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Brett A. Neilan - One of the best experts on this subject based on the ideXlab platform.
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Insertions within the Saxitoxin Biosynthetic Gene Cluster Result in Differential Toxin Profiles
ACS chemical biology, 2018Co-Authors: Alescia Cullen, Susanna A Wood, Paul M. D’agostino, Rabia Mazmouz, Russell Pickford, Brett A. NeilanAbstract:The neurotoxin Saxitoxin and related paralytic shellfish toxins are produced by multiple species of cyanobacteria and dinoflagellates. This study investigates the two Saxitoxin-producing strains of Scytonema crispum, CAWBG524 and CAWBG72, isolated in New Zealand. Each strain was previously reported to have a distinct paralytic shellfish toxin profile, a rare observation between strains within the same species. Sequencing of the Saxitoxin biosynthetic clusters (sxt) from S. crispum CAWBG524 and S. crispum CAWBG72 revealed the largest sxt gene clusters described to date. The distinct toxin profiles of each strain were correlated to genetic differences in sxt tailoring enzymes, specifically the open-reading frame disruption of the N-21 sulfotransferase sxtN, adenylylsulfate kinase sxtO, and the C-11 dioxygenase sxtDIOX within S. crispum CAWBG524 via genetic insertions. Heterologous overexpression of SxtN allowed for the proposal of Saxitoxin and 3′-phosphoadenosine 5′-phosphosulfate as substrate and cofactor...
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Insertions within the Saxitoxin Biosynthetic Gene Cluster Result in Differential Toxin Profiles
2018Co-Authors: Alescia Cullen, Paul M. D’agostino, Rabia Mazmouz, Russell Pickford, Susanna Wood, Brett A. NeilanAbstract:The neurotoxin Saxitoxin and related paralytic shellfish toxins are produced by multiple species of cyanobacteria and dinoflagellates. This study investigates the two Saxitoxin-producing strains of Scytonema crispum, CAWBG524 and CAWBG72, isolated in New Zealand. Each strain was previously reported to have a distinct paralytic shellfish toxin profile, a rare observation between strains within the same species. Sequencing of the Saxitoxin biosynthetic clusters (sxt) from S. crispum CAWBG524 and S. crispum CAWBG72 revealed the largest sxt gene clusters described to date. The distinct toxin profiles of each strain were correlated to genetic differences in sxt tailoring enzymes, specifically the open-reading frame disruption of the N-21 sulfotransferase sxtN, adenylylsulfate kinase sxtO, and the C-11 dioxygenase sxtDIOX within S. crispum CAWBG524 via genetic insertions. Heterologous overexpression of SxtN allowed for the proposal of Saxitoxin and 3′-phosphoadenosine 5′-phosphosulfate as substrate and cofactor, respectively, using florescence binding assays. Further, catalytic activity of SxtN was confirmed by the in vitro conversion of Saxitoxin to the N-21 sulfonated analog gonyautoxin 5, making this the first known report to biochemically confirm the function of a sxt tailoring enzyme. Further, SxtN could not convert neoSaxitoxin to its N-21 sulfonated analog gonyautoxin 6, indicating paralytic shellfish toxin biosynthesis most likely occurs along a predefined route. In this study, we identified key steps toward the biosynthetic conversation of Saxitoxin to other paralytic shellfish toxins
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elevated na and ph influence the production and transport of Saxitoxin in the cyanobacteria anabaena circinalis awqc131c and cylindrospermopsis raciborskii t3
Environmental Microbiology, 2016Co-Authors: Sarah E Ongley, Jasper J L Pengelly, Brett A. NeilanAbstract:Saxitoxins (STX), neurotoxic alkaloids, fall under the umbrella of paralytic shellfish toxins produced by marine dinoflagellates and freshwater cyanobacteria. The genes responsible for the production of STX have been proposed, but factors that influence their expression and induce toxin efflux remain unclear. Here we characterize the putative STX NorM-like MATE transporters SxtF and SxtM. Complementation of the antibiotic-sensitive strain Escherichia coli KAM32 with these transporters decreased fluoroquinolone sensitivity, indicating that while becoming evolutionary specialized for STX transport these transporters retain relaxed specificity typical of this class. The transcriptional response of STX biosynthesis (sxtA) along with that of the STX transporters (sxtM and sxtF from Cylindrospermopsis raciborskii T3, and sxtM from Anabaena circinalis AWQC131C) were assessed in response to ionic stress. These data, coupled with a measure of toxin intracellular to extracellular ratios, provide an insight into the physiology of STX export. Cylindrospermopsis raciborskii and Anabaena circinalis exhibited opposing responses under conditions of ionic stress. High Na(+) (10 mM) induced moderate alterations of transcription and STX localization, whereas high pH (pH 9) stimulated the greatest physiological response. Saxitoxin production and cellular localization are responsive to ionic strength, indicating a role of this molecule in the maintenance of cellular homeostasis.
