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Brett A. Neilan - One of the best experts on this subject based on the ideXlab platform.
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re evaluation of paralytic Shellfish Toxin profiles in cyanobacteria using hydrophilic interaction liquid chromatography tandem mass spectrometry
Toxicon, 2019Co-Authors: Tim Harwood, Wayne W. Carmichael, Brett A. Neilan, Paul M Dagostino, Michael J Boundy, Susanna A WoodAbstract:Abstract To date Paralytic Shellfish Toxin (PST) variants in cyanobacteria have primarily been characterized using high performance liquid chromatography coupled with fluorescence detection. In this study we re-evaluated the PST profiles of five cyanobacterial cultures (Dolichospermum circinale AWQC131C, Aphanizomenon sp. NH-5, Raphidiopsis raciborskii T3, Scytonema cf. crispum CAWBG524 and CAWBG72) and one environmental sample (Microseria wollei) using hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry. A total of 35 different PST variants were detected. D. circinale contained the highest number of variants (23), followed by S. cf. crispum CAWBG72 (21). Many of the variants detected in the cultures/environmental sample had not been reported from these strains previously: D. circinale (14 variants), S. cf. crispum CAWBG72 (16), S. cf. crispum CAWBG524 (9), Aphanizomenon sp. (9), R. raciborskii (7), and M. wollei (7). Of particular interest was the detection of M-Toxins (Aphanizomenon sp., R. raciborskii, D. circinale). These have previously only been identified from Shellfish where they were thought to be metabolites. Well-characterized PST variant profiles are essential for research investigating the genetic basis of PST production, and given that the toxicity of each variants differs, it will assist in refining risk assessments.
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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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A putative gene cluster from a Lyngbya wollei bloom that encodes paralytic Shellfish Toxin biosynthesis.
PLOS ONE, 2011Co-Authors: Troco Kaan Mihali, Wayne W. Carmichael, Brett A. NeilanAbstract: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.
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Characterisation of the paralytic Shellfish Toxin biosynthesis gene clusters in Anabaena circinalis AWQC131C and Aphanizomenon sp. NH-5
BMC Biochemistry, 2009Co-Authors: Troco Kaan Mihali, Ralf Kellmann, Brett A. NeilanAbstract: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.
Yasukatsu Oshima - One of the best experts on this subject based on the ideXlab platform.
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synthesis of a tricyclic bisguanidine compound structurally related to saxiToxin and its identification in paralytic Shellfish Toxin producing microorganisms
Chemistry: A European Journal, 2015Co-Authors: Shigeki Tsuchiya, Yasukatsu Oshima, Yuko Cho, Keiichi Konoki, Kazuo Nagasawa, Mari YotsuyamashitaAbstract:We recently reported the chemical synthesis and identification of the genetically predicted biosynthetic intermediates of saxiToxin (STX), including a 2-aminoimidazole-bearing monoguanidine compound (Int-C'2) in two paralytic Shellfish Toxin (PST)-producing microorganisms. In this study, we achieved the direct conversion of Int-C'2 into a tricyclic bisguanidine compound (called Cyclic-C'), which is structurally related to STX, through oxidative intramolecular guanidine transfer to 2-aminoimidazole catalyzed by Pd/C under basic conditions in air. By using HPLC-MS analysis, Cyclic-C' was also identified in the PST-producing microorganisms, suggesting that Cyclic-C' is either another biosynthetic intermediate or a shunt product of PSTs. In addition, a weak inhibitory activity of Cyclic-C' to the voltage-gated sodium channels was detected by using a cell-based assay.
