium ion channels and as a pharmaceutical agent. Established chemical strategies can provide access to the natural product; however, a chemoenzymatic route to saxitoxin that could provide expedited access to related compounds has not been devised. The first step toward realizing a chemoenzymatic approach toward this class of molecules is the elucidation of the saxitoxin biosynthetic pathway. To date, a biochemical link between STX and its putative biosynthetic enzymes has not been demonstrated. Herein, we report the first biochemical characterization of any enzyme involved in STX biosynthesis. Specifically, the chemical functions of a polyketide-like synthase, SxtA, from the cyanobacteria Cylindrospermopsis raciborskii T3 are elucidated. This unique megasynthase is comprised of four domains: methyltransferase (MT), GCN5-related N-acetyltransferase (GNAT), acyl carrier protein (ACP), and the first example of an 8-amino-7-oxononanoate synthase (AONS) associated with a multidomain synthase. We have established that this single polypeptide carries out the formation of two carbon-carbon bonds, two decarboxylation events and a stereospecific protonation to afford the linear biosynthetic precursor to STX (4). The synthetic utility of the SxtA AONS is demonstrated by the synthesis of a suite of α-amino ketones from the corresponding α-amino acid in a single step.

Variability in the sxt Gene Clusters of PSP Toxin Producing Aphanizomenon gracile Strains from Norway, Spain, Germany and North America.

Paralytic shellfish poisoning (PSP) toxin production has been detected worldwide in the cyanobacterial genera Anabaena, Lyngbya, Scytonema, Cuspidothrix and Aphanizomenon. In Europe Aphanizomenon gracile and Cuspidothrix issatschenkoi are the only known producers of PSP toxins and are found in Southwest and Central European freshwater bodies. In this study the PSP toxin producing Aphanizomenon sp. strain NIVA-CYA 851 was isolated from the Norwegian Lake Hillestadvannet. In a polyphasic approach NIVA-CYA 851 was morphologically and phylogenetically classified, and investigated for toxin production. The strain NIVA-CYA 851 was identified as A. gracile using 16S rRNA gene phylogeny and was confirmed to produce neosaxitoxin, saxitoxin and gonyautoxin 5 by LC-MS. The whole sxt gene clusters (circa 27.3 kb) of four A. gracile strains: NIVA-CYA 851 (Norway); NIVA-CYA 655 & NIVA-CYA 676 (Germany); and UAM 529 (Spain), all from latitudes between 40° and 59° North were sequenced and compared with the sxt gene cluster of reference strain A. gracile NH-5 from the USA. All five sxt gene clusters are highly conserved with similarities exceeding 99.4%, but they differ slightly in the number and presence of single nucleotide polymorphisms (SNPs) and insertions/deletions (In/Dels). Altogether 178 variable sites (44 SNPs and 4 In/Dels, comprising 134 nucleotides) were found in the sxt gene clusters of the Norwegian, German and Spanish strains compared to the reference strain. Thirty-nine SNPs were located in 16 of the 27 coding regions. The sxt gene clusters of NIVA-CYA 851, NIVA-CYA 655, NIVA-CYA 676 and UAM 529, were characterized by 15, 16, 19 and 23 SNPs respectively. Only the Norwegian strain NIVA-CYA 851 possessed an insertion of 126 base pairs (bp) in the noncoding area between the sxtA and sxtE genes and a deletion of 6 nucleotides in the sxtN gene. The sxtI gene showed the highest variability and is recommended as the best genetic marker for further phylogenetic studies of the sxt gene cluster of A. gracile. This study confirms for the first time the role of A. gracile as a PSP toxin producer in Norwegian waters, representing the northernmost occurrence of PSP toxin producing A. gracile in Europe known so far.

Matrix effects on a cell-based assay used for the detection of paralytic shellfish toxins in bivalve shellfish samples.

