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Roles of Nickel Binding Proteins in Helicobacter Species

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Roles of Nickel Binding Proteins in Helicobacter Species ROLES OF NICKEL BINDING PROTEINS IN HELICOBACTER SPECIES by SUSMITHA SESHADRI (Under the Direction of Robert J. Maier) ABSTRACT Proteins Hpn and Hpn-like of the gastric pathogen H. pylori were hypothesized to bind nickel since histidine residues make up 45% and 27% of their amino acid content, respectively. Characterization of an hpn, an hpn-like and an hpn, hpn-like double mutant revealed novel functions for these gene products in nickel detoxification and storage. Compared to the wild-type parent, mutant strains were more sensitive to elevated concentrations of nickel, cobalt, and cadmium, indicating roles for the two proteins in surviving metal toxicity. Under low nickel conditions, the mutants exhibited higher urease activities and had increased amount of Ni- associated with urease; but similar urease apo-protein levels to wild-type. The parent achieved mutant level urease activities under nickel supplementation and lower pH conditions while growth with a nickel chelator decreased mutant but not wild-type urease activities. These results strongly imply a role for these proteins as nickel reservoirs/storage proteins. H. hepaticus colonizes a non-acidic niche, hence nickel metabolism may be different than in H. pylori. A nikR mutant exhibited higher urease and hydrogenase activities under all supplemental nickel conditions, but there was no change in urease expression, and NikR did not bind pUreA or pHydA. Higher total nickel levels (detected by ICP-MS) in the nikR mutant implied possible higher nickel transporter (NikA) levels, which was later verified by qRT-PCR and binding of NikR to pNikA. Periplasmic nitrate reductase (NapA) was upregulated in the NikR strain. Wild-type H. hepaticus had increased H2 oxidation levels in the presence of nitrate compared to O2 provided as a terminal acceptor, suggesting the importance of anaerobic respiration for this bacterium. The NikR strain showed higher H2 oxidation levels than the parent - with either O2/NO3 as electron acceptor. Also, the rate of nitrate disappearance was higher in a nikR mutant. Collectively, these results suggest NikR modulates the nickel-enzymes via regulating the nickel transporter and plays important roles other than in nickel metabolism. My studies aimed at characterizing nickel binding proteins by studying the mutant strains in two different organisms Helicobacter species. The data indicate that these proteins play vital functions to specifically suit the needs of each pathogen. INDEX WORDS: Nickel, Nickel Metabolism, Nickel homeostasis, Hpn, Hpn-like, Urease, Hydrogenase, NikA, NikR, Helicobacter pylori, Helicobacter hepaticus ROLES OF NICKEL BINDING PROTEINS IN HELICOBACTER SPECIES by SUSMITHA SESHADRI B.S., UNIVERSITY OF MADRAS, INDIA, 2002 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2009 © 2009 Susmitha Seshadri All Rights Reserved ROLES OF NICKEL BINDING PROTEINS IN HELICOBACTER SPECIES by SUSMITHA SESHADRI Major Professor: Robert J. Maier Committee: Michael W.W. Adams Duncan C. Krause Juergen Wiegel Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia August, 2009 DEDICATION I dedicate this work to my family- my parents, my husband, my brother, my grandmother and my daughter. I dedicate this dissertation to my dad, for instilling a love for science and books in me, for believing in me and for listening to me and for being a great friend. I dedicate this to my mother, for helping me when I needed it the most, and for encouraging me to make important life decisions- at the right time, that have brought me immense joy. I dedicate this work to my husband Ramesh, without whose support and encouragement, and numerous compromises, I could not have realized this lifelong ambition. I dedicate this work to my brother Krishna and my grandmother for cheering me up and showering me with their affection. Lastly, I dedicate this work to my daughter Shriya; whose arrival brought me indescribable happiness, the vigor and motivation to complete what I had started, when it was needed the most. I dedicate my dissertation to all of you for always being there for me and for unconditionally loving me. iv ACKNOWLEDGEMENTS First and foremost, I would like to thank Dr. Robert Maier for giving me the opportunity to do research in his lab. His enthusiasm for research is highly contagious. I would like to thank him for being an approachable, understanding, and encouraging and excellent mentor. I appreciate all the help and guidance he has given me throughout my graduate studies. I would also like to thank my doctoral committee members Dr. Krause, Dr. Adams and Dr. Wiegel for their timely support and suggestions and encouragement. I would especially like to thank Dr. Stephane Benoit, Dr. Nalini Mehta, Dr. Praveen Alamuri, Adriana Olczak, and Dr. Guy Wang for answering innumerable questions with great patience and for being excellent mentors. I would like to thank all of them and Sue Maier, Dr. Andrea Zbell, Andrew Burk, Rory Eutsey, Dr. Mike Wineberg, Mykeshia McNorton, Erica Miller and Dr. Manish Mahawar for being wonderful colleagues and making the lab a happy place to work in. I have made many great friends in Athens (You know who you are☺). I would like to thank each and every one of them for the wonderful times we have shared and the golden memories we have created together in Athens. Athens would not have been home without their help, humor, company and friendship. v TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .............................................................................................................v LIST OF TABLES ....................................................................................................................... viii LIST OF FIGURES ....................................................................................................................... ix CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW .....................................................1 Nickel metabolism and homeostasis .........................................................................1 Mechanism of nickel toxicity ....................................................................................2 Nickel transport in bacteria .......................................................................................3 Nickel enzymes, their accessory proteins and maturation factors .............................9 Regulators of nickel metabolism .............................................................................21 Helicobacter pylori and Helicobacter hepaticus- Model organisms of study ........24 Microbiology of Helicobacter pylori ......................................................................27 Microbiology of Helicobacter hepaticus ................................................................43 Nickel metabolism in Helicobacter pylori and Helicobacter hepaticus .................48 Scope of this study ..................................................................................................56 References ...............................................................................................................58 2 ROLES OF HIS-RICH HPN AND HPN-LIKE PROTEINS IN HELICOBACTER PYLORI NICKEL PHYSIOLOGY .........................................................................92 Abstract ..................................................................................................................93 vi Introduction .............................................................................................................94 Materials and methods .............................................................................................96 Results .....................................................................................................................99 Discussion .............................................................................................................104 Acknowledgements ...............................................................................................108 References .............................................................................................................108 3 HELICOBACTER HEPATICUS NIKR- ROLES IN NICKEL METABOLISM AND POSSIBLE ROLE IN ANAEROBIC RESPIRATION .........................................120 Abstract ................................................................................................................121 Introduction ...........................................................................................................122 Materials and methods ...........................................................................................124 Results ...................................................................................................................132 Discussion .............................................................................................................138 Acknowledgements ...............................................................................................143 References .............................................................................................................143 4 DISSERTATION SUMMARY AND FUTURE DIRECTIONS ..............................159 Roles of the his-rich Hpn and Hpn-like proteins in H. pylori nickel physiology..159 H. hepaticus NikR modulates urease and hydrogenase ........................................161 References
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