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single cell analysis of paralytic Shellfish Toxins in alexandrium tamarense by hplc with post column fluorescent derivatization
Harmful Algae, 2013Co-Authors: Ryoko Ozeki, Mari Yotsuyamashita, Yasukatsu OshimaAbstract:Abstract We developed a methodology for analyzing the C-Toxin (C2) content in single Alexandrium tamarense cells; this method was based on high performance liquid chromatography (HPLC). C2 is the main paralytic Shellfish Toxin (PST) detected in a clonal culture of A. tamarense , which is a common causative organism in cases of paralytic Shellfish poisoning in Japan. This HPLC method employs post-column fluorescent derivatization (FL). Mobile phase, column size, flow rate, reagent concentrations, and lamp type for the fluorescent detector were all optimized for the detection of C2. With this improved methodology, we could measure 1 fmol of C2 with a signal to noise ratio (S/N) = 2. Clonal heterogeneity within the toxic strain, which was maintained for 13 years after re-isolation from the original clonal culture, ranged from −1 . This report is the first to demonstrate definitively that PST content varies on a cell-by-cell basis in a clonal culture of a dinoflagellate that causes paralytic Shellfish poisoning.
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effects of mitomycin c and colchicine on Toxin production and cell cycle regulation in the dinoflagellate alexandrium tamarense
Harmful Algae, 2011Co-Authors: Motoo Ogawa, Mayumi Hirota, Yasukatsu OshimaAbstract:Abstract Paralytic Shellfish Toxin (PST) production and cell proliferation in the dinoflagellate Alexandrium tamarense were investigated under the influence of two metabolic inhibitors (mitomycin C and colchicine) that have different mechanisms of action. Intracellular PST levels in cells treated with 2 μM mitomycin C increased gradually, reaching a maximum of 176 fmol/cell (a 6-fold increase). High concentrations of colchicine prolonged G 1 phase in A. tamarense cells, even though colchicine arrests several other eukaryotic cell types in M phase. The cells in prolonged G 1 phase under the influence of colchicine were apparently unable to produce PST. Cell proliferation and Toxin production recovered after removal of colchicine; in contrast, the effect of mitomycin C was irreversible. While the timing of Toxin production within the cell cycle was not conclusively determined, A. tamarense cells in S phase were able to produce PST.
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purification and characterization of paralytic Shellfish Toxin transforming enzyme sulfocarbamoylase i from the japanese bivalve peronidia venulosa
Biochimica et Biophysica Acta, 2008Co-Authors: Yuko Cho, Noriyuki Ogawa, Miyako Takahashi, Hsipin Lin, Yasukatsu OshimaAbstract:The Japanese bivalve Peronidia venulosa contains paralytic Shellfish Toxin (PST)-transforming enzymes that convert the weakly toxic C-Toxins to the more potent decarbamoyl Toxins. The enzyme was purified 154-fold with a yield of 0.26% and was named sulfocarbamoylase I. It was found to be a protein with an estimated molecular weight of 300 kDa by gel filtration column chromatography. Observation of a single band equivalent to 150 kDa on SDS-PAGE with or without reducing agents suggested it to be a homodimer with ionically bound subunits. The enzyme catalyzes the hydrolysis of the carboxyl bond in the N-sulfocarbamoyl moiety of PSP-Toxins. The sulfonyl moiety in the carbamoyl side chain of substrates is essential for enzyme recognition. The N-terminal amino acid sequences of nine tryptic peptides were determined by the Edman degradation method. In a database search using the BLAST program, no protein that shows remarkable homology was retrieved. Several characteristics of the enzyme were also compared with those of another PST-transforming enzyme, carbamoylase I, which was previously isolated from the Japanese clam Mactra chinensis.