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proteogenomics of a Saxitoxin producing and non toxic strain of anabaena circinalis cyanobacteria in response to extracellular nacl and phosphate depletion
Environmental Microbiology, 2016Co-Authors: Brett A. Neilan, Xiaomin Song, Paul M Dagostino, Michelle C. MoffittAbstract:In Australia, Saxitoxin production is strain dependent within the bloom-forming freshwater cyanobacterium Anabaena circinalis. Freshwater cyanobacteria are exposed to rapid fluctuations in environmental nutrient concentrations, and their adaption is vital for competition, succession and dominance. Two elements of environmental significance, phosphorus and sodium chloride, are proposed to play a role in bloom development and Saxitoxin biosynthesis respectively. The aim of our study was to comparatively analyse the model Saxitoxin-producing A. circinalis AWQC131C and non-toxic A. circinalis AWQC310F at the genomic level and proteomic level, in response to phosphate depletion and increased extracellular NaCl. When challenged, photosynthesis, carbon/nitrogen metabolisms, transcription/translation, oxidative stress and nutrient transport functional categories demonstrated the largest changes in protein abundance. In response to increased NaCl, SxtC, a protein conserved in all known Saxitoxin biosynthetic pathways, was downregulated. Additionally, toxin quantification revealed a decrease in total Saxitoxin and decarbomoyl-gonyautoxin2/3 content in response to the NaCl treatment. In response to phosphate depletion, the toxic and non-toxic strain displayed similar proteomic profiles, although the toxic strain did not alter the abundance of as many proteins as the non-toxic strain. These findings have important implications for the future, since response and adaption mechanisms are directly related to in situ dominance of cyanobacteria.
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Comparative Proteomics Reveals That a Saxitoxin-Producing and a Nontoxic Strain of Anabaena circinalis Are Two Different Ecotypes
Journal of proteome research, 2014Co-Authors: Paul M. D’agostino, Brett A. Neilan, Xiaomin Song, Michelle C. MoffittAbstract: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-o...
Susanna A Wood - One of the best experts on this subject based on the ideXlab platform.
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Insertions within the Saxitoxin Biosynthetic Gene Cluster Result in Differential Toxin Profiles
ACS chemical biology, 2018Co-Authors: Alescia Cullen, Susanna A Wood, Paul M. D’agostino, Rabia Mazmouz, Russell Pickford, Brett A. NeilanAbstract:The neurotoxin Saxitoxin and related paralytic shellfish toxins are produced by multiple species of cyanobacteria and dinoflagellates. This study investigates the two Saxitoxin-producing strains of Scytonema crispum, CAWBG524 and CAWBG72, isolated in New Zealand. Each strain was previously reported to have a distinct paralytic shellfish toxin profile, a rare observation between strains within the same species. Sequencing of the Saxitoxin biosynthetic clusters (sxt) from S. crispum CAWBG524 and S. crispum CAWBG72 revealed the largest sxt gene clusters described to date. The distinct toxin profiles of each strain were correlated to genetic differences in sxt tailoring enzymes, specifically the open-reading frame disruption of the N-21 sulfotransferase sxtN, adenylylsulfate kinase sxtO, and the C-11 dioxygenase sxtDIOX within S. crispum CAWBG524 via genetic insertions. Heterologous overexpression of SxtN allowed for the proposal of Saxitoxin and 3′-phosphoadenosine 5′-phosphosulfate as substrate and cofactor...
