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Avances en el estudio de cilindrospermopsina: detección de cianobacterias potencialmente productoras de la toxina y prospección de las etapas iniciales de su síntesis

  • Autores: Angel Barón Sola
  • Directores de la Tesis: Francisca Fernández del Campo (dir. tes.), Soledad Sanz Alférez (dir. tes.)
  • Lectura: En la Universidad Autónoma de Madrid ( España ) en 2013
  • Idioma: español
  • Tribunal Calificador de la Tesis: Vitor Manuel Oliveira Vasconcelos (presid.), Antonio Quesada (secret.), Silvia Pichardo Sánchez (voc.), Francisco Leganés Nieto (voc.), María F. Fillat (voc.)
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  • Resumen
    • The occurrence of toxic cyanobacterial blooms is increasing world-wide in the last decades. A good number of cyanotoxins have been already described, the best known among them being microcystins (MC) and cylindrospermopsin (CYN), due to their high abundance and global distribution. It is frequent to find in blooms mixed populations of cyanobacteria producing MCs and CYN. In many cases, the known cyanotoxins contained cannot explain by themselves the toxicity of the blooms; therefore, other toxic compounds are to be present.

      In this work, different approaches were used to get insight into cyanotoxicity: i) a new methodology was developed to detect simultaneously CYN and MC-producing strains; ii) a new cyanotoxic candidate has been found to accumulate in cyanobacteria, guanidinoacetate (GAA); iii) the enzyme responsible for the first reaction of CYN synthesis pathway has been characterized in a strain of Aphanizomenon ovalisporum; and iv) in this strain, it was analyzed the relationship between the production of CYN and the expression of some key genes considered to codify enzymes of CYN synthesis.

      Several studies have revealed that the genes mcy and aoa/cyr are involved in MC and CYN synthesis, respectively. Based on the identification of those gene sequences, we developed a multiplex PCR assay that allows simultaneous detection of CYN+ and MC+ strains in mixed populations of cyanobacteria. For that, various primer sets were designed, by using mcy and aoa gene sequences. Purified DNA as well as cultured cell mixtures and field samples with MC and CYN-producing strains were suitable as DNA template. The method could be applied to environmental samples, implying a rapid and low-cost test to jointly detect the presence of CYN+ and MC+ cyanobacteria in sestonic fractions of water samples.

      In the last years, several CYN+ strains of A.ovalisporum have been isolated in water bodies from different geographical regions, leading to a rise in ecological and health risks. According to the models proposed, an amidinotransferase (AMDT) codified by the aoaA gene is the first enzyme involved in CYN synthesis. We have cloned and overexpressed the aoaA gene from the CYN+ strain A.ovalisporum UAM-MAO, isolated in the laboratory. The recombinant purified AoaA was biochemically characterized, confirming that it is amidinotransferase. AoaA is similar in many aspects to the previously reported AMDT of Cylindrospermopsis raciborskii, CyrA: high substrate specificity for Arg and Gly, and a mixed sequential/ping-pong kinetic mechanism in its activity. We have further observed that AoaA is inhibited by GAA in a non-competitive manner.

      GAA is a precursor of CYN, being formed in the first step of the toxin synthesis pathway. On the other hand, it is one of the most extensively studied toxic guanidine compounds. GAA changes can affect the nervous system and induce hyperhomo-cysteinemia, representing a risk factor for cardiovascular diseases. In spite of the evidence supporting GAA toxicity and its role in CYN synthesis, no data have been reported on the accumulation of GAA in any cyanobacterium. We have analyzed and compared the content of GAA in cultures of diverse cyanobacteria types, both CYN-producing (CYN+) and non-producing (CYN-). GAA was present in the majority of the strains tested, although the highest content was found in the CYN+ strain, A. ovalisporum UAM-MAO. Therefore, GAA appears to be a general cyanobacterial metabolite that due to its proven toxic should be considered when studying cyanobacterial toxicity, and in cyanotoxicity management.

      In the last years, several highly homologous gene clusters related to CYN synthesis, aoa and cyr, have been described in different cyanobacteria genera. We have studied aoaA-C and cyrJ gene expression by real time qPCR in A. ovalisporum UAM-MAO grown under optimal conditions. A good correlation between the expression of those genes and CYN production was found. Furthermore, taking into account the CYN high nitrogen content, a possible relation of the nitrogen master controller NtcA with CYN synthesis was explored, by following the expression kinetics of the codifying gene ntcA. The tendency of ntcA relative expression looks similar to that of aoaA-C and cyrJ. Besides, three putative NtcA binding sites have been localized within the aoaA-C sequence. These findings support the idea of CYN production be regulated by NtcA.

      Since arginine and glycine seem to be the only substrates accepted by the AMDT related to CYN synthesis, we studied the effect of these amino acids in cultures of A. ovalisporum UAM-MAO. Arg clearly caused an increase in CYN, but Gly a decrease. The decrease appears not to result from inhibiting the activity of the genes considered to be involved in CYN synthesis, since Gly, as Arg, enhanced the transcription of aoaA-C and cyrJ genes. On the other hand, culture growth was affected by Arg and Gly in a similar way to CYN production, Arg stimulating and Gly impairing it. Therefore, the influences of both Arg and Gly on CYN oscillations during growth seem not to be due to a specific effect on the first step of CYN synthesis, but to changes in the physiological cell status.

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