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

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Date
2001-05
Authors
Botes, Elsabé
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University of the Free State
Abstract
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.
Afrikaans: Daar is gepoog om 'n molekulêre siftingsmetode te ontwikkel vir natuurlike opbloeie van M. aeruginosa, gebasseer op die aan- of afwesigheid van die geen mcyB. Hierdie peptied-sintetase is voorheen geïmpliseer in toksienproduksie in M. aeruginosa (Dittmann et al., 1997) Geografies onverwante stamme van M. aeruginosa is bekom vanaf die Pasteur Instituut, Frankryk; die National Institute for Environmental Studies, Japan; die Insitiute for Freshwater Ecology, Verenigde Koninkryk; en die Universiteit van die Vrystaat kultuurversamelings . Vier inleierpare is ontwerp gebasseer op gekonserveerde gedeeltes aanwesig in bekende basispaarvolgordes van mcyB. Die stamme is gegroei by standaard toestande en totale genomiese DNS is geëkstraheer vanuit toksienproduserende stamme PCC 7813 en UV 027 en die nie-toksienproduserende stam CCAP 1450/1. PKR reaksies is gedoen en die fragmente wat gegenereer is met die onderskeie priemstukke was vergelyk met die verwagte fragmentgroottes. PKR-produkte van die verwagte grootte is geamplifiseer in beide toksienproduserende stamme met al vier inleierpare, wat aangedui het dat hierdie stamme 'n kopie van mcyB bevat het. Dit was ook moontlik om PKR-fragmente te genereer met drie inleierpare vanuit die nie-toksienproduserende stam CCAP 1450/1. Hierdie resultate het aangedui dat hierdie stam ten minste gedeeltelike elemente van 'n kopie van mcyB bevat het. Fragmente wat deur PKR geamplifiseer is vanuit toksienproduserende gekloneer en die stamme is in pGemT®-Easy basispaaropeenvolging is (Promega) bepaal. Basispaarvolgorde en getranslerde aminosuurinlynstelling van die saamgevoegde fragmente het 'n hoë mate van homologie getoon met voorheen-gedeponeerde basispaarvolgordes in die Genbank databasis. 'n Fragment geamplifiseer deur PKR vanuit stam PCC 7813 met inleierpaar Tox 7P/3M is lukraak gemerk en gebruik as 'n peiler om onverwante stamme van M. aeruginosa vir die teenwoordigheid van mcyB te sif. Hierdie peilstuk het aan 'n fragment van die verwagte grootte in al die toksienproduserende stamme gehibridiseer asook aan die genomiese DNS van die nie-toksienproduserende stam. Dit bevestig PKR resultate dat alle stamme hierdie spesifieke gedeelte van mcyB bevat. 'n Tweede peilstuk is gemaak van 'n PKR-produk van stam PCC 7813 met inleierpaar Tox 1P/1M. Hierdie fragment verteenwoordig die gedeelte van mcyB in stam CCAP 1450/1 wat nie met PKR geamplifiseer is nie. Hierdie peilstuk het aan 'n fragment van die verwagte grootte gehibridiseer in alle toksienproduserende stamme asook die nie-toksien-produserende stam CCAP 1450/1. Hibridisering van hierdie peilstuk aan PvuII-gesnyde genomiese DNS van CCAP 1450/1 het aangedui dat daar genoeg teikenvolgorde-DNS aanwesig was in die CCAP 1450/1-genoom vir die Tox 1P/1M/PCC 7813 peilstuk om te kon hibridiseer, wat ook kon aangedui het dat hierdie stam selfs 'n volledige kopie van die mcyB geen kon bevat het. Kru sel-ekstrakte is gemaak van alle stamme wat ondersoek is en dit is geanaliseer deur HDVC vir die aanwesigheid van mikrosistien-LR. Mikrosistien-LR is gevind in alle toksienproduserende stamme asook produserende' stam CCAP 1450/1. die 'nie-toksien- Die Institute of Freshwater Ecology, waarvandaan die stam verkry is, is gekontak en navraag is gedoen. Met terugvoer wat verkry is, het die aan die lig gekom dat eerstens, die Instituut nog nooit die stam getoets het vir mikrosistien-LR produksie nie, en tweedens dat die stamme nie reinkulture is nie. Die waarskynlikste verklaring vir die onreëlmatige resultate waargeneem van stam CCAP 1450/1 is dat 'n toksienproduserende M. aeruginosa tipe dominant was vir die duur van hierdie studie.
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Cyanobacterial toxins, Microcystins, Freshwater microbiology, Dissertation (M.Sc. (Botany and Genetics))--University of the Free State, 2001
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