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changes in Saxitoxin production through growth phases in the metaphytic cyanobacterium scytonema cf crispum
Toxicon, 2015Co-Authors: Francine M J Harland, Susanna A Wood, Paul A Broady, Wendy M WilliamsonAbstract:The cyanobacterium Scytonema cf. crispum produces a range of Saxitoxins. Previous studies on other Saxitoxin-producing cyanobacteria have shown that toxin production can vary throughout the growth cycle. Monitoring cyanotoxin-production in S. cf. crispum is challenging because it is metaphytic and has a very slow growth rate (ca. 6 months to reach stationary phase). In this study, a new method was developed to track growth and toxin production in S. cf. crispum. Samples were collected once a week for 131 days, and cell concentrations and Saxitoxin quotas determined. Cells in the lag and exponential growth phases had significantly (P < 0.05) higher Saxitoxin quotas (162 ± 37 fg cell−1 and 139 ± 32 fg cell−1, respectively) than the stationary phases (83 ± 19 fg cell−1). Extracellular Saxitoxin concentrations were present at low concentrations (2–16 ng mL−1 of culture medium) throughout the experiment. The proportion of extracellular Saxitoxin to total Saxitoxin decreased throughout the experiment. New knowledge on growth and Saxitoxin variability will assist in improving monitoring, risk assessment and management of this species.
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survey of scytonema cyanobacteria and associated Saxitoxins in the littoral zone of recreational lakes in canterbury new zealand
Phycologia, 2012Co-Authors: Francine M J Smith, Susanna A Wood, Taryn Wilks, David W Kelly, Paul A Broady, Wendy M WilliamsonAbstract:Smith F.M.J., Wood S.A., Wilks T., Kelly D., Broady P.A., Williamson W. and Gaw S. 2012. Survey of Scytonema (Cyanobacteria) and associated Saxitoxins in the littoral zone of recreational lakes in Canterbury (New Zealand). Phycologia 51: 542–551. DOI: 10.2216/11-84.1 The recent identification of Saxitoxin-producing Scytonema cf. crispum triggered a survey of metaphyton and periphyton for Scytonema spp. in 34 high-use recreational lakes across Canterbury, New Zealand. Scytonema was observed in 10 of the lakes surveyed. Three morphospecies were identified: Scytonema cf. crispum, Scytonema cf. chiastum and Scytonema cf. fritschii. Environmental samples containing Scytonema were analysed for Saxitoxins using the Jellett rapid test for paralytic shellfish poisoning, and Saxitoxin variants were identified in positive samples using high-performance liquid chromatography with fluorescence detection (HPLC–FD). Cultures were established from selected sites and their phylogeny investigated using partial 16S rRNA gen...
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first report of Saxitoxin production by a species of the freshwater benthic cyanobacterium scytonema agardh
Toxicon, 2011Co-Authors: Francine M J Smith, Susanna A Wood, Roel Van Ginkel, Paul A BroadyAbstract:Abstract Saxitoxins or paralytic shellfish poisons (PSP) are neurotoxins produced by some species of freshwater cyanobacteria and marine dinoflagellates. Samples collected from the metaphyton of a drinking-water supply’s pre-treatment reservoir and a small eutrophic lake in New Zealand returned positive results when screened using a Jellett PSP Rapid Test Kit. The dominant species in the sample was identified as Scytonema cf. crispum. A non-axenic clonal culture (UCFS10) was isolated from the lake. The partial 16S rRNA gene sequence shared only a 91% or less sequence similarity with other Scytonema species, indicating that it is unlikely that this genus is monophyletic and that further in-depth phylogenetic re-evaluation is required. The sxtA gene, which is known to be involved in Saxitoxin production, was detected in UCFS10. Saxitoxin concentrations were determined from the lake samples and from UCFS10 using pre-column oxidation high performance liquid chromatography with fluorescence detection. Saxitoxin was the only variant detected and this was found at concentrations of 65.6 μg g−1 dry weight in the lake sample and 119.4 μg g−1 dry weight or 1.3 pg cell−1 in UCFS10. This is the first confirmation of a Saxitoxin-producing species in New Zealand and the first report of Saxitoxin production by a species of Scytonema.
