Biosynthesis of Cylindrospermopsin and 7-Epicylindrospermopsin in Oscillatoria sp. Strain PCC 6506: Identification of the cyr Gene Cluster and Toxin Analysis

Abstract

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 g/mg and 0.29 g/mg, respectively) and cylindrospermopsin (0.10 g/mg and 0.11 g/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.Cyanobacteria are known to produce a wide range of secondary metabolites (19), some of which are toxic to humans and animals, and cyanobacterial blooms are frequently associated with human-or animal-poisoning events. Several classes of cyanobacterial toxins have so far been identified, including hepatotoxins, neurotoxins, cytotoxins, and irritants (42). Cases of animal intoxication due to cyanobacterial toxin exposure are regularly reported in different places around the world, and it is now recognized that the release of cyanobacterial toxins in water reservoirs and water supplies has major implications for public health and for the environment (8). For instance, anatoxin-a and homoanatoxin-a, two cyanobacterial neurotoxins, provoke the rapid death of animals by acute asphyxia when ingested, because these alkaloids are potent agonists of the nicotinic acetylcholine receptor (6,11,43,44).In 1979, a significant human-poisoning incident was reported on Palm Island in Australia (14, 16). Afterwards, it was shown that the compound responsible for that poisoning was cylindrospermopsin (CYN) (Fig. 1) and that this cytotoxic molecule was produced by a cyanobacterium, Cylindrospermopsis raciborskii (28). Since then, it was shown that this molecule is also produced by cyanobacteria belonging to other genera such as Anabaena (38), Aphanizomenon (1, ...

Isolation and preliminary characterization of cyanobacteria strains from freshwaters of Greece

“…However, cylindrospermopsin genes have been reported from Oscillatoria sp. PCC6506 [41]. Genetic analyses have shown that the CYN genes may have been horizontally transferred [40].…”

On the occurrence and toxicity of Cylindrospermopsis raciborskii in Poland

“…Aphanizomenon gracile in Poland or Oscillatoria sp. in France (Mazmouz et al 2010). Although one recent study has reported the alleged production of CYN by C. raciborskii blooming in Lake Aleksandrovac in Serbia, the whole conclusion was not based on any molecular and/or analytical investigation of the isolated strain (Dorđević et al 2015).…”

Análise genômica de isolados brasileiros de Cylindrospermopsis raciborskii com enfoque em cilindrospermopsina e fixação biológica de N2

“…Estas adaptações incluem uma separação temporal durante o fotoperíodo, onde a fixação do N 2 ocorre durante a fase escura e a fotossíntese durante a fase clara, e em outros casos ocorre uma segregação espacial, na qual a fixação de N 2 encontra-se confinada em uma célula especializada, chamada heterócito (BERMAN-FRANK et al, 2011 NEILAN et al, 2003;CHONUDOMKUL et al, 2004;HUMPAGE, 2006), confirmado em inúmeros estudos realizados em linhagens de C. raciborskii de vários países (HAWKINS et al, 1997;NORTON, 1999;LI et al, 2001b;BERNARD et al, 2003;FASTNER et al, 2003;SAKER et al, 2003;WOOD;STIRLING 2003;CHONUDOMKUL et al, 2004 (ROUHIAINEN et al, 2000;TOOMING-KLUNDERUD et al, 2007;ROUNGE et al, 2007), aeruginosina (ISHIDA et al, 2009), STX , CYN (MIHALI et al, 2008;MAZMOUZ et al, 2010;JAKOBSEN, 2010), entre outros compostos sintetizados por cianobactérias.…”

“…Esta técnica permite providenciar uma informação quantitativa e qualitativa, contudo, para tal análise são necessários padrões da toxina purificada, os quais por vezes são difíceis de serem obtidos, além de necessitar de grandes volumes de amostra quando a toxina se encontra em baixas concentrações, e de passos para purificação das amostras (RIVASSEAU et al, 1999;RAPALA et al, 2002;MATHYS;SURHOLT, 2004;SANGOLKAR;MASKE;CHAKRABARTI, 2006;. A cromatografia líquida acoplada à espectrometria de massas (LC-MS) e a LC-MS/MS também são métodos muito difundidos que fornecem resultados mais precisos sobre a presença desta toxina, bem como disponibilizam dados referentes aos picos gerados pela ionização e fragmentação da molécula (CHISWELL et al, 1999;FALCONER et al, 1999;LI et al, 2001a;LI et al, 2001b;FASTNER et al, 2003;HUMPAGE et al, 2005;FROSCIO et al, 2008;RASMUSSEN et al, 2008;YLMAZ et al, 2008;BLÁHOVÁ et al, 2009;GALLO et al, 2009;PREUβEL et al, 2009;MAZMOUZ et al, 2010;MEREL et al, 2010;ANTAL et al, 2011;BALLOT et al, 2011;EVERSON et al, 2011;MAZMOUZ et al, 2011;Mc GREGOR et al, 2011). IV) Utilizar os dados gerados no sequenciamento gênomico das duas linhagens para mapeamentos e buscas por genes relacionados à síntese de metabólitos secundários, com foco principal na CYN e genes relacionados à fixação biológica do N 2 e diferenciação dos heterócitos.…”

“…Estudos visando a amplificação do gene cyrJ nas linhagens C. raciborskii CENA302 e CENA303 foram realizados previamente, no entanto, nenhum fragmento deste gene foi amplificado . De acordo com vários autores, a ausência do gene cyrJ no agrupamento gênico cyr pode estar relacionado com a não produção de toxina MAZMOUZ et al, 2010;BALLOT et al, 2011). Este gene codifica para uma enzima responsável pela sulfatação da molécula de CYN mediada pela atividade de CyrN, no entanto, o sulfato presente na estrutura parece não estar relacionado com a toxicidade de CYN .…”