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purification and characterization of paralytic Shellfish Toxin transforming enzyme from mactra chinensis
Toxicon, 2004Co-Authors: Hsipin Lin, Yuko Cho, Hitoshi Yashiro, Tomoko Yamada, Yasukatsu OshimaAbstract:Abstract A carbamoylase, which catalyzes hydrolysis of the carbamoyl (or N -sulfocarbamoyl) moiety of paralytic Shellfish Toxins, was purified from the digestive glands of the Japanese clam Mactra chinensis . Using five steps of column chromatography, 290 μg of Carbamoylase I showing homogeneity on SDS-PAGE was obtained. Carbamoylase I was revealed to be a glycoprotein, having estimated molecular weight of 190 kDa. Observation of single band equivalent to 94 kDa on SDS-PAGE under reducing conditions suggested it to be a homodimer. The optimal temperature and pH were 20 °C and 7.0. Carbamoylase I did not require a divalent cation and its activity was inhibited by the serine proteinase inhibitors, benzenesulfonyl fluoride and 4-(2-aminoethyl)-benzenesulfonyl fluoride. Carbamoylase I hydrolyzed both carbamate and N -sulfocarbamate Toxins. The presence or absence of a hydroxyl moiety at the N-1 position of the substrate Toxins did not significantly alter the reaction rate, but the stereochemistry of sulfate esters at C-11 greatly affected it. The K m was 3.02 μM for saxiToxin as a substrate. Nineteen amino acids of the N-terminal sequence were identified by the Edman method. MALDI-TOF-MS/MS spectra of 18 O-labeled tryptic peptides indicated the possible internal amino acid sequences of five peptides.
Christopher J S Bolch - One of the best experts on this subject based on the ideXlab platform.
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qpcr assays for the detection and quantification of multiple paralytic Shellfish Toxin producing species of alexandrium
Frontiers in Microbiology, 2018Co-Authors: Rendy Ruvindy, Christopher J S Bolch, Lincoln Mackenzie, Kirsty F Smith, Shauna A MurrayAbstract:Paralytic Shellfish Toxin producing dinoflagellates have negatively impacted the Shellfish aquaculture industry worldwide, including in Australia and New Zealand. Morphologically identical cryptic species of dinoflagellates that may differ in toxicity, in particular, species of the former Alexandrium tamarense species complex, co-occur in Australia, as they do in multiple regions in Asia and Europe. To understand the dynamics and the ecological drivers of the growth of each species in the field, accurate quantification at the species level is crucial. We have developed the first quantitative polymerase chain reaction (qPCR) primers for A. australiense, and new primers targeting A. ostenfeldii, A. catenella, and A. pacificum. We showed that our new primers for A. pacificum are more specific than previously published primer pairs. These assays can be used to quantify planktonic cells and cysts in the water column and in sediment samples with limits of detection of 2 cells/L for the A. catenella and A. australiense assays, 2 cells/L and 1 cyst/mg sediment for the A. pacificum assay, and 1 cells/L for the A. ostenfeldii assay, and efficiencies of >90%. We utilized these assays to discriminate and quantify co-occurring A. catenella, A. pacificum, and A. australiense in samples from the east coast of Tasmania, Australia.
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marinobacter algicola sp nov isolated from laboratory cultures of paralytic Shellfish Toxin producing dinoflagellates
International Journal of Systematic and Evolutionary Microbiology, 2006Co-Authors: David H Green, Tony Gutierrez, J P Bowman, Elizabeth A Smith, Christopher J S BolchAbstract:Phylogenetic and phenotypic analysis of cultivable marine bacteria isolated from laboratory cultures of two paralytic Shellfish Toxin-producing dinoflagellates, Gymnodinium catenatum and Alexandrium tamarense, showed the presence of a novel group of Gram-negative, aerobic, moderately halophilic and hydrocarbon-degrading bacteria, related to the genus Marinobacter. The strains, designated DG893T, DG1136 and ATAM407-13, grew optimally in media with 3-6 % NaCl and at 25-30 degrees C, and all could utilize n-hexadecane and n-tetradecane as the sole carbon source. The strains had a 16S rRNA gene sequence similarity of 94.2-94.3 % to Marinobacter hydrocarbonoclasticus ATCC 27132, and a similarity of 97.5-97.8 % to the closest phylogenetically related type strain, Marinobacter flavimaris DSM 16070T. DNA-DNA hybridization levels to M. flavimaris and other Marinobacter type strains were or = 83 %. The DNA G + C content was 54-55 mol% and the major isoprenoid quinone was ubiquinone-9. On the basis of phenotypic, chemotaxonomic, DNA-DNA hybridization and phylogenetic analysis, it is proposed that these three strains represent a novel species, Marinobacter algicola sp. nov. The type strain is DG893T (= DSM 16394T = NCIMB 14009T).