Paul A Broady - One of the best experts on this subject based on the ideXlab platform.
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changes in Saxitoxin production through growth phases in the metaphytic cyanobacterium scytonema cf crispum
Toxicon, 2015Co-Authors: Francine M J Harland, Susanna A Wood, Paul A Broady, Wendy M WilliamsonAbstract:The cyanobacterium Scytonema cf. crispum produces a range of Saxitoxins. Previous studies on other Saxitoxin-producing cyanobacteria have shown that toxin production can vary throughout the growth cycle. Monitoring cyanotoxin-production in S. cf. crispum is challenging because it is metaphytic and has a very slow growth rate (ca. 6 months to reach stationary phase). In this study, a new method was developed to track growth and toxin production in S. cf. crispum. Samples were collected once a week for 131 days, and cell concentrations and Saxitoxin quotas determined. Cells in the lag and exponential growth phases had significantly (P < 0.05) higher Saxitoxin quotas (162 ± 37 fg cell−1 and 139 ± 32 fg cell−1, respectively) than the stationary phases (83 ± 19 fg cell−1). Extracellular Saxitoxin concentrations were present at low concentrations (2–16 ng mL−1 of culture medium) throughout the experiment. The proportion of extracellular Saxitoxin to total Saxitoxin decreased throughout the experiment. New knowledge on growth and Saxitoxin variability will assist in improving monitoring, risk assessment and management of this species.
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survey of scytonema cyanobacteria and associated Saxitoxins in the littoral zone of recreational lakes in canterbury new zealand
Phycologia, 2012Co-Authors: Francine M J Smith, Susanna A Wood, Taryn Wilks, David W Kelly, Paul A Broady, Wendy M WilliamsonAbstract:Smith F.M.J., Wood S.A., Wilks T., Kelly D., Broady P.A., Williamson W. and Gaw S. 2012. Survey of Scytonema (Cyanobacteria) and associated Saxitoxins in the littoral zone of recreational lakes in Canterbury (New Zealand). Phycologia 51: 542–551. DOI: 10.2216/11-84.1 The recent identification of Saxitoxin-producing Scytonema cf. crispum triggered a survey of metaphyton and periphyton for Scytonema spp. in 34 high-use recreational lakes across Canterbury, New Zealand. Scytonema was observed in 10 of the lakes surveyed. Three morphospecies were identified: Scytonema cf. crispum, Scytonema cf. chiastum and Scytonema cf. fritschii. Environmental samples containing Scytonema were analysed for Saxitoxins using the Jellett rapid test for paralytic shellfish poisoning, and Saxitoxin variants were identified in positive samples using high-performance liquid chromatography with fluorescence detection (HPLC–FD). Cultures were established from selected sites and their phylogeny investigated using partial 16S rRNA gen...
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first report of Saxitoxin production by a species of the freshwater benthic cyanobacterium scytonema agardh
Toxicon, 2011Co-Authors: Francine M J Smith, Susanna A Wood, Roel Van Ginkel, Paul A BroadyAbstract:Abstract Saxitoxins or paralytic shellfish poisons (PSP) are neurotoxins produced by some species of freshwater cyanobacteria and marine dinoflagellates. Samples collected from the metaphyton of a drinking-water supply’s pre-treatment reservoir and a small eutrophic lake in New Zealand returned positive results when screened using a Jellett PSP Rapid Test Kit. The dominant species in the sample was identified as Scytonema cf. crispum. A non-axenic clonal culture (UCFS10) was isolated from the lake. The partial 16S rRNA gene sequence shared only a 91% or less sequence similarity with other Scytonema species, indicating that it is unlikely that this genus is monophyletic and that further in-depth phylogenetic re-evaluation is required. The sxtA gene, which is known to be involved in Saxitoxin production, was detected in UCFS10. Saxitoxin concentrations were determined from the lake samples and from UCFS10 using pre-column oxidation high performance liquid chromatography with fluorescence detection. Saxitoxin was the only variant detected and this was found at concentrations of 65.6 μg g−1 dry weight in the lake sample and 119.4 μg g−1 dry weight or 1.3 pg cell−1 in UCFS10. This is the first confirmation of a Saxitoxin-producing species in New Zealand and the first report of Saxitoxin production by a species of Scytonema.
