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H. Volcanii Deletion Strains CHARACTERIZATION OF GLYCEROL METABOLISM AND RELATED METABOLIC PATHWAYS IN THE HALOARCHAEAON Haloferax volcanii By KATHERINE SHERWOOD RAWLS A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010 1 © 2010 Katherine Sherwood Rawls 2 To my husband, Colin Rawls, who has been my guiding light, and to my parents, Steve and Francy Sherwood, for their unconditional love and support 3 ACKNOWLEDGMENTS I express my deepest gratitude to my academic advisor, Dr. Julie Maupin-Furlow, for challenging my intellectual capabilities and for expanding my scientific curiosity. Her guidance during my graduate career has been crucial toward successful completion of my degree. I also wish to thank the additional members of my graduate supervisory committee, Drs. Linda Bloom, Graciela Lorca, Lonnie Ingram and K.T. Shanmugam for their project insight and for generous use of their equipment. Special thanks are extended to current and former graduate students, post- doctoral students, laboratory technicians, and undergraduate researchers of the Maupin-Furlow research laboratory for creating a stimulating, pleasant environment in which to work. Specifically, I would like to acknowledge David Cano for his help in generating the glycerol kinase mutant; Shalane Yacovone and Mario Corro for their assistance in generating several knockout strains including the phosphotransferase system mutants, the dihydroxyacetone kinase mutants, and the GlpR repressor mutant; and Mayra Souza for her additional assistance in generating the GlpR repressor mutant as well as the transcriptional promoter-reporter fusion constructs. Also, I wish to thank Gosia Gil-Ramadas for her guidance as my undergraduate research mentor. Special thanks are also extended to Dr. Sivakumar Uthandi and Nikita Nembhard for their insightful advice and support, especially during my last semester of graduate school. Thank you to all graduate funding agencies including the University of Florida Alumni Foundation, the Doris Lowe and Verna and Earl Lowe Scholarship Fund, the National Institute of Health, and the Department of Energy for their financial support of my dissertation project. 4 I finally wish to acknowledge my immediate family members: my parents, Francy and Steve Sherwood; my sister, Kelley Sherwood; my niece, Reagan Timmons; my grandmothers Johnnie Sherwood and Helen Glenn; and my late grandfathers Jim Sherwood and Bill Glenn, who have always graciously provided their moral and financial support throughout my academic career. I most importantly thank my husband, Colin Rawls, for providing unwavering love and encouragement throughout this challenging process. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF ABBREVIATIONS ........................................................................................... 14 ABSTRACT ................................................................................................................... 20 CHAPTER 1 LITERATURE REVIEW .......................................................................................... 22 Introduction ............................................................................................................. 22 An Overview of Glycerol Metabolism ...................................................................... 22 Biosynthesis of Glycerol ................................................................................... 23 Glycerol-3-phosphate dehydrogenase ....................................................... 24 Glycerol-3-phosphate phosphatase ........................................................... 25 Glycerol Dissimilation to Dihydroxyacetone Phosphate ................................... 25 Glycerol kinase .......................................................................................... 27 Glycerol-3-phosphate dehydrogenase ....................................................... 28 Glycerol dehydrogenase ............................................................................ 30 Dihydroxyacetone kinase ........................................................................... 30 Anaerobic Glycerol Dissimilation Pathways ...................................................... 32 Glycerol dehydratase ................................................................................. 33 1,3-Propanediol dehydrogenase ................................................................ 35 Glycerol Transport Across a Biological Membrane ................................................. 35 Intrinsic Permeability ........................................................................................ 36 Aquaglyceroporins ............................................................................................ 37 Glycerol facilitator protein GlpF .................................................................. 38 Fps1p ......................................................................................................... 39 Additional Aquaglyceroporins ........................................................................... 41 Active Transport of Glycerol ............................................................................. 44 Transcriptional and Post-Translational Control of Glycerol Metabolism .................. 45 Transcriptional Regulators of Glycerol Metabolism .......................................... 45 cAMP-cAMP receptor protein ..................................................................... 46 FNR ........................................................................................................... 48 CcpA .......................................................................................................... 50 GlpR ........................................................................................................... 51 DhaR .......................................................................................................... 54 ArcA/ArcB .................................................................................................. 55 Additional transcriptional regulators of glycerol metabolism ...................... 57 Allosteric and Post-translational Regulation of Glycerol Kinase Activity ........... 58 Hpr- and EI-dependent phosphorylation of glycerol kinase ........................ 59 Fructose 1,6-bisphosphate......................................................................... 60 6 EIIAGlc ........................................................................................................ 61 Industrial and Biological Significance of Glycerol .................................................... 63 Glycerol as a Feedstock for Bioconversion ...................................................... 63 Therapeutic and Diagnostic Uses of Glycerol ................................................... 65 Biological Relevance of Glycerol to Halophilic Communities ............................ 66 Osmoprotectant properties of glycerol ....................................................... 66 Glycerol production by the halotolerant green algae Dunaliella ................. 68 Glycerol metabolism in halophilic bacteria ................................................. 70 Glycerol metabolism in haloarchaea .......................................................... 71 An Overview of Glucose and Fructose Metabolism in Haloarchaea ....................... 72 Transport of Fructose and Glucose Across a Biological Membrane ....................... 73 Overview of the Phosphotransferase System ................................................... 73 General carrier protein EI ........................................................................... 75 General carrier protein Hpr ........................................................................ 76 Sugar-specific component EII .................................................................... 76 ATP Binding Cassette Transporters ................................................................. 77 Secondary Transporters ................................................................................... 79 Fructose and Glucose Metabolism and Their Regulation in Haloarchaea .............. 79 Glucose Degradation through a Modified Entner-Doudoroff Pathway .............. 80 Fructose Degradation through a Modified Embden-Meyerhof-Parnas Pathway ........................................................................................................ 82 Regulation of Glucose and Fructose Metabolism in Haloarchaea .................... 83 TrmB .......................................................................................................... 83 General transcription factors TATA binding protein and transcription factor B ................................................................................................... 84 Project Rationale and Design ................................................................................. 86 2 METHODS AND MATERIALS ................................................................................ 91 Chemicals, Media, and Strains ............................................................................... 91 Chemicals and Reagents ................................................................................. 91 Strains, Plasmids, and Culture Conditions ....................................................... 92 High Performance Liquid Chromatography
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