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Characterisation of the Tailoring and Transport Enzymes Involved in the Microcystin Biosynthesis Pathway

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Characterisation of the Tailoring and Transport Enzymes Involved in the Microcystin Biosynthesis Pathway Characterisation of the Tailoring and Transport Enzymes involved in the Microcystin Biosynthesis Pathway Leanne A. Pearson A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy School of Biotechnology and Biomolecular Science The University of New South Wales Sydney, Australia July, 2006 ACKNOWLEDGEMENTS Many thanks to all whom contributed to this work. I would like to extend my sincere thanks to Tom Börner and Elke Dittmann for allowing me the opportunity to conduct part of my research in their laboratories in Berlin, and for donating many of the cyanobacterial strains and DNA samples used throughout this study. I would also like to thank The CRC for Water Quality, The Australian Research Council, and The Deutscher Akademisher Austausch Dienst for funding this research. My thanks also go out to Anne Poljak and Mark Raftery from the Bioanalytical Mass Spectrometry Facility (UNSW) for their help with the MALDI-TOF work. I would also like to thank Wendy Glenn for her assistance with the ultra centrifuge and FPLC, and Andy Netting for his help editing this manuscript. Many thanks to Michelle Moffitt, Bradley Moore, and Hans von Döhren for their advice regarding the biochemistry of McyI. Likewise, I wish to thank Tony George for his advice regarding the expression and characterisation of McyH. Many thanks to Ian McFarlane for his advice and co-supervision over the years. A big thank you to my family and friends for their encouragement and support throughout my university career, and to all the members of the BGGM for making the lab a great place to work. Finally, I wish to thank my supervisors Brett Neilan and Kevin Barrow for their wisdom, patience and inspiration. It has been an honour and a privilege working with you. 2 TABLE OF CONTENTS ABSTRACT 8 CHAPTER 1. INTRODUCTION 9 1.0 Cyanobacteria 10 1.1 Cyanotoxins 11 1.2 Secondary metabolite biosynthesis 15 1.2.0 Non-ribosomal peptide biosynthesis 16 1.2.1 Polyketide biosynthesis 17 1.2.2 Mixed NRP/PK biosynthesis 18 1.2.3 Combinatorial biosynthesis 19 1.2.4 NRPS and PKS associated tailoring and transport enzymes 20 1.2.5 Biosynthesis of microcystin and nodularin 33 1.2.6 Regulation of microcystin biosynthesis 37 1.3 Aims 40 CHAPTER 2. MATERIALS AND METHODS 41 2.0 Bacterial strains and Culturing 42 2.0.0 Cyanobacteria 42 2.0.1 Escherichia coli 42 2.1 Nucleic Acid Extraction 43 2.1.0 Extraction of genomic DNA 43 2.1.1 Extraction of plasmid DNA 44 2.2 Polymerase chain reaction (PCR) 44 2.3 Purification of Nucleic Acids 46 2.3.0 Ethanol and isopropanol precipitation of DNA solutions 46 2.3.1 Column purification 46 2.4 Automated Sequencing and Analysis 47 2.5 Agarose gel electrophoresis 47 2.6 Modification of DNA fragments 48 2.6.0 Digestion of DNA with restriction endonucleases 48 2.6.1 End-filling DNA overhangs 48 2.6.2 Dephosphorylation of DNA with shrimp alkaline phosphatase (SAP) 49 2.6.3 Ligation of DNA fragments 49 2.7 Genetic manipulation of bacteria 50 2.7.0 Preparation of competent E. coli cells and transformation 50 2.8 Total Protein Assays 51 2.8.0 Bio-Rad Dc protein assay 51 2.8.1 Folin protein assay 51 2.9 Protein Electrophoresis 51 3 2.10 Ammonium sulphate precipitation of proteins 52 2.11 Immunodetection of proteins with Ni-NTA conjugates 52 CHAPTER 3. CHARACTERISATION OF THE ABC TRANSPORTER, MCYH 53 3.0 OVERVIEW 54 3.1 EXPERIMENTAL DESIGN AND METHODOLOGY 57 3.1.0 Screening various strains of cyanobacteria for mcyH orthologues 57 3.1.1 Sequence analysis 57 3.1.2 Phylogenetic analysis 58 3.1.3 Heterologous expression and purification of McyH and component peptides 59 Engineering the McyH expression constructs for the expression of full-length McyH 59 Engineering the McyH expression constructs for the expression of the individual membrane and ATPase domains of McyH 60 Engineering expression constructs for the expression of N-terminally truncated McyH peptides 61 Over expression of recombinant peptides 63 Purification of recombinant peptides 63 3.1.4 Construction of everted membrane vesicles from E. coli expression strains 64 3.1.5 Enzyme assays 65 ATPase assays 65 Microcystin transport in everted membrane vesicles 66 3.1.6 Mutagenesis of mcyH 67 3.1.7 Regulation of McyH expression 68 Regulation of McyH expression under different light conditions 69 Regulation of McyH expression in different mcy mutants 71 3.2 RESULTS 72 3.2.0 Distribution of mcyH orthologues in various species of cyanobacteria 72 3.2.1 Sequence analysis of mcyH 72 3.2.2 Over-expression and Purification of histidine-tagged McyH peptides 75 3.2.3 Enzyme assays 78 ATPase assays 78 Microcystin transport in everted membrane vesicles 78 3.2.4 Mutagenesis