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Annick Mejean - One of the best experts on this subject based on the ideXlab platform.
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Characterization of CyrI, the hydroxylase involved in the last step of cylindrospermopsin biosynthesis: Binding studies, site-directed mutagenesis and stereoselectivity
Archives of Biochemistry and Biophysics, 2018Co-Authors: Rabia Mazmouz, Insaf Essadik, Annick Mejean, Djemel Hamdane, Olivier PlouxAbstract:Abstract Cylindrospermopsin, a cytotoxin from cyanobacteria, is biosynthesized by a complex pathway, which involves CyrI, an iron and 2-oxoglutarate dependent hydroxylase that transforms 7-deoxy-cylindrospermopsin into cylindrospermopsin and its epimer, 7- epi -cylindrospermopsin, in the last step. The activity of CyrI from Oscillatoria sp. PCC 7926 depends on Fe(II) ( K m = 2.1 μM), and 2-oxoglutarate ( K m = 3.2 μM), and is strongly inhibited by 7-deoxy-cylindrospermopsin at concentration higher than 1 μM. Using tryptophan fluorescence, we measured the binding to CyrI of Fe(II) ( K D = 0.02 μM) and 2-oxoglutarate ( K D = 53 μM and K D = 1.1 μM in the absence or presence of 10 μM Fe(II), respectively). The Oscillatoria sp. PCC 6506 CyrI mutants H157A, D159A, H247A, and R257A were all inactive, and impaired in the binding of Fe(II) or 2-oxoglutarate, confirming the identity of the iron ligands and the role of R257 in the binding of 2-oxoglutarate. We constructed several chimeric enzymes using the Oscillatoria sp. PCC 7926 CyrI protein (stereoselective) and that from Oscillatoria sp. PCC 6506 (not stereoselective) to help understanding the structural factors that influence the stereoselectivity of the hydroxylation. Our data suggest that a predicted α-helix in CyrI (positions 87–108) seems to modulate the stereoselectivity of the reaction.
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biosynthesis of cylindrospermopsin and 7 epicylindrospermopsin in Oscillatoria sp strain pcc 6506 identification of the cyr gene cluster and toxin analysis
Applied and Environmental Microbiology, 2010Co-Authors: Rabia Mazmouz, Florence Chapuishugon, Stephane Mann, Annick Mejean, Valérie Pichon, Olivier PlouxAbstract:Cylindrospermopsin is a cytotoxin produced by Cylindrospermopsis raciborskii and other cyanobacteria that has been implicated in human intoxications. We report here the complete sequence of the gene cluster responsible for the biosynthesis of this toxin in Oscillatoria sp. strain PCC 6506. This cluster of genes was found to be homologous with that of C. raciborskii but with a different gene organization. Using an enzyme-linked immunosorbent assay and an optimized liquid chromatography analytical method coupled to tandem mass spectrometry, we detected 7-epicylindrospermopsin, cylindrospermopsin, and 7-deoxycylindrospermopsin in the culture medium of axenic Oscillatoria PCC 6506 at the following relative concentrations: 68.6%, 30.2%, and 1.2%, respectively. We measured the intracellular and extracellular concentrations, per mg of dried cells of Oscillatoria PCC 6506, of 7-epicylindrospermopsin (0.18 μg/mg and 0.29 μg/mg, respectively) and cylindrospermopsin (0.10 μg/mg and 0.11 μg/mg, respectively). We showed that these two toxins accumulated in the culture medium of Oscillatoria PCC 6506 but that the ratio (2.5 ± 0.3) was constant with 7-epicylindrospermopsin being the major metabolite. We also determined the concentrations of these toxins in culture media of other Oscillatoria strains, PCC 6407, PCC 6602, PCC 7926, and PCC 10702, and found that, except for PCC 6602, they all produced 7-epicylindrospermopsin and cylindrospermopsin, with the former being the major toxin, except for PCC 7926, which produced very little 7-epicylindrospermopsin. All the cylindrospermopsin producers studied gave a PCR product using specific primers for the amplification of the cyrJ gene from genomic DNA.