Detecting marine biotoxins such as paralytic shellfish toxins (PSTs) is essential to ensuring the safety of seafood. The mouse bioassay is the internationally accepted method for monitoring PSTs, but technical and ethical issues have led to a search for new detection methods. The mouse neuroblastoma cell-based assay (Neuro-2a CBA) using ouabain and veratridine (O/V) has proven useful for the detection of PSTs. However, CBAs are sensitive to shellfish-associated matrix interferences. As the extraction method highly influences matrix interferences, this study compared three extraction protocols: Association of Official Analytical Chemists (AOAC) 2005.06, AOAC 2011.02 and an alternative liquid-liquid method. These methods were used to assess the matrix effect of extracts from four commercially important bivalve species (Chilean mussel, Magellan mussel, clam and Pacific oyster) in Neuro-2a CBA. Extracts from all three protocols caused a toxic effect in Neuro-2a cells (without O/V) when tested at a concentration of 25 mg of tissue-equivalent (TE) ml(-1). The greatest toxicity was obtained through the AOAC 2011.02 protocol, especially for the Chilean mussel and Pacific oyster extracts. Similar toxicity levels (less than 15%) were observed in all extracts at 3.1 mg TE ml(-1). When assessed in Neuro-2a CBA, AOAC 2005.06 extracts presented the lowest matrix interferences, while the highest interferences were observed for AOAC 2011.02 in Magellan mussel and clam extracts. Finally, the AOAC 2005.06 and alternative protocols were compared using Chilean mussel samples fortified with 40 and 80 µg STX per 100 g meat. The AOAC 2005.06 method demonstrated better results. In conclusion, the AOAC 2005.06 extracts exhibited the fewest interferences in the Neuro-2a CBA. Therefore, this extraction method should be considered for the implementation of Neuro-2a CBA as a high-throughput screening methodology for PST detection.

Comparative proteomics reveals that a saxitoxin-producing and a nontoxic strain of Anabaena circinalis are two different ecotypes.

In Australia, saxitoxin production is restricted to the cyanobacterial species Anabaena circinalis and is strain-dependent. We aimed to characterize a saxitoxin-producing and nontoxic strain of A. circinalis at the proteomic level using iTRAQ. Seven proteins putatively involved in saxitoxin biosynthesis were identified within our iTRAQ experiment for the first time. The proteomic profile of the toxic A. circinalis was significantly different from the nontoxic strain, indicating that each is likely to inhabit a unique ecological niche. Under control growth conditions, the saxitoxin-producing A. circinalis displayed a higher abundance of photosynthetic, carbon fixation and nitrogen metabolic proteins. Differential abundance of these proteins suggests a higher intracellular C:N ratio and a higher concentration of intracellular 2-oxoglutarate in our toxic strain compared with the nontoxic strain. This may be a novel site for posttranslational regulation because saxitoxin biosynthesis putatively requires a 2-oxoglutarate-dependent dioxygenase. The nontoxic A. circinalis was more abundant in proteins, indicating cellular stress. Overall, our study has provided the first insight into fundamental differences between a toxic and nontoxic strain of A. circinalis, indicating that they are distinct ecotypes.

Paralytic shellfish poisoning toxin-producing cyanobacterium Aphanizomenon gracile in northeast Germany.

Neurotoxic paralytic shellfish poisoning (PSP) toxins, anatoxin-a (ATX), and hepatotoxic cylindrospermopsin (CYN) have been detected in several lakes in northeast Germany during the last 2 decades. They are produced worldwide by members of the nostocalean genera Anabaena, Cylindrospermopsis, and Aphanizomenon. Although no additional sources of PSP toxins and ATX have been identified in German water bodies to date, the observed CYN concentrations cannot be produced solely by Aphanizomenon flos-aquae, the only known CYN producer in Germany. Therefore, we attempted to identify PSP toxin, ATX, and CYN producers by isolating and characterizing 92 Anabaena, Aphanizomenon, and Anabaenopsis strains from five lakes in northeast Germany. In a polyphasic approach, all strains were morphologically and phylogenetically classified and then tested for PSP toxins, ATX, and CYN by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA) and screened for the presence of PSP toxin- and CYN-encoding gene fragments. As demonstrated by ELISA and LC-MS, 14 Aphanizomenon gracile strains from Lakes Melang and Scharmützel produced four PSP toxin variants (gonyautoxin 5 [GTX5], decarbamoylsaxitoxin [dcSTX], saxitoxin [STX], and neosaxitoxin [NEO]). GTX5 was the most prevalent PSP toxin variant among the seven strains from Lake Scharmützel, and NEO was the most prevalent among the seven strains from Lake Melang. The sxtA gene, which is part of the saxitoxin gene cluster, was found in the 14 PSP toxin-producing A. gracile strains and in 11 non-PSP toxin-producing Aphanizomenon issatschenkoi, A. flos-aquae, Anabaena planktonica, and Anabaenopsis elenkinii strains. ATX and CYN were not detected in any of the isolated strains. This study is the first confirming the role of A. gracile as a PSP toxin producer in German water bodies.

