Biochemical Characterisation of DMSP Lyases in Marine Bacteria and Phytoplankton Simone Emily Newton-Payne A Thesis submitted to the University of East Anglia in accordance with the requirements of the degree of Doctor of Philosophy School of Biological Sciences September 2015 ©This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. i Abstract Dimethylsulphoniopropionate (DMSP) is the most abundant sulphur molecule in the oceans. Catabolism of DMSP by marine organisms is important both for the global movement of sulphur and as a carbon and sulphur source for microbial life. The molecular basis of DMSP catabolism had been revealed by the discovery of a DMSP demethylase and six different lyases in marine bacteria. However, at the start of this study in 2009 no eukaryotic DMSP lyase had been isolated, purified or characterized from axenic cultures, and there was little information on the enzymology of any of the bacterial DMSP lyase enzymes. The work presented in this thesis identifies the requirement of the three bacterial cupin DMSP lyases DddL, DddQ and DddW for metal cofactors, and also establishes the enzymology and biochemistry of these cupin-containing DMSP lyases. The presence of a typical DMSP lyase in the coccolithophore Emiliania huxleyi RCC1217 is also demonstrated for the first time from bacteria-free cultures by application of previously successful techniques used in the purification of bacterial ddd genes. Combined these findings provide new insights into the mechanisms of cleavage of DMSP by bacteria and phytoplankton and expand our understanding of the enzyme diversity involved in this process. ii List of Contents Title page i Abstract ii List of contents iii List of figures iv List of tables viii Acknowledgements x Abbreviations xi 1: Introduction 1 1.1 Dimethylsulphioniopropionate 2 1.2 DMSP – Why is it important? 3 1.3 Why do organisms synthesise DMSP? 4 1.3.1 DMSP as an osmolyte 4 1.3.2 DMSP as a cryoprotectant 6 1.3.3 DMSP as an antioxidant 6 1.3.4 DMSP as a grazing deterrent 7 1.3.5 DMSP as a sulphur storage molecule 7 1.4 The roles of Dimethylsulphide in the environment 8 1.4.1 DMS: Climate cooling, Cloud Condensation Nuclei and the CLAW hypothesis 9 1.4.2 DMS and its role as a chemoattractant 10 1.4.3 DMSP and DMS as key nutrients 11 1.5 Biosynthesis of DMSP 11 1.5.1 Biosynthesis of DMSP in angiosperms (s-methylation) 15 1.5.2 Biosynthesis of DMSP via the transamination pathway 16 1.5.3 Molecular identification of DMSP synthesis genes of the transamination pathway 18 1.5.4 The decarboxylation pathway in the dinoflagellate Crypthecodinium cohnii 18 1.6 DMSP biosynthesis in corals 21 1.7 DMS production from marine eukaryotes 21 1.7.1 Algal DMSP lyases 21 1.8 DMSP catabolism 23 1.8.1 DMSP Demethylation 23 1.8.2 DMSP cleavage pathway 26 1.8.3 DddD 27 1.8.3.1 DddD distribution 28 1.8.4 DddL 30 1.8.4.1 Cupins 31 1.8.5 DddP 34 1.8.5.1 Characterising DddP 34 1.8.5.2 DddP distribution 35 1.8.6 DddQ 37 1.8.7 DddW 38 1.8.8 DddY 38 iii 1.8.8.1 Distribution of DddY in other bacteria 38 1.8.8.2 DddY is a periplasmic lyase 40 1.9 Transport of DMSP 40 1.10 Introduction to the Roseobacters 43 1.10.1 Ruegeria pomeroyi DSS-3 43 1.11 Marine Phytoplankton and their importance 45 1.12 Phytoplankton and their effects on the Environment 46 1.12.1 The Organic Carbon Pump 47 1.12.2 The Carbonate Counter Pump 47 1.13 Phytoplankton and the sulphur cycle 48 1.13.1 The role of the Coccolithophores in the Marine carbon and sulphur cycles. 49 1.14 The major objetives of this study 49 2: Materials and Methods 51 2.1 Bacterial methods 54 2.1.1 Media, growth conditions, Strains and Plasmids 54 2.1.1.1 Preparation of culture media 54 2.2 Antibiotic Concentrations 55 2.3 Inducer Concentrations 56 2.4 Carbon Source 57 2.5 Long term storage of bacterial strains 57 2.6 Nucleic acid preparation techniques 57 2.6.1 Mini preparation of Plasmid DNA using QIAGEN Mini kit spin columns 57 2.6.2 Midi preparation of Plasmid DNA using QIAGEN Midi Kit spin columns 59 2.6.3 Extraction of plasmid DNA by alkaline lysis and phenol chloroform 60 2.6.4 Preparation of genomic DNA using the Wizard Genomic DNA purification kit 61 2.6.5 Ethanol precipitation of DNA/RNA 62 2.6.6 DNA quantification 63 2.7 Transfer of genetic material 63 2.7.1 Transformations 63 2.7.1.1 Preparation of competent cells 63 2.7.1.2 Transformations using heat shock 63 2.7.2 Bacterial Conjugations 64 2.7.2.1 Patch cross 64 2.7.2.2 Filter cross 64 2.8 Polymerase