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identification of a polyketide synthase coding sequence specific for anatoxin a producing Oscillatoria cyanobacteria
Applied and Environmental Microbiology, 2009Co-Authors: Sabrina Cadelsix, Stephane Mann, Olivier Ploux, Annick Mejean, Isabelle Iteman, Caroline PeyraudthomasAbstract:We report the identification of a sequence from the genome of Oscillatoria sp. strain PCC 6506 coding for a polyketide synthase. Using 50 axenic cyanobacteria, we found this sequence only in the genomes of Oscillatoria strains producing anatoxin-a or homoanatoxin-a, indicating its likely involvement in the biosynthesis of these toxins.
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Different genotypes of anatoxin-producing cyanobacteria coexist in the Tarn river, France.
Applied and Environmental Microbiology, 2007Co-Authors: Sabrina Cadel-six, Caroline Peyraud-thomas, Luc Brient, Nicole Tandeau De Marsac, Rosmarie Rippka, Annick MejeanAbstract:Repeated dog deaths occurred in 2002, 2003, and 2005 after the animals drank water from the shoreline of the Tarn River in southern France. Signs of intoxication indicated acute poisoning due to a neurotoxin. Floating scum and biofilms covering pebbles were collected in the summers of 2005 and 2006 from six different sites along 30 km from the border of this river. The cyanobacterial neurotoxic alkaloid anatoxin-a and/or its methyl homolog, homoanatoxin-a, was detected in the extracts of most samples examined by gas chromatography-mass spectrometry. Fifteen filamentous cyanobacteria of the order Oscillatoriales were isolated and displayed four distinct phenotypes based on morphological characteristics and pigmentation. Three of the phenotypes can be assigned to the genus Oscillatoria or Phormidium, depending on the taxonomic treatises (bacteriological/botanical) employed for identification. The fourth phenotype is typical of the genus Geitlerinema Anagnostidis 1989. Eight strains rendered axenic were analyzed for production of anatoxin-a and homoanatoxin-a, and all strains of Oscillatoria/Phormidium proved to be neurotoxic. The genetic relatedness of the new isolates was evaluated by comparison of the intergenic transcribed spacer sequences with those of six Oscillatorian strains from the Pasteur Culture Collection of Cyanobacteria. These analyses showed that the neurotoxic representatives are composed of five different genotypes, three of which correspond to phenotypes isolated in this study. Our findings prove that neurotoxic Oscillatorian cyanobacteria exist in the Tarn River and thus were most likely implicated in the reported dog poisonings. Furthermore, they reemphasize the importance of monitoring benthic cyanobacteria in aquatic environments to fully assess the health risks associated with these organisms.
Jun Chen - One of the best experts on this subject based on the ideXlab platform.
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Physiological differences between free-floating and periphytic filamentous algae, and specific submerged macrophytes induce proliferation of filamentous algae: A novel implication for lake restoration.
Chemosphere, 2019Co-Authors: Weizhen Zhang, Hong Shen, Jia Zhang, Ping Xie, Jun ChenAbstract:Abstract Restoration of submerged macrophytes is widely applied to counteract eutrophication in shallow lakes. However, proliferation and accumulation of filamentous algae (possessing free-floating and periphytic life forms) hamper growth of submerged macrophytes. Here, we explored factors triggering the excessive proliferation of filamentous algae during lake restoration using field investigations and laboratory experiments. Results showed that, compared with free-floating Oscillatoria sp. (FO), periphytic Oscillatoria sp. (PO) showed faster growth rate, greater photosynthetic capacities and higher phosphorus (P) affinity. Therefore, PO was physiologically competitively superior to FO under low P concentration and improved light conditions. And proliferation of filamentous algae was mainly manifested in periphytic life form. Besides, field results showed that density of filamentous algae in water column might be related to substrate types. Some macrophyte (Ceratophyllum oryzetorum and Potamogeton crispus) might provide proper substrates for proliferation of filamentous algae. Further physiological experiments found that Oscillatoria showed specific eco-physiological responses to different macrophyte species. Hydrilla verticillata and C. oryzetorum promoted growth and photosynthetic activity of Oscillatoria, while Potamogeton malaianus inhibited growth and P uptake of PO. Myriophyllum spicatum exhibited no impact on growth of Oscillatoria. Our results revealed the intrinsic (physiological differences between free-floating and periphytic life forms of filamentous algae) and extrinsic (different macrophytes) factors affect the proliferation of filamentous algae, which are important for guidance on planting of submerged macrophytes during lake restoration.