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marinobacter algicola sp nov isolated from laboratory cultures of paralytic Shellfish Toxin producing dinoflagellates
International Journal of Systematic and Evolutionary Microbiology, 2006Co-Authors: David H Green, Tony Gutierrez, J P Bowman, Elizabeth A Smith, Christopher J S BolchAbstract:Phylogenetic and phenotypic analysis of cultivable marine bacteria isolated from laboratory cultures of two paralytic Shellfish Toxin-producing dinoflagellates, Gymnodinium catenatum and Alexandrium tamarense, showed the presence of a novel group of Gram-negative, aerobic, moderately halophilic and hydrocarbon-degrading bacteria, related to the genus Marinobacter. The strains, designated DG893T, DG1136 and ATAM407-13, grew optimally in media with 3–6 % NaCl and at 25–30 °C, and all could utilize n-hexadecane and n-tetradecane as the sole carbon source. The strains had a 16S rRNA gene sequence similarity of 94·2–94·3 % to Marinobacter hydrocarbonoclasticus ATCC 27132, and a similarity of 97·5–97·8 % to the closest phylogenetically related type strain, Marinobacter flavimaris DSM 16070T. DNA–DNA hybridization levels to M. flavimaris and other Marinobacter type strains were ⩽42 %, while DNA–DNA reassociation values among DG893T, DG1136 and ATAM407-13 were ⩾83 %. The DNA G+C content was 54–55 mol% and the major isoprenoid quinone was ubiquinone-9. On the basis of phenotypic, chemotaxonomic, DNA–DNA hybridization and phylogenetic analysis, it is proposed that these three strains represent a novel species, Marinobacter algicola sp. nov. The type strain is DG893T (=DSM 16394T=NCIMB 14009T).
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Toxin composition of resting cysts of alexandrium tamarense dinophyceae
Toxicon, 1992Co-Authors: Yasukatsu Oshima, Christopher J S Bolch, Gustaaf M. HallegraeffAbstract:Paralytic Shellfish Toxin composition in the resting cysts of the dinoflagellate Alexandrium tamarense was investigated by means of high performance liquid chromatography. A comparison was made between cysts collected from ship ballast tank sediments, natural population of motile vegetative cells collected from the area where ballast water was taken, as well as cultured vegetative cells established from the cysts and the natural plankton bloom. Total Toxin concentration of the cysts (595 fmole/cell) was six-fold higher than that of the natural population of vegetative cells. They contained the same ten toxic components but in different relative abundances. The higher proportion of 11-α-hydroxysulfate epimers in the cysts suggests that the biosynthesis of Toxins is halted at an early stage in cyst formation.
Antonio Quesada - One of the best experts on this subject based on the ideXlab platform.
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phylogeography of cylindrospermopsin and paralytic Shellfish Toxin producing nostocales cyanobacteria from mediterranean europe spain
Applied and Environmental Microbiology, 2014Co-Authors: Samuel Cirés, Ramsy Agha, Mar Ia Cristina Casero, Claudia Wiedner, Andreas Ballot, Lars Wörmer, David Velázquez, Antonio QuesadaAbstract:Planktonic Nostocales cyanobacteria represent a challenge for microbiological research because of the wide range of cyanoToxins that they synthesize and their invasive behavior, which is presumably enhanced by global warming. To gain insight into the phylogeography of potentially toxic Nostocales from Mediterranean Europe, 31 strains of Anabaena (Anabaena crassa, A. lemmermannii, A. mendotae, and A. planctonica), Aphanizomenon (Aphanizomenon gracile, A. ovalisporum), and Cylindrospermopsis raciborskii were isolated from 14 freshwater bodies in Spain and polyphasically analyzed for their phylogeography, cyanoToxin production, and the presence of cyanoToxin biosynthesis genes. The potent cytoToxin cylindrospermopsin (CYN) was produced by all 6 Aphanizomenon ovalisporum strains at high levels (5.7 to 9.1 μg CYN mg−1 [dry weight]) with low variation between strains (1.5 to 3.9-fold) and a marked extracellular release (19 to 41% dissolved CYN) during exponential growth. Paralytic Shellfish poisoning (PSP) neuroToxins (saxiToxin, neosaxiToxin, and decarbamoylsaxiToxin) were detected in 2 Aphanizomenon gracile strains, both containing the sxtA gene. This gene was also amplified in non-PSP Toxin-producing Aphanizomenon gracile and Aphanizomenon ovalisporum. Phylogenetic analyses supported the species identification and confirmed the high similarity of Spanish Anabaena and Aphanizomenon strains with other European strains. In contrast, Cylindrospermopsis raciborskii from Spain grouped together with American strains and was clearly separate from the rest of the European strains, raising questions about the current assumptions of the phylogeography and spreading routes of C. raciborskii. The present study confirms that the nostocalean genus Aphanizomenon is a major source of CYN and PSP Toxins in Europe and demonstrates the presence of the sxtA gene in CYN-producing Aphanizomenon ovalisporum.
