A Thesis Submitted for the Degree of PhD at the University of Warwick Permanent WRAP URL: http://wrap.warwick.ac.uk/89711 Copyright and reuse: This thesis is made available online and is protected by original copyright. Please scroll down to view the document itself. Please refer to the repository record for this item for information to help you to cite it. Our policy information is available from the repository home page. For more information, please contact the WRAP Team at: [email protected] warwick.ac.uk/lib-publications Zinc on the Move: Insights Towards Understanding Zinc Homeostasis in the Open Ocean Cyanobacterium Synechococcus sp. WH8102 By Amira Zahir Ksibe A thesis submitted for the fulfilment of the degree of Doctor of Philosophy in Chemistry Department of Chemistry and School of Life Sciences February 2017 Table of Contents Table of Contents ....................................................................................................................... i List of Figures ........................................................................................................................ viii List of Tables .......................................................................................................................... xiii Acknowledgments .................................................................................................................. xiv Declaration .............................................................................................................................. xv Dedications ............................................................................................................................. xvi Abstract ................................................................................................................................. xvii List of Abbreviations ............................................................................................................. xix Amino Acid Abbreviations .................................................................................................. xxii Chapter 1 General Introduction ............................................................................................. 1 1.1 Zinc Hypothesis in the Open Ocean ................................................................................. 1 1.1.1 Evidence Points to Bioactive Zinc Cycles ................................................................. 2 1.1.2 Properties of Organic Zinc Ligands .......................................................................... 4 1.2 Elemental Composition of Life ........................................................................................ 5 1.3 Metal Cofactors in Biology .............................................................................................. 7 1.4 Zinc in Biological Systems ............................................................................................... 8 1.4.1 Zinc is Impressive .................................................................................................. 9 1.5 Systems for Zinc Homeostasis ....................................................................................... 11 1.6 Families of Zinc Transporters ........................................................................................ 14 1.6.1 Efflux Transporters .................................................................................................. 14 1.6.2 Uptake Transporters ................................................................................................ 16 1.6.2.1 High-affinity Uptake Systems .......................................................................... 16 1.6.2.2 Low-affinity Uptake Systems ........................................................................... 18 1.7 Zinc Responsive Transcription Regulators..................................................................... 20 1.7.1 The Fur Family of Metal Sensing Proteins .............................................................. 20 1.7.2 Zinc Uptake Regulators: The Zur Sensor ................................................................ 26 1.7.2.1 Metal Sites in Zur Proteins ............................................................................... 27 i 1.7.3 The Functions of Zur ............................................................................................... 29 1.7.3.1 Zur as a Repressor ............................................................................................ 29 1.7.3.2 Zur as an Activator ........................................................................................... 30 1.8 Zincophores: Molecules whose Time Has Come ........................................................... 31 1.9 Metals in Cyanobacteria ................................................................................................. 34 1.9.1 Zinc Homeostasis in Cyanobacteria ........................................................................ 35 1.9.1.1 The Zinc Uptake Regulator, Zur in Cyanobacteria .......................................... 36 1.10 Research Motivation and Aims .................................................................................... 40 Chapter 2 General Materials and Experimental Methods ................................................. 43 2.1 Chemicals and Reagents ................................................................................................. 43 2.2 Media, Bacterial Growth and Incubation Conditions ..................................................... 43 2.2.1 Cyanobacterial Media and Growth Conditions ....................................................... 43 2.2.2 Contamination Plates ............................................................................................... 45 2.3 Analysis of Culture Supernatant ..................................................................................... 46 2.3.1 Cell Pre-concentration and Ligand Extraction Procedures...................................... 46 2.3.2 Chromatography Analysis ....................................................................................... 48 2.4 Physiological Methods ................................................................................................... 49 2.4.1 Flow Cytometry ....................................................................................................... 49 2.4.2 Growth Rate Analysis .............................................................................................. 50 2.4.3 Chlorophyll a Extraction ......................................................................................... 50 2.5 Identification of Proteins using MALDI-TOF ............................................................... 51 2.5.1 Sample Preparation .................................................................................................. 51 2.5.2 Band Preparation and Destaining ............................................................................ 51 2.5.3 Peptide Mass Fingerprinting (PMF) ........................................................................ 53 2.5.4 Data Interpretation and Database Searching ........................................................... 53 2.6 Molecular Biology Procedures ....................................................................................... 53 2.6.1 Genomic DNA Extraction from Synechococcus sp. WH8102 ................................ 53 2.6.2 Synthetic synw_2401 gene ...................................................................................... 54 2.6.3 Gene Amplification by PCR .................................................................................... 55 2.6.4 Agarose Gel Electrophoresis ................................................................................... 56 2.6.5 Purification of PCR Product .................................................................................... 56 2.6.6 Gene Cloning into the TOPO® vector ...................................................................... 57 ii 2.6.7 Transformation of Competent Cells and Plasmid Purification ................................ 57 2.7 Overproduction and Purification of Zur Protein ............................................................ 58 2.7.1 Production of Recombinant Zur Protein .................................................................. 58 2.7.2 Immobilized Metal Affinity Chromatography ........................................................ 59 2.7.3 TEV Protease Purification ....................................................................................... 60 2.8 Protein Identification ...................................................................................................... 61 2.8.1 SDS-polyacrylamide Gel Electrophoresis ............................................................... 61 2.8.2 Native Polyacrylamide Gel Electrophoresis (NP-AGE) ......................................... 61 2.8.3 Detection of Protein by Western Blotting ............................................................... 61 2.9 Estimation of Zur Protein Concentration ....................................................................... 62 2.9.1 Ellmanʼs Test, Thiol Quantification ........................................................................ 62 2.9.2 Inductively Coupled Plasma-Optical Emission Spectroscopy ................................ 63 2.10 Protein Biophysical Techniques ................................................................................... 63 2.10.1 Electrospray Ionization Mass