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

[摘要] English: The main aim of this study was to attempt to develop amolecular screening tool for naturally occurring blooms ofM. aeruginosa based on the presence or absence of the genemcyB. This peptide synthetase has previously beenimplicated in toxin production in M. aeruginosa (Dittmann etal., 1997).Geographically unrelated strains of M. aeruginosa wereobtained from the Pasteur Institute, France; the NationalInstitute for Environmental Studies, Japan; the Instituteof Freshwater Ecology, UK; and the University of the FreeState culture collections. Based on conserved regionspresent in known sequences of mcyB four primer pairs weredesigned. The strains were maintained under standardPCR reactions were performed and the fragments generatedwith the various primer pairs were compared with expectedfragment sizes. PCR products of the expected size wereamplified in both toxin-produêing strains with all fourprimer pairs, signifying that these toxin-producing strainspossess a copy of mcyB. It was also possible to generatePCR fragments with three primer pairs from the nontoxin-producing strain CCAP 1450/1. These results indicatedthat this strain contained at least partial elements ofmcyB.Fragments amplified by PCR from toxin-producing strains werecloned into pGemT®-Easy (Promega) and sequenced. Basepairand translated amino acid alignment of the assembledfragments showed a high degree of homology with previouslydeposited sequences of mcyB in the Genbank database.A fragment amplified by PCR from strain PCC 7813 with primerpair Tax 7P/3M was randomly labelled and used as a probe toscreen unrelated strains of M. aeruginosa for the presenceof mcyB. This probe hybridised to a fragment of the expected size in all toxin-producing strains as well as thenon toxin-producing strain confirming PCR results that allstrains contain this particular portion of mcyB.A second probe generated from strain PCC 7813 with primerpair Tox 1P/1M representing the fragment of mcyB notamplified by PCR in strain CCAP 1450/1 was synthesised.This probe hybridised to a fragment of the expected size inall toxin-producing strains and the non toxin-producingstrain. Hybridisation of this probe to PvuII digested DNAfrom CCAP 1450/1 indicated that there was enough target DNAin the CCAP 1450/1 genome for the Tox 1P/1M/PCC 7813 probeto hybridise to, hinting at the possibility that this strainalso possess a complete copy of the gene.Crude cell extracts were made from all strains investigatedand analysed by HPLC for the presence of microcystin-LR.Microcystin-LR was detected in all toxin-producing strainsas well as the 'non toxin-producing' strain CCAP 1450/1.The Institute of Freshwater Ecology where this strain wasobtained from was contacted and enquiries made. Fromreplies received it became known that firstly, the Institutehas never tested the strain for microcystin-LR productionand that secondly, the strains are not monocul tures. Themost probable explanation for the anomalous results gatheredfrom strain CCAP 1450/1 is that a toxin-producingM. aeruginosa type dominated in the culture for the durationof this study.

[发布机构] University of the Free State