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phylogeography of cylindrospermopsin and paralytic Shellfish Toxin producing nostocales cyanobacteria from mediterranean europe spain
Applied and Environmental Microbiology, 2014Co-Authors: Samuel Cirés, Ramsy Agha, Mar Ia Cristina Casero, Claudia Wiedner, Andreas Ballot, Lars Wörmer, David Velázquez, Antonio QuesadaAbstract:Planktonic Nostocales cyanobacteria represent a challenge for microbiological research because of the wide range of cyanoToxins that they synthesize and their invasive behavior, which is presumably enhanced by global warming. To gain insight into the phylogeography of potentially toxic Nostocales from Mediterranean Europe, 31 strains of Anabaena (Anabaena crassa, A. lemmermannii, A. mendotae, and A. planctonica), Aphanizomenon (Aphanizomenon gracile, A. ovalisporum), and Cylindrospermopsis raciborskii were isolated from 14 freshwater bodies in Spain and polyphasically analyzed for their phylogeography, cyanoToxin production, and the presence of cyanoToxin biosynthesis genes. The potent cytoToxin cylindrospermopsin (CYN) was produced by all 6 Aphanizomenon ovalisporum strains at high levels (5.7 to 9.1 μg CYN mg−1 [dry weight]) with low variation between strains (1.5 to 3.9-fold) and a marked extracellular release (19 to 41% dissolved CYN) during exponential growth. Paralytic Shellfish poisoning (PSP) neuroToxins (saxiToxin, neosaxiToxin, and decarbamoylsaxiToxin) were detected in 2 Aphanizomenon gracile strains, both containing the sxtA gene. This gene was also amplified in non-PSP Toxin-producing Aphanizomenon gracile and Aphanizomenon ovalisporum. Phylogenetic analyses supported the species identification and confirmed the high similarity of Spanish Anabaena and Aphanizomenon strains with other European strains. In contrast, Cylindrospermopsis raciborskii from Spain grouped together with American strains and was clearly separate from the rest of the European strains, raising questions about the current assumptions of the phylogeography and spreading routes of C. raciborskii. The present study confirms that the nostocalean genus Aphanizomenon is a major source of CYN and PSP Toxins in Europe and demonstrates the presence of the sxtA gene in CYN-producing Aphanizomenon ovalisporum.
Lars Wörmer - One of the best experts on this subject based on the ideXlab platform.
