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Microcystins and Nodularin) by Novel Gram-Positive Bacteria

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Microcystins and Nodularin) by Novel Gram-Positive Bacteria OpenAIR@RGU The Open Access Institutional Repository at Robert Gordon University http://openair.rgu.ac.uk Citation Details Citation for the version of the work held in ‘OpenAIR@RGU’: WELGAMAGE DON, A. C. D., 2012. An investigation into the biodegradation of peptide cyanotoxins (microcystins and nodularin) by novel gram-positive bacteria. Available from OpenAIR@RGU. [online]. Available from: http://openair.rgu.ac.uk Copyright Items in ‘OpenAIR@RGU’, Robert Gordon University Open Access Institutional Repository, are protected by copyright and intellectual property law. If you believe that any material held in ‘OpenAIR@RGU’ infringes copyright, please contact [email protected] with details. The item will be removed from the repository while the claim is investigated. AN INVESTIGATION INTO THE BIODEGRADATION OF PEPTIDE CYANOTOXINS (MICROCYSTINS AND NODULARIN) BY NOVEL GRAM-POSITIVE BACTERIA By Welgamage Don Aakash Channa Dharshan A thesis submitted in partial fulfilment for the degree of Doctor of Philosophy Robert Gordon University February 2012 i TABLE OF CONTENTS Title page .............................................................................................. i Contents ............................................................................................... ii Declaration .......................................................................................... iii Dedication ........................................................................................... iv Acknowledgements ................................................................................ v Abbreviations ....................................................................................... vi Abstract ............................................................................................... 1 Chapter 1 Introduction ....................................................................... 2 Chapter 2 Culturing and processing of cyanobacteria ............................ 34 Chapter 3 Bacterial metabolism and biodegradation of microcystins and nodularin ......................................................................... 67 Chapter 4 Unravelling the inducible peptide degradation mechanisms of novel MC degraders ......................................................... 124 Chapter 5 Elucidating the microcystinase gene(s) involved in the bacterial degradation of MC-LR ...................................................... 184 Chapter 6 Discussion ...................................................................... 255 Chapter 7 References ..................................................................... 260 Appendices ...................................................................................... 289 ii DECLARATION I declare that the work presented in this thesis is my own, except where otherwise acknowledged, and has not been submitted in any form for another degree or qualification at any other academic institution. Information derived from the published or unpublished work of others has been acknowledged in the text and a list of references is given. ……………………………………………. Aakash Welgamage Don iii DEDICATION FOR MY MUM AND DAD iv ACKNOWLEDGEMENTS I would like to sincerely thank my supervisors Prof. Linda Lawton and Dr. Christine Edwards for their immense support, guidance and wonderful friendship throughout this project. I also extend my sincere thanks to Prof. Elke Dittmann (University of Potsdam, Germany) for the great support, hospitality and friendship during my research visit to Germany. I am also very grateful to the Institute for Innovation, Design & Sustainability Research for their financial assistance. Thank you very much Eoin, for the friendship and support through my teaching and demonstrating career. Thank you very much Heather, Mairi, John, Kirsten and Beth for your support. Thanks also to the many people in the department who helped and advised me over the years, especially Kostas, Carlos, Vijith, Luca, Shaista, Radisti, Arif, Ania, James, Olivier and all my lab colleagues from Germany; Jan, Eva, Kathrin, Arthur, Douglas, Katarina, Sven and Chen-Lin. I am indebted to Mum, Dad and my Brother, not only for their love but for the opportunities they have given me along the way. Thank you very much Evie for your love, courage and support; that kept me going through all the tough times. Thank you very much for everything. v ABBREVIATIONS aa Amino acid ABPNB Anabaenopeptin B Amp Ampicillin ANGTN Angiotensin III AT Aminotransferase ATP Adenosin triphosphate BLAST Basic Local Alignment Search Tool bp Base pair(s) BSA Bovine serum albumin cDNA Complementary deoxyribonucleic acid CY Cyclosporin A d Days Da Daltons