STUDYING ZINC HOMEOSTASIS IN ESCHERICHIA COLI USING CARBONIC ANHYDRASE-BASED RATIOMETRIC SENSORS by Da Wang A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Chemical Biology) in the University of Michigan 2012 Doctoral Committee: Professor Carol A. Fierke, Chair Professor Raoul Kopelman Professor James E. Penner-Hahn Professor Joel A. Swanson © Da Wang 2012 DEDICATION To my parents. ii ACKNOWLEDGEMENTS There are many people I would like to thank through my graduate school. First, I would like to thank my advisor Dr. Carol Fierke for giving me great guidance and support through my graduate schooling. She has always been encouraging, understanding, helpful and supportive in both science and my personal life. I have learnt so much from her that will benefit me lifelong wherever I go. I would like to thank my committee, Prof. James Penner-Hahn, Prof. Raoul Kopelman, and Prof. Joel Swanson. I have benefited greatly from the helpful advice and insightful comments about my work over the past years. I truly appreciate the help. I would also like to thank my collaborators Dr. Richard Thompson and Dr. Bryan McCranor at the University of Maryland School Of Medicine. Dr. Thompson has been very instrumental in solving many of the practical issues of sensor development, and Dr. McCranor kindly provided assistance in sensor development for mammalian cell imaging. I appreciate their input that helped move the project forward. I would like to thank all my labmates for creating such a great and wonderful lab culture. It has been a delightful experience working with you. I would like to thank Dr. Tamiika Hurst for her hard work that laid the groundwork for the sensor development, and mentoring me when I first started working on this project. I greatly appreciate Andrea Stoddard’s generous help throughout the years and her contribution to the CA team. I would like to thank Dr. Sam Gattis for mentoring me through my first project in iii the lab. I had many inspiring discussions about my work with Dr. Terry Watt, Dr. Nathan Zahler, Dr. John Hsieh and Dr. Lubomir Dostal, and I would like to thank them for sharing their wisdom. I appreciate Corissa Lamphear, Noah Wolfson, and Carol Ann Pitcairn’s initiatives and efforts for organizing all the lab activities. I had many fun memories working with Caleb Joseph, Xiaomu Guan, Wan Lim, Elaina Zverina, Xin Liu, Byung Chul Kim, Mike Howe, Yu Chen and Elia Wright. Thank you all for being such wonderful colleagues and friends. I also enjoyed working with the rotating students: Chenxi Shen, Si Yang, Kristin Ko, and Olijahwon Hosteen. I could not possibly express enough of my gratitude to my family, who has always been the strongest support for me. Even though we have been globes away during my graduate study, their love and support have always provided strength for me to overcome obstacles and move forward. Live long and prosper. iv TABLE OF CONTENTS DEDICATION .................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................... iii LIST OF FIGURES ......................................................................................................... viii LIST OF TABLES .............................................................................................................. x LIST OF ABBREVIATIONS ............................................................................................ xi ABSTRACT ..................................................................................................................... xiii CHAPTER I.INTRODUCTION 1.1 Zinc in Biology..................................................................................................... 1 1.1.1 Cellular Functions of Zinc ............................................................................ 1 1.1.2 Zinc and Human Health ................................................................................ 3 1.2 Cellular Zinc Homeostasis ................................................................................... 4 1.2.1 Intracellular Zinc Concentrations ................................................................. 5 1.2.2 Zinc Homeostasis in Eukaryotic Cells .......................................................... 6 1.2.3 Zinc Homeostasis in Prokaryotes................................................................ 14 1.3 Zinc Quantification And Sensor Development .................................................. 21 1.3.1 Methods for Quantifying Total Zinc ........................................................... 21 1.3.2 Methods for Detecting and Quantifying Free Zinc ..................................... 22 1.4 Carbonic Anhydrase-based Zinc Sensors ........................................................... 36 1.4.1 Zinc Binding Properties of Carbonic Anhydrase ........................................ 36 1.4.2 Development of Carbonic Anhydrase-based Zinc Sensors ........................ 39 II. DEVELOPMENT OF GENETICALLY ENCODED ZINC SENSORS AND INTRACELLULAR FREE ZINC MEASURMENTS IN E.COLI 2.1 Introduction ........................................................................................................ 43 2.2 Materials and Methods ....................................................................................... 45 2.2.1 Construction of CA_RFP Sensors .............................................................. 45 2.2.2 Protein Expression and Purification............................................................ 46 2.2.3 Synthesis of Dapoxyl Sulfonamide:............................................................ 47 2.2.4 In Vitro Characterization of CA_RFPs ....................................................... 48 v 2.2.5 Growth Conditions of E. coli ...................................................................... 49 2.2.6 Fluorescence Microscopy and Data Analysis ............................................. 49 2.3 Results ................................................................................................................ 51 2.3.1 Construction of CA_RFP Zinc Sensors ...................................................... 51 2.3.2 In Vitro Calibration of CA_RFP Sensors.................................................... 53 2.3.3 Applying CA_RFP Zinc Sensors in E. coli ................................................ 56 2.3.4 In Situ Calibration of CA_RFP Sensors...................................................... 61 2.3.5 Measurement of Intracellular Free Zinc Concentration in E. coli .............. 64 2.4 Discussion .......................................................................................................... 67 2.4.1 The Development of CA_RFP Sensors and Application in E. coli ............ 67 2.4.2 Intracellular Free Zinc Concentration in E. coli ......................................... 70 III. REGULATORY MECHANISM OF ZINC DETOXIFICATION IN E. COLI UNDER ZINC SHOCK 3.1 Introduction ........................................................................................................ 73 3.2 Materials and Methods ....................................................................................... 76 3.2.1 E. coli Strains and Growth Conditions ....................................................... 76 3.2.2 Measurement of Intracellular Total Zinc .................................................... 77 3.2.3 Measurement of Intracellular Free Zinc ..................................................... 77 3.2.4 mRNA Preparation and Quantification ....................................................... 79 3.3 Results ................................................................................................................ 80 3.3.1 Growth Rates of WT, ∆zntA and ∆zitB ....................................................... 80 3.3.2 Accumulation of Total Zinc in ∆zntA ......................................................... 81 3.3.3 Intracellular Free Zinc in WT, ∆zntA and ∆zitB Cells ................................ 83 3.3.4 Role of zntA and zitB in Regulating Intracellular Free Zinc ....................... 84 3.3.5 Transcriptional Response of Zinc Exporters and Importers ....................... 90 3.4 Discussion .......................................................................................................... 92 3.4.1 Regulation of Intracellular Free Zinc Fluctuations ..................................... 92 3.4.2 Role of ZntA and ZitB in Zinc Detoxification after Zinc Shock ................ 93 3.4.3 Intracellular Free Zinc Spikes Accelerate a Transcriptional Response ...... 95 3.4.4 The In Vivo Zinc Affinity of ZntR .............................................................. 96 IV. FUNCTIONS OF BAE S/R REGULON IN ZINC HOMEOSTASIS IN E. COLI 4.1 Introduction ...................................................................................................... 100 4.2 Methods and Materials ..................................................................................... 105 4.2.1 E. coli Strains and Growth Conditions ..................................................... 105 4.2.2 Measurement of Intracellular Total Zinc .................................................. 106 4.2.3 Measurement of Intracellular Free Zinc ................................................... 107 4.2.4 mRNA Preparation and Quantification ..................................................... 108 4.3 Results .............................................................................................................