Molecular characterisation of toxin-producing and non toxin-producing strains of Microcystis aeruginosa

English: The main aim of this study was to attempt to develop a molecular screening tool for naturally occurring blooms of M. aeruginosa based on the presence or absence of the gene mcyB. This peptide synthetase has previously been implicated in toxin production in M. aeruginosa (Dittmann et al., 1997). Geographically unrelated strains of M. aeruginosa were obtained from the Pasteur Institute, France; the National Institute for Environmental Studies, Japan; the Institute of Freshwater Ecology, UK; and the University of the Free State culture collections. Based on conserved regions present in known sequences of mcyB four primer pairs were designed. The strains were maintained under standard PCR reactions were performed and the fragments generated with the various primer pairs were compared with expected fragment sizes. PCR products of the expected size were amplified in both toxin-produêing strains with all four primer pairs, signifying that these toxin-producing strains possess a copy of mcyB. It was also possible to generate PCR fragments with three primer pairs from the non toxin-producing strain CCAP 1450/1. These results indicated that this strain contained at least partial elements of mcyB. Fragments amplified by PCR from toxin-producing strains were cloned into pGemT®-Easy (Promega) and sequenced. Basepair and translated amino acid alignment of the assembled fragments showed a high degree of homology with previously deposited sequences of mcyB in the Genbank database. A fragment amplified by PCR from strain PCC 7813 with primer pair Tax 7P/3M was randomly labelled and used as a probe to screen unrelated strains of M. aeruginosa for the presence of mcyB. This probe hybridised to a fragment of the expected size in all toxin-producing strains as well as the non toxin-producing strain confirming PCR results that all strains contain this particular portion of mcyB. A second probe generated from strain PCC 7813 with primer pair Tox 1P/1M representing the fragment of mcyB not amplified by PCR in strain CCAP 1450/1 was synthesised. This probe hybridised to a fragment of the expected size in all toxin-producing strains and the non toxin-producing strain. Hybridisation of this probe to PvuII digested DNA from CCAP 1450/1 indicated that there was enough target DNA in the CCAP 1450/1 genome for the Tox 1P/1M/PCC 7813 probe to hybridise to, hinting at the possibility that this strain also possess a complete copy of the gene. Crude cell extracts were made from all strains investigated and analysed by HPLC for the presence of microcystin-LR. Microcystin-LR was detected in all toxin-producing strains as well as the 'non toxin-producing' strain CCAP 1450/1. The Institute of Freshwater Ecology where this strain was obtained from was contacted and enquiries made. From replies received it became known that firstly, the Institute has never tested the strain for microcystin-LR production and that secondly, the strains are not monocul tures. The most probable explanation for the anomalous results gathered from strain CCAP 1450/1 is that a toxin-producing M. aeruginosa type dominated in the culture for the duration of this study.