DNA Deoxyribonucleic acid dNTP Deoxynucleoside triphosphate DTT 1,4-dithiothreitole E. coli Escherichia coli EDTA Ethylene diamine tetra-acetic acid EMBL European Molecular Biology Laboratory ESI Electrospray ionization GRA Gramicidin A vi h Hours HPLC High performance liquid chromatography IPTG Isopropyl β-D-1-thiogalactopyranoside kb kilo base pairs kDa kilo Dalton LB Lysogeny broth LNOD Linear nodularin MC Microcystin ME Microcystinase enzyme min Minutes MPG Microcystinase producing genes MS Mass spectrometry MW Molecular weight NA Nutrient agar NB Nurtrient broth NCBI The National Center for Biotechnology Information NOD Nodularin OD Optical density ORF Open reading frame PAA Polyacrylamide PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffered saline PCC Pasteur Culture Collection vii PCR Polymerase chain reaction PDA Photodiode array PEG Polyethylenglycol PLP Pyridoxal-phosphate PMSF Phenyl-methyl-sulphonyl-fluoride POLYB Polymyxin B RNA Ribonucleic acid RP Reversed phase rpm Rounds per minute RT Room temperature SDS Sodium dodecyl sulfate sec Seconds TAE Tris-acetate-EDTA buffer TFA Trifluoroacetic acid Tm Primer melting temperature UPLC Ultra performance liquid chromatography UV Ultraviolet viii Abstract Cyanobacterial secondary metabolites, microcystins (MC) and nodularin (NOD) have become common contaminants in most aquatic ecosystems over recent years presenting a hazard to animal and human health. Unfortunately, these chemically diverse peptide hepatotoxins remain a challenge to most conventional water treatments due to their stable cyclic structures. Over recent years, bioremediation of MC and NOD has become one of the most exciting areas that holds promise for a successful and cost effective solution for water treatment process. The current work presents the biodegradation of MCs and NOD by bacterial isolates from three different bacteria genus Arthrobacter, Brevibacterium and Rhodococcus belonging to Actinobacteria. A total of five isolates representing the three genera have demonstrated an overall metabolism of MC-LR, -LF, -LY, -LW, -RR and NOD in a Biolog MT2 assay. Subsequently, these bacteria were reported to degrade the range of toxins in a separate batch experiment. The bacterial degradation rate of the above cyanobacterial peptides were found to decrease with the multiple sub- culturing of the bacteria. However, a rapid degradation was discovered when the bacteria were re-exposed to MC or other prokaryotic peptides demonstrating an inducible bacterial biodegradation. Utilising latest molecular biology techniques, the gene responsible for production of MC degrading enzymes was successfully elucidated and its activity was evaluated. Analysis of the degradation products of MC-LR revealed a glutathione conjugate detoxification mechanism involved during the degradation of MC-LR by Rhodococcus sp. (C1). A novel MC degradation pathway was proposed. Further studies were suggested to fully characterise the degradation pathway and to evaluate the MC detoxification mechanism in bacteria. 1 CHAPTER 1 INTRODUCTION 2 TABLE OF CONTENTS 1 INTRODUCTION ............................................................................................ 4 1.1 Origin and distribution of cyanobacteria ................................................... 4 1.2 Occurrence of cyanobacterial blooms ....................................................... 6 1.3 Cyanotoxins .......................................................................................... 8 1.3.1 Cyclic peptide hepatotoxins - microcystins and nodularin ....................10 1.3.2 Toxic Alkaloids ...............................................................................15 1.3.2.1 Neurotoxin alkaloids - anatoxin-a and anatoxin-a(S) ....................15 1.3.2.2 Saxitoxins ...............................................................................16 1.3.2.3 Hepatotoxic cylindrospermopsin .................................................18 1.4 Persistence and degradation of microcystins ............................................20 1.5 Removal of microcystin from drinking water ............................................22 1.6 Aims and Objectives .............................................................................30 3 1 Introduction 1.1 Origin and distribution of cyanobacteria Cyanobacteria are a diverse group of prokaryotes that occupy a broad range of ecological niches. They are the Earth’s oldest known oxygen-producing organisms, having first appeared billions of years ago (Adams, 1997). Cyanobacteria represent some of the most primitive cells to have appeared in the early Precambrian period, with fossils remaining dating back 3.5 billion years ago. As the progenitor of oxygenic photosynthesis, cyanobacteria are believed to have transformed the evolution of biology and geochemistry of Earth (Shi and Falkowski,
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