Wendy M Williamson - One of the best experts on this subject based on the ideXlab platform.
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changes in Saxitoxin production through growth phases in the metaphytic cyanobacterium scytonema cf crispum
Toxicon, 2015Co-Authors: Francine M J Harland, Susanna A Wood, Paul A Broady, Wendy M WilliamsonAbstract:The cyanobacterium Scytonema cf. crispum produces a range of Saxitoxins. Previous studies on other Saxitoxin-producing cyanobacteria have shown that toxin production can vary throughout the growth cycle. Monitoring cyanotoxin-production in S. cf. crispum is challenging because it is metaphytic and has a very slow growth rate (ca. 6 months to reach stationary phase). In this study, a new method was developed to track growth and toxin production in S. cf. crispum. Samples were collected once a week for 131 days, and cell concentrations and Saxitoxin quotas determined. Cells in the lag and exponential growth phases had significantly (P < 0.05) higher Saxitoxin quotas (162 ± 37 fg cell−1 and 139 ± 32 fg cell−1, respectively) than the stationary phases (83 ± 19 fg cell−1). Extracellular Saxitoxin concentrations were present at low concentrations (2–16 ng mL−1 of culture medium) throughout the experiment. The proportion of extracellular Saxitoxin to total Saxitoxin decreased throughout the experiment. New knowledge on growth and Saxitoxin variability will assist in improving monitoring, risk assessment and management of this species.
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survey of scytonema cyanobacteria and associated Saxitoxins in the littoral zone of recreational lakes in canterbury new zealand
Phycologia, 2012Co-Authors: Francine M J Smith, Susanna A Wood, Taryn Wilks, David W Kelly, Paul A Broady, Wendy M WilliamsonAbstract:Smith F.M.J., Wood S.A., Wilks T., Kelly D., Broady P.A., Williamson W. and Gaw S. 2012. Survey of Scytonema (Cyanobacteria) and associated Saxitoxins in the littoral zone of recreational lakes in Canterbury (New Zealand). Phycologia 51: 542–551. DOI: 10.2216/11-84.1 The recent identification of Saxitoxin-producing Scytonema cf. crispum triggered a survey of metaphyton and periphyton for Scytonema spp. in 34 high-use recreational lakes across Canterbury, New Zealand. Scytonema was observed in 10 of the lakes surveyed. Three morphospecies were identified: Scytonema cf. crispum, Scytonema cf. chiastum and Scytonema cf. fritschii. Environmental samples containing Scytonema were analysed for Saxitoxins using the Jellett rapid test for paralytic shellfish poisoning, and Saxitoxin variants were identified in positive samples using high-performance liquid chromatography with fluorescence detection (HPLC–FD). Cultures were established from selected sites and their phylogeny investigated using partial 16S rRNA gen...
Kjetill S. Jakobsen - One of the best experts on this subject based on the ideXlab platform.
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Evolution and Distribution of Saxitoxin Biosynthesis in Dinoflagellates
Marine Drugs, 2013Co-Authors: Anke Stüken, Shauna A. Murray, Kjetill S. JakobsenAbstract:Numerous species of marine dinoflagellates synthesize the potent environmental neurotoxic alkaloid, Saxitoxin, the agent of the human illness, paralytic shellfish poisoning. In addition, certain freshwater species of cyanobacteria also synthesize the same toxic compound, with the biosynthetic pathway and genes responsible being recently reported. Three theories have been postulated to explain the origin of Saxitoxin in dinoflagellates: The production of Saxitoxin by co-cultured bacteria rather than the dinoflagellates themselves, convergent evolution within both dinoflagellates and bacteria and horizontal gene transfer between dinoflagellates and bacteria. The discovery of cyanobacterial Saxitoxin homologs in dinoflagellates has enabled us for the first time to evaluate these theories. Here, we review the distribution of Saxitoxin within the dinoflagellates and our knowledge of its genetic basis to determine the likely evolutionary origins of this potent neurotoxin.