of mcyH 79 3.2.5 Regulation of McyH expression 79 Regulation of McyH expression under different light conditions 79 Regulation of mcyH expression in mcy knock-out mutants 79 3.3 DISCUSSION 81 4 CHAPTER 4. CHARACTERISATION OF THE 2-HYDROXYACID DEHYDROGENASE, MCYI 92 4.0 OVERVIEW 93 4.1 EXPERIMENTAL DESIGN 97 4.1.0 Screening various strains of cyanobacteria for mcyI orthologues 97 4.1.1 Sequence analysis 97 4.1.2 Phylogenetic analysis 98 4.1.3 Complementation of E. coli auxotrophs with mcyI 98 Construction of the complementation plasmids pDRIVE(T7/mcyI) and pIN-III(lac/mcyI) 98 Complementation experiments 99 4.1.4 Heterologous expression and purification of McyI 100 Engineering the pET30(mcyI) expression construct 100 Overexpression of histidine-tagged McyI 100 Purification of histidine-tagged McyI 101 4.1.5 Determining the subunit organisation of native McyI 102 4.1.6 Enzyme assays 102 Dehydratase assays 102 Chemical synthesis of 2-Hydroxy-3-methylsuccinic acid 103 Oxidoreductase assays 104 End-product analysis 105 Determining the optimal pH of the McyI OAA reductase assay 106 Determining the optimal temperature and thermostability of the McyI OAA reductase assay 106 McyI specificity 106 McyI Kinetic analysis 107 Inhibition assays 107 Cell extract studies 108 4.2 RESULTS 109 4.2.0 Distribution of mcyI orthologues in various species of cyanobacteria 109 4.2.1 Sequence analysis of mcyI 109 4.2.2 Mutant complementation studies 113 4.2.3 Over-expression and Purification of histidine-tagged McyI 114 4.2.4 Subunit composition of McyI 115 4.2.5 Biochemical and kinetic properties of McyI 115 [MeDha7]microcystin-LR hydratase assays 115 Oxidoreductase assays 115 End-product analysis 117 Inhibition assays 117 4.3 DISCUSSION 118 5 CHAPTER 5. CHARACTERISATION OF THE O-METHYLTRANSFERASE, MCYJ 134 5.0 OVERVIEW 135 5.1 EXPERIMENTAL DESIGN 138 5.1.0 Screening various strains of cyanobacteria for mcyJ orthologues 138 5.1.1 Sequence analysis 138 5.1.2 Phylogenetic analysis 139 5.1.3 Heterologous expression and purification of McyJ 139 Engineering the pET30(mcyJ) expression construct 139 Overexpression of histidine-tagged McyJ 140 Purification of histidine-tagged McyJ 140 5.1.4 Enzyme assays 141 5.2 RESULTS 143 5.2.0 Distribution of mcyJ orthologues in various species of cyanobacteria 143 5.2.1 Sequence analysis of mcyJ 143 5.2.2 Overexpression and Purification of histidine-tagged McyJ 146 5.2.3 Enzyme assays 147 5.3 DISCUSSION 148 CHAPTER 6. GENERAL DISCUSSION 156 6.0 Major results 157 6.0.0 McyH 157 6.0.1 McyI 159 6.0.2 McyJ 160 6.1 Overall significance 162 REFERENCES 166 6 APPENDIX 198 A1. Media for culturing E. coli 198 A1.0 Luria Bertaini medium 198 A1.1 Tryptone phosphate medium 198 A1.2 Superbroth 198 A1.3 M9 salts 199 A2. Sequence alignments for the generation of phylogenetic trees 200 A2.0 McyH homologous sequence alignment 200 A2.1 McyH ATPase domain homologous sequence alignment 200 A2.2 McyI homologous sequence alignment 200 A2.3 McyJ homologous sequence alignment 200 7 ABSTRACT The cyanobacterium Microcystis aeruginosa is widely known for its production of the potent hepatotoxin microcystin. This cyclic heptapeptide is synthesised non-ribosomally by the thiotemplate function of a large, modular enzyme complex encoded within the 55 kb microcystin synthetase (mcy) gene cluster. The mcy gene cluster also encodes several stand-alone enzymes, putatively involved in the tailoring and export of microcystin. This thesis describes the characterisation of the Adda O-methyltransferase, McyJ, the 2-hydroxy-3-methylsuccinic acid dehydrogenase, McyI, and the ABC transporter, McyH. A combination of bioinformatic, molecular, and biochemical approaches have been used to elucidate the structure, function, regulation and evolution of these microcystin synthetase gene cluster encoded enzymes. Extensive sequence analyses are reported, including phylogenetic and structural studies. The distribution of mcyH, mcyI and mcyJ orthologues in different species of cyanobacteria has been investigated via genetic screening with M. aeruginosa specific, and degenerate oligonucleotide primers. McyH, McyI and McyJ have been heterologosly over-expressed in E. coli and enzymatically assayed. Finally, an McyH antibody has been engineered and used to investigate the regulation of the McyH ABC transporter in wild-type (WT) M. aeruginosa, and in various non-toxic engineered mutant strains. The results of these experiments are discussed with respect to the roles of McyH, McyI and McyJ in microcystin biosynthesis, and their relevance to the fields of water quality management and rational drug design and production. 8 CHAPTER 1. INTRODUCTION 9 1.0 Cyanobacteria The cyanobacteria or “blue-green algae” as they are commonly termed, comprise a diverse group of oxygenic photosynthetic bacteria that possess the ability to synthesise chlorophyll-a and the phycobilin protein, phycocyanin.
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