Chain Reaction 65 2.8.1 Colony PCR 65 2.9 Purification of PCR products 66 2.10 Restriction Enzyme digestion of DNA 66 2.11 Alkaline Dephosphorylation of Plasmid DNA 66 2.12 Ligation reactions 67 2.13 DNA Gel electrophoresis 67 2.14 Gel Extractions 67 2.15 Mutagenesis 68 2.15.1 Insertional Mutations in Ruegeria Pomeroyi 68 2.15.2 Site directed mutagenesis 68 2.16 Protein purification 69 2.16.1 Over-expression and purification of DddQ protein 70 iv 2.16.2 Over-expression and purification of DddL protein 71 2.17 Separation of protein by SDS-PAGE 71 2.18 Bradford’s Assay 73 2.19 Assays for DMS production via gas chromatography 73 2.19.1 Assays in vivo 74 2.19.2 Assays in vitro 74 2.20 β-galactosidase assays 74 2.21 Oligonucleotide design 75 2.22 DNA sequencing 75 2.23 in silico Analyses 76 2.23.1 Sequence Alignment 76 2.23.2 Phylogenetic Trees 76 2.23.4 Database Searches 76 2.24 Enzymatic Properties of DddL, DddQ and DddW in vitro 80 2.24.1 Establishing pH optima 80 2.24.2 Chelation of metal co-factors 80 2.24.3 Establishing Km and Vmax of DddL, DddQ and DddW 80 2.25 Establishing identity of metal cofactors in as purified protein 80 2.25.1 Inductively Couple Plasma, Optical Emission Spectroscopy (ICP-OES) 80 2.26 Analytical Ultracentrifugation 81 2.27 Methods employed for Emiliania huxleyi DMSP lyase studies 81 2.28 Media and growth conditions 81 2.28.1 Growth in liquid media 81 2.28.1.1 Preparation of Aquil media 81 2.28.1.1.1 Preparation of synthetic ocean water (SOW) 81 2.28.1.1.2 Preparation of Concentrated Stock Solutions (CSS) of major nutrients 82 2.28.1.1.3 Preparation of Aquil Vitamin Stock solutions 83 2.28.1.1.4 Final preparation of Aquil media 84 2.29 Culturing of Emiliania huxleyi strain RCC1217 84 2.30 Growth rate determination 84 2.31 Identification of 14C Acrylate via High Performance Liquid Chromatography (HPLC) 85 2.32 Testing of E. huxleyi culture axenicity by DAPI staining 85 2.33 Isolation of RNA from Emiliania huxleyi 85 3: Expression, Purification and Biochemical analysis of the DMSP lyases DddL and DddQ 87 3.1 Aims of this chapter 88 3.2 DddL: Preliminary identification and cloning of the dddL lyase by Curson et al. (2008) 88 3.2.1 Overexpression of DddL in E. coli 89 3.2.2 Purification of DddL from E. coli 90 3.2.3 DddL is a cytosolic protein 90 3.2.4 Purification of DddL protein 91 3.3 Identification and cloning of DddQ by Todd et al. (2011) 93 3.3.1 Purification of DddQ 94 3.4 Association state of DddL 96 3.4.1 Analytical Ultracentrifugation 96 3.5 Enzyme kinetics of DddL and DddQ 97 v 3.6 Metal binding studies 99 3.6.1 EDTA experiments 99 3.6.2 Metal cofactor determination by Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) 105 3.7 Site-directed mutagenesis of DddL/DddQ active site residues 108 3.8 Summary 111 4: Identification and characterization of the cupin-containing DMSP lyase DddW 112 4.1 Overview 113 4.2 Distribution and characteristics of DddW homologues 114 4.3 Microarray analysis of R. pomeroyi DSS-3 in response to DMSP addition 114 4.4 What is DddW? 114 4.5 Confirmation that dddW is induced by DMSP 117 4.5.1 Assaying dddW transcription using lacZ reporter fusions 117 4.5.2 qRT-PCR analysis of dddW transcription 119 4.6 DddW encodes a functional DMSP lyase enzyme 119 4.7 DddW encodes a typical DMSP lyase 121 4.8 SPO0454 is responsible for the DMSP-regulated transcription of dddW 122 4.9 Insertional mutation of dddW and its effect on DMS production 123 4.10 Purification of DddW 125 4.10.1 Overexpression and protein purification of DddW 126 4.11 Enzyme kinetics of DddW 127 4.12 Effect of metals on DMS production / Metal binding studies 129 4.13 Metal cofactor determination by Inductively Coupled Plasma-Optical Emission Spectroscopy (ICP-OES) 132 4.14 Determining DddW association state by analytical ultracentrifugation 133 4.15 Site-directed mutagenesis of DddW active site residues 135 4.16 Summary 137 5: Biochemical Characterisation of DMSP Lyase Enzymes from the coccolithophore Emiliania huxleyi 138 5.1 Biochemical characterization of DMSP Lyase enzymes from Emiliania huxleyi 139 5.2 Coccolithophores 139 5.2.1 Emiliania huxleyi 140 5.3 Eukaryotic DMSP-dependent DMS production 143 5.4 Algal DMSP lyases in history 143 5.5 Attempts to identify eukaryotic DMSP lyases 146 5.5.1 Bioinformatics analysis of Eukaryotic genomes for potential DMSP lyases 146 5.5.1.1 DddY 146 5.5.1.2 DddP 147 5.5.1.3 DddQ 147 5.5.1.4 DddL 147 5.5.1.5 DddW 148 5.5.1.6 DddD 148 vi 5.6 The E.