Olivier Ploux - One of the best experts on this subject based on the ideXlab platform.
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Characterization of CyrI, the hydroxylase involved in the last step of cylindrospermopsin biosynthesis: Binding studies, site-directed mutagenesis and stereoselectivity
Archives of Biochemistry and Biophysics, 2018Co-Authors: Rabia Mazmouz, Insaf Essadik, Annick Mejean, Djemel Hamdane, Olivier PlouxAbstract:Abstract Cylindrospermopsin, a cytotoxin from cyanobacteria, is biosynthesized by a complex pathway, which involves CyrI, an iron and 2-oxoglutarate dependent hydroxylase that transforms 7-deoxy-cylindrospermopsin into cylindrospermopsin and its epimer, 7- epi -cylindrospermopsin, in the last step. The activity of CyrI from Oscillatoria sp. PCC 7926 depends on Fe(II) ( K m = 2.1 μM), and 2-oxoglutarate ( K m = 3.2 μM), and is strongly inhibited by 7-deoxy-cylindrospermopsin at concentration higher than 1 μM. Using tryptophan fluorescence, we measured the binding to CyrI of Fe(II) ( K D = 0.02 μM) and 2-oxoglutarate ( K D = 53 μM and K D = 1.1 μM in the absence or presence of 10 μM Fe(II), respectively). The Oscillatoria sp. PCC 6506 CyrI mutants H157A, D159A, H247A, and R257A were all inactive, and impaired in the binding of Fe(II) or 2-oxoglutarate, confirming the identity of the iron ligands and the role of R257 in the binding of 2-oxoglutarate. We constructed several chimeric enzymes using the Oscillatoria sp. PCC 7926 CyrI protein (stereoselective) and that from Oscillatoria sp. PCC 6506 (not stereoselective) to help understanding the structural factors that influence the stereoselectivity of the hydroxylation. Our data suggest that a predicted α-helix in CyrI (positions 87–108) seems to modulate the stereoselectivity of the reaction.
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biosynthesis of cylindrospermopsin and 7 epicylindrospermopsin in Oscillatoria sp strain pcc 6506 identification of the cyr gene cluster and toxin analysis
Applied and Environmental Microbiology, 2010Co-Authors: Rabia Mazmouz, Florence Chapuishugon, Stephane Mann, Annick Mejean, Valérie Pichon, Olivier PlouxAbstract:Cylindrospermopsin is a cytotoxin produced by Cylindrospermopsis raciborskii and other cyanobacteria that has been implicated in human intoxications. We report here the complete sequence of the gene cluster responsible for the biosynthesis of this toxin in Oscillatoria sp. strain PCC 6506. This cluster of genes was found to be homologous with that of C. raciborskii but with a different gene organization. Using an enzyme-linked immunosorbent assay and an optimized liquid chromatography analytical method coupled to tandem mass spectrometry, we detected 7-epicylindrospermopsin, cylindrospermopsin, and 7-deoxycylindrospermopsin in the culture medium of axenic Oscillatoria PCC 6506 at the following relative concentrations: 68.6%, 30.2%, and 1.2%, respectively. We measured the intracellular and extracellular concentrations, per mg of dried cells of Oscillatoria PCC 6506, of 7-epicylindrospermopsin (0.18 μg/mg and 0.29 μg/mg, respectively) and cylindrospermopsin (0.10 μg/mg and 0.11 μg/mg, respectively). We showed that these two toxins accumulated in the culture medium of Oscillatoria PCC 6506 but that the ratio (2.5 ± 0.3) was constant with 7-epicylindrospermopsin being the major metabolite. We also determined the concentrations of these toxins in culture media of other Oscillatoria strains, PCC 6407, PCC 6602, PCC 7926, and PCC 10702, and found that, except for PCC 6602, they all produced 7-epicylindrospermopsin and cylindrospermopsin, with the former being the major toxin, except for PCC 7926, which produced very little 7-epicylindrospermopsin. All the cylindrospermopsin producers studied gave a PCR product using specific primers for the amplification of the cyrJ gene from genomic DNA.