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phylogeography of cylindrospermopsin and paralytic Shellfish Toxin producing nostocales cyanobacteria from mediterranean europe spain
Applied and Environmental Microbiology, 2014Co-Authors: Samuel Cirés, Ramsy Agha, Mar Ia Cristina Casero, Claudia Wiedner, Andreas Ballot, Lars Wörmer, David Velázquez, Antonio QuesadaAbstract:Planktonic Nostocales cyanobacteria represent a challenge for microbiological research because of the wide range of cyanoToxins that they synthesize and their invasive behavior, which is presumably enhanced by global warming. To gain insight into the phylogeography of potentially toxic Nostocales from Mediterranean Europe, 31 strains of Anabaena (Anabaena crassa, A. lemmermannii, A. mendotae, and A. planctonica), Aphanizomenon (Aphanizomenon gracile, A. ovalisporum), and Cylindrospermopsis raciborskii were isolated from 14 freshwater bodies in Spain and polyphasically analyzed for their phylogeography, cyanoToxin production, and the presence of cyanoToxin biosynthesis genes. The potent cytoToxin cylindrospermopsin (CYN) was produced by all 6 Aphanizomenon ovalisporum strains at high levels (5.7 to 9.1 μg CYN mg−1 [dry weight]) with low variation between strains (1.5 to 3.9-fold) and a marked extracellular release (19 to 41% dissolved CYN) during exponential growth. Paralytic Shellfish poisoning (PSP) neuroToxins (saxiToxin, neosaxiToxin, and decarbamoylsaxiToxin) were detected in 2 Aphanizomenon gracile strains, both containing the sxtA gene. This gene was also amplified in non-PSP Toxin-producing Aphanizomenon gracile and Aphanizomenon ovalisporum. Phylogenetic analyses supported the species identification and confirmed the high similarity of Spanish Anabaena and Aphanizomenon strains with other European strains. In contrast, Cylindrospermopsis raciborskii from Spain grouped together with American strains and was clearly separate from the rest of the European strains, raising questions about the current assumptions of the phylogeography and spreading routes of C. raciborskii. The present study confirms that the nostocalean genus Aphanizomenon is a major source of CYN and PSP Toxins in Europe and demonstrates the presence of the sxtA gene in CYN-producing Aphanizomenon ovalisporum.
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phylogeography of cylindrospermopsin and paralytic Shellfish Toxin producing nostocales cyanobacteria from mediterranean europe spain
Applied and Environmental Microbiology, 2014Co-Authors: Samuel Cirés, Ramsy Agha, Mar Ia Cristina Casero, Claudia Wiedner, Andreas Ballot, Lars Wörmer, David Velázquez, Antonio QuesadaAbstract:Planktonic Nostocales cyanobacteria represent a challenge for microbiological research because of the wide range of cyanoToxins that they synthesize and their invasive behavior, which is presumably enhanced by global warming. To gain insight into the phylogeography of potentially toxic Nostocales from Mediterranean Europe, 31 strains of Anabaena (Anabaena crassa, A. lemmermannii, A. mendotae, and A. planctonica), Aphanizomenon (Aphanizomenon gracile, A. ovalisporum), and Cylindrospermopsis raciborskii were isolated from 14 freshwater bodies in Spain and polyphasically analyzed for their phylogeography, cyanoToxin production, and the presence of cyanoToxin biosynthesis genes. The potent cytoToxin cylindrospermopsin (CYN) was produced by all 6 Aphanizomenon ovalisporum strains at high levels (5.7 to 9.1 μg CYN mg−1 [dry weight]) with low variation between strains (1.5 to 3.9-fold) and a marked extracellular release (19 to 41% dissolved CYN) during exponential growth. Paralytic Shellfish poisoning (PSP) neuroToxins (saxiToxin, neosaxiToxin, and decarbamoylsaxiToxin) were detected in 2 Aphanizomenon gracile strains, both containing the sxtA gene. This gene was also amplified in non-PSP Toxin-producing Aphanizomenon gracile and Aphanizomenon ovalisporum. Phylogenetic analyses supported the species identification and confirmed the high similarity of Spanish Anabaena and Aphanizomenon strains with other European strains. In contrast, Cylindrospermopsis raciborskii from Spain grouped together with American strains and was clearly separate from the rest of the European strains, raising questions about the current assumptions of the phylogeography and spreading routes of C. raciborskii. The present study confirms that the nostocalean genus Aphanizomenon is a major source of CYN and PSP Toxins in Europe and demonstrates the presence of the sxtA gene in CYN-producing Aphanizomenon ovalisporum.