University of Montana ScholarWorks at University of Montana Graduate Student Theses, Dissertations, & Professional Papers Graduate School 1998 Development of a system for genetic manipulation of Bartonella bacilliformis James M. Battisti The University of Montana Follow this and additional works at: https://scholarworks.umt.edu/etd Let us know how access to this document benefits ou.y Recommended Citation Battisti, James M., "Development of a system for genetic manipulation of Bartonella bacilliformis" (1998). Graduate Student Theses, Dissertations, & Professional Papers. 10534. https://scholarworks.umt.edu/etd/10534 This Dissertation is brought to you for free and open access by the Graduate School at ScholarWorks at University of Montana. It has been accepted for inclusion in Graduate Student Theses, Dissertations, & Professional Papers by an authorized administrator of ScholarWorks at University of Montana. For more information, please contact [email protected]. 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Battisti B.A. Carroll College, Helena, Montana 1991 Presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy The University of Montana 1998 Vc-LaiC ^•"vl c -- —Chairman, Board of Examiners Dean, Graduate School Date Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Maureen and Mike MANSFIELD LIBRARY The University of1 V I O N T A N A Permission is granted by the author to reproduce this material in its entirety, provided that this material is used for scholarly purposes and is properly cited in published works and reports. * * Please check "Yes" or "No" and provide signature ** Y es, I grant permission x T No, I do not grant permission Author's Signature D a te ____________ Any copying for commercial purposes or financial gain may be undertaken only with the author's explicit consent. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 9840731 UMI Microform 9840731 Copyright 1998, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS I am grateful to Michael F. Minnick for his unparalleled mentorship on both professional and personal levels, for the tools of molecular biology, and the mastery of nymphing. The guidance and instruction of professors Scott Samuels, George Card, Bill Granath, Ralph Judd, Scott Manning, Keith Parker, Bill Holben, Barb Wright, Dan Deborde and Jim Gannon was integral to the completion of this study, as was the assistance and furnishings from; Dr. Sam Mitchell, Helen Grasseschi, David Scherer, Paul Dexter, Sherry Coleman, Steve McAllister, Joan Strange, Pat Ball, Dr. Tim Gsell, Jim Driver, Dr. Dennis Reshke, Dr. M.E. Kovach, Dr. Jean Pfau, Dr. Angelica Longacre and Deborah Kane. I was especially lucky to be assisted by Laura Smitherman in the gyrB section of this dissertation (Chapter four), her laboratory and intellectual skills are superb. Over the years I have found many friends throughout the division with whom I have shared science, laughter, anticipation, confusion, and frustration, all of which has had an impact on this study in many ways that I am aware of as well as many that I am not To my good friends Scott, Brian, Pete, Shane, Warren, Craig, Dave, Mike, Eric, Larry, Jen, Abbie, and Sheila for all of the smiles and laughs. To my Mom and Dad, grandparents, and family for their love and support over the years. To the New York Yankees, Grandpa’s J.D. Lamp, and the rivers and lakes and the beautiful fish in them. Finally, I dedicate this work to Tracy, Julia, and John, for without their love, understanding, time, and confidence, none of this would have been possible. iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS Chapter Page 1 Introduction to the Bartonella and Genetic Manipulation 1 I. The Bartonella 1 A. Introduction 1 B. Phylogeny 2 C. History and Epidemiology 5 1. B. bacillifoTtms 5 2. B. quinlana 8 3. B. henselae 10 4. Other Bartonella 11 D. Physical Characteristics 12 E. Pathogenesis 13 1. Carri6n’s disease: Oroya Fever and Verruga peruana 14 2. Trench Fever 15 3. Cat Scratch Disease (CSD) 16 4. Endocarditis 17 5. Bacillary Angiomatosis (BA) 17 F. Virulence Determinants 18 1. Adhesion to Host Cell 20 2. Invasion of Host Cell 22 3. Angiogenic Factor 25 4. Immune Evasion Factors and Strategies 26 5. Stress Response Factors 27 6. Hemin Binding Factor 27 G. Diagnosis, Treatment and Prevention 28 1. Diagnosis 28 2. Treatment 28 3. Prevention 29 II. Genetic Manipulation 30 A. Introduction and Background 30 1. Plasmid Transformation 33 2. Random Mutagenesis 34 3. Site-Specific Mutagenesis:Insertional Recombination 35 4. Site-Specific Mutagenesis:Replacement Recombination 36 5. Natural Mutations 36 B . Homologous Recombination 37 III. Research Significance and Goals 38 2. Optimization of the Conditions for Homologous Recombination 42 I. Introduction 42 II. Materials and Methods 42 III. Results 59 IV. Discussion 59 iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3. Site-Directed Mutagenesis and Transcomplementation of the 62 Bartonella baciltiformis Fiagellin Gene I. Abstract 62 II. Introduction 63 III. Materials and Methods 65 IV. Results 71 V. Discussion 90 VI. Acknowledgments 98 VII. References 98 4 . Mutations in Bartonella bacilliformis gyrB confer resistance 104 to coumermycin Aj I. Abstract 104 II. Introduction 105 III. Materials and Methods 107 IV. Results 113 V. Discussion 126 VI. Acknowledgments 129 VII. References 130 5. General Discussion 137 References for Chapters One, Two, and Five 143 v Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES Chapter Table Description Page 1 1 Significant Historical Moments 6 2 The Bartonella genus 9 3 1 Bacterial strains and plasmids 66 4 1 Bacterial strains and plasmids 108 2 Genotypic analysis of natural mutants 124 3 Complementation with B. bacilliformis gyrB 125 vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. l i s t o f f i g u r e s Chapter Figure Description Page I 1 Phylogeny of the Bartonella 3 2 Strategies of genetic manipulation 31 2 1 Obstacles impeding homologous recombination 43 2 Flagellin O-type gene targeting constructs 51 3 Flagellin Q-Type gene targeting construct 53 4 GyrB Q-Type gene targeting construct 55 3 1 Diagram of site-directed flagellin disruption 73 2 PCR analysis of flagellin mutants 77 3 SDS-PAGE and immunoblot analysis of mutants 79 4 PCR analysis of transcomplements 83 5 SDS-PAGE, immunoblot of transcomplements 85 6 Phenotypic analysis by electron microscopy 87 7 Phenotypic analysis of motility 91 8 Human erythrocyte adhesion analysis 93 4 1 gyrB nucleotide and predicted amino acid sequence 115 2 GyrB multiple alignment 117 3 gyrB DNA hybridization 120 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. CHAPTER ONE Introduction to the Bartonella and Genetic Manipulation I. THE BARTONELLA A. INTRODUCTION The bartonellae are a unique group of gram negative intracellular bacteria that employ arthropod-mediated transmission and hemotrophy as common parasitic strategies. Phylogenetically, recent taxonomic re-classifications have expanded the number of Bartonella species from one, B. bacilliformis, to 11 species,