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identification of a polyketide synthase coding sequence specific for anatoxin a producing Oscillatoria cyanobacteria
Applied and Environmental Microbiology, 2009Co-Authors: Sabrina Cadelsix, Stephane Mann, Olivier Ploux, Annick Mejean, Isabelle Iteman, Caroline PeyraudthomasAbstract:We report the identification of a sequence from the genome of Oscillatoria sp. strain PCC 6506 coding for a polyketide synthase. Using 50 axenic cyanobacteria, we found this sequence only in the genomes of Oscillatoria strains producing anatoxin-a or homoanatoxin-a, indicating its likely involvement in the biosynthesis of these toxins.
Muriel Gugger - One of the best experts on this subject based on the ideXlab platform.
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anatoxin a synthetase gene cluster of the cyanobacterium anabaena sp strain 37 and molecular methods to detect potential producers
Applied and Environmental Microbiology, 2011Co-Authors: Anne Rantalaylinen, Leo Rouhiainen, Muriel Gugger, Suvi Kana, Hao Wang, Matti Wahlsten, Ermanno Rizzi, Katri A BergAbstract:Cyanobacterial mass occurrences are common in fresh and brackish waters. They pose a threat to water users due to toxins frequently produced by the cyanobacterial species present. Anatoxin-a and homoanatoxin-a are neurotoxins synthesized by various cyanobacteria, e.g., Anabaena, Oscillatoria, and Aphanizomenon. The biosynthesis of these toxins and the genes involved in anatoxin production were recently described for Oscillatoria sp. strain PCC 6506 (A. Mejean et al., J. Am. Chem. Soc. 131:7512-7513, 2009). In this study, we identified the anatoxin synthetase gene cluster (anaA to anaG and orf1; 29 kb) in Anabaena sp. strain 37. The gene (81.6% to 89.2%) and amino acid (78.8% to 86.9%) sequences were highly similar to those of Oscillatoria sp. PCC 6506, while the organization of the genes differed. Molecular detection methods for potential anatoxin-a and homoanatoxin-a producers of the genera Anabaena, Aphanizomenon, and Oscillatoria were developed by designing primers to recognize the anaC gene. Anabaena and Oscillatoria anaC genes were specifically identified in several cyanobacterial strains by PCR. Restriction fragment length polymorphism (RFLP) analysis of the anaC amplicons enabled simultaneous identification of three producer genera: Anabaena, Oscillatoria, and Aphanizomenon. The molecular methods developed in this study revealed the presence of both Anabaena and Oscillatoria as potential anatoxin producers in Finnish fresh waters and the Baltic Sea; they could be applied for surveys of these neurotoxin producers in other aquatic environments.
Weizhen Zhang - One of the best experts on this subject based on the ideXlab platform.
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Physiological differences between free-floating and periphytic filamentous algae, and specific submerged macrophytes induce proliferation of filamentous algae: A novel implication for lake restoration.
Chemosphere, 2019Co-Authors: Weizhen Zhang, Hong Shen, Jia Zhang, Ping Xie, Jun ChenAbstract:Abstract Restoration of submerged macrophytes is widely applied to counteract eutrophication in shallow lakes. However, proliferation and accumulation of filamentous algae (possessing free-floating and periphytic life forms) hamper growth of submerged macrophytes. Here, we explored factors triggering the excessive proliferation of filamentous algae during lake restoration using field investigations and laboratory experiments. Results showed that, compared with free-floating Oscillatoria sp. (FO), periphytic Oscillatoria sp. (PO) showed faster growth rate, greater photosynthetic capacities and higher phosphorus (P) affinity. Therefore, PO was physiologically competitively superior to FO under low P concentration and improved light conditions. And proliferation of filamentous algae was mainly manifested in periphytic life form. Besides, field results showed that density of filamentous algae in water column might be related to substrate types. Some macrophyte (Ceratophyllum oryzetorum and Potamogeton crispus) might provide proper substrates for proliferation of filamentous algae. Further physiological experiments found that Oscillatoria showed specific eco-physiological responses to different macrophyte species. Hydrilla verticillata and C. oryzetorum promoted growth and photosynthetic activity of Oscillatoria, while Potamogeton malaianus inhibited growth and P uptake of PO. Myriophyllum spicatum exhibited no impact on growth of Oscillatoria. Our results revealed the intrinsic (physiological differences between free-floating and periphytic life forms of filamentous algae) and extrinsic (different macrophytes) factors affect the proliferation of filamentous algae, which are important for guidance on planting of submerged macrophytes during lake restoration.