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

BACKGROUND: 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. RESULTS: We describe the identification, annotation and bioinformatic characterisation of the putative paralytic shellfish toxin biosynthesis clusters in an Australian isolate of Anabaena circinalis and an American isolate of Aphanizomenon sp., both members of the Nostocales. 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 Anabaena circinalis 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. CONCLUSION: 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 sxt 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.

Identification of a saxitoxin biosynthesis gene with a history of frequent horizontal gene transfers.

The paralytic shellfish poisoning (PSP) toxins, saxitoxin, and its derivatives, are produced by a complex and unique biosynthetic pathway. It involves reactions that are rare in other metabolic pathways, however, distantly related organisms, such as dinoflagellates and cyanobacteria, produce these toxins by an identical pathway. Speculative explanations for the unusual phylogenetic distribution of this metabolic pathway have been proposed, including a polyphyletic origin, the involvement of symbiotic bacteria, and horizontal gene transfer. This study describes for the first time the identity of one gene, sxt1, that is involved in the biosynthesis of saxitoxin in cyanobacteria. It encoded an O-carbamoyltransferase (OCTASE) that was proposed to carbamoylate the hydroxymethyl side chain of saxitoxin precursor. Orthologues of sxt1 were exclusively present in PSP-toxic strains of cyanobacteria and had a high sequence similarity to each other. L. wollei had a naturally mutated sxt1 gene that encoded an inactive enzyme, and was incapable of producing carbamoylated PSP-toxin analogues, supporting the proposed function of Sxt1. Phylogenetic analysis revealed that OCATSE genes were present exclusively in prokaryotic organisms and were characterized by a high rate of horizontal gene transfer. OCTASE has most likely evolved from an ancestral O-sialoglycoprotein endopeptidase from proteobacteria, whereas the most likely phylogenetic origin of sxt1 was an ancestral alpha-proteobacterium. The phylogeny of sxt1 suggested that the entire set of genes required for saxitoxin biosynthesis may spread by horizontal gene transfer.

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

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.

The loss of PSP toxin production in a formerly toxic Alexandrium lusitanicum clone.

Toxin production has always been considered a constitutive characteristic of dinoflagellates in the genus Alexandrium. Here we demonstrate that saxitoxin production can be lost by an Alexandrium species during routine culture maintenance. This is the first report of any marine saxitoxin-producing alga ever to have completely lost the ability to produce toxins. A clonal toxic isolate of Alexandrium lusitanicum from Portugal has been maintained in culture since 1962. In 1992, a subculture was established and sent to a different laboratory. Recent comparisons of the parental strain and the subculture revealed that the former had lost its toxicity, whereas the latter still produces saxitoxins. This loss of toxicity was confirmed by three independent toxin detection methods: mouse bioassay, mouse neuroblastoma assay and HPLC. Sequence analyses of different rRNA domains demonstrated that the toxic and non-toxic cultures are genetically identical for those markers. Morphological analysis showed that both cultures have the same plate tabulation and are A. lusitanicum. These results strongly argue that the loss of toxicity is not a result of a culturing artifact or mistake, such as mislabeling or contamination. The clonal cultures also show a significant difference in growth. Possible explanations for the change include genetic mutations or the effects of prolonged treatment of the non-toxic culture with antibiotics.

Identification of thioether intermediates in the reductive transformation of gonyautoxins into saxitoxins by thiols.

O-Sulfate group of gonyautoxin I and IV is transformed into methylene to form neosaxitoxin by thiols such as glutathione, a common cellular scavenger, in mild conditions. We isolated the intermediate of this conversion and propose that this reaction proceeds through formation of thiohemiketal, 1,2 shift to form stable thioether intermediate, and then redox exchange at sulfur atom to form the final product.