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Evolutionary Acquisition and Loss of Saxitoxin Biosynthesis in Dinoflagellates: the Second “Core” Gene, sxtG
Applied and environmental microbiology, 2013Co-Authors: Russell J. S. Orr, Anke Stüken, Shauna A. Murray, Kjetill S. JakobsenAbstract:Saxitoxin and its derivatives are potent neurotoxins produced by several cyanobacteria and dinoflagellate species. SxtA is the initial enzyme in the biosynthesis of Saxitoxin. The dinoflagellate full mRNA and partial genomic sequences have previously been characterized, and it appears that sxtA originated in dinoflagellates through a horizontal gene transfer from a bacterium. So far, little is known about the remaining genes involved in this pathway in dinoflagellates. Here we characterize sxtG, an amidinotransferase enzyme gene that putatively encodes the second step in Saxitoxin biosynthesis. In this study, the entire sxtG transcripts from Alexandrium fundyense CCMP1719 and Alexandrium minutum CCMP113 were amplified and sequenced. The transcripts contained typical dinoflagellate spliced leader sequences and eukaryotic poly(A) tails. In addition, partial sxtG transcript fragments were amplified from four additional Alexandrium species and Gymnodinium catenatum. The phylogenetic inference of dinoflagellate sxtG, congruent with sxtA, revealed a bacterial origin. However, it is not known if sxtG was acquired independently of sxtA. Amplification and sequencing of the corresponding genomic sxtG region revealed noncanonical introns. These introns show a high interspecies and low intraspecies variance, suggesting multiple independent acquisitions and losses. Unlike sxtA, sxtG was also amplified from Alexandrium species not known to synthesize Saxitoxin. However, amplification was not observed for 22 non-Saxitoxin-producing dinoflagellate species other than those of the genus Alexandrium or G. catenatum. This result strengthens our hypothesis that Saxitoxin synthesis has been secondarily lost in conjunction with sxtA for some descendant species.
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Discovery of Nuclear-Encoded Genes for the Neurotoxin Saxitoxin in Dinoflagellates
PloS one, 2011Co-Authors: Anke Stüken, Brett A. Neilan, Shauna A. Murray, Russell J. S. Orr, Ralf Kellmann, Kjetill S. JakobsenAbstract:Saxitoxin is a potent neurotoxin that occurs in aquatic environments worldwide. Ingestion of vector species can lead to paralytic shellfish poisoning, a severe human illness that may lead to paralysis and death. In freshwaters, the toxin is produced by prokaryotic cyanobacteria; in marine waters, it is associated with eukaryotic dinoflagellates. However, several studies suggest that Saxitoxin is not produced by dinoflagellates themselves, but by co-cultured bacteria. Here, we show that genes required for Saxitoxin synthesis are encoded in the nuclear genomes of dinoflagellates. We sequenced >1.2×106 mRNA transcripts from the two Saxitoxin-producing dinoflagellate strains Alexandrium fundyense CCMP1719 and A. minutum CCMP113 using high-throughput sequencing technology. In addition, we used in silico transcriptome analyses, RACE, qPCR and conventional PCR coupled with Sanger sequencing. These approaches successfully identified genes required for Saxitoxin-synthesis in the two transcriptomes. We focused on sxtA, the unique starting gene of Saxitoxin synthesis, and show that the dinoflagellate transcripts of sxtA have the same domain structure as the cyanobacterial sxtA genes. But, in contrast to the bacterial homologs, the dinoflagellate transcripts are monocistronic, have a higher GC content, occur in multiple copies, contain typical dinoflagellate spliced-leader sequences and eukaryotic polyA-tails. Further, we investigated 28 Saxitoxin-producing and non-producing dinoflagellate strains from six different genera for the presence of genomic sxtA homologs. Our results show very good agreement between the presence of sxtA and Saxitoxin-synthesis, except in three strains of A. tamarense, for which we amplified sxtA, but did not detect the toxin. Our work opens for possibilities to develop molecular tools to detect Saxitoxin-producing dinoflagellates in the environment.
