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Characterization of Seca-Sod Operon in Borrellia Burgdorferi

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Characterization of Seca-Sod Operon in Borrellia Burgdorferi University of Louisville ThinkIR: The University of Louisville's Institutional Repository Electronic Theses and Dissertations 12-2001 Characterization of secA-sod operon in Borrellia burgdorferi. Tonya Lynn Nichols University of Louisville Follow this and additional works at: https://ir.library.louisville.edu/etd Part of the Medical Immunology Commons, and the Medical Microbiology Commons Recommended Citation Nichols, Tonya Lynn, "Characterization of secA-sod operon in Borrellia burgdorferi." (2001). Electronic Theses and Dissertations. Paper 2881. https://doi.org/10.18297/etd/2881 This Doctoral Dissertation is brought to you for free and open access by ThinkIR: The University of Louisville's Institutional Repository. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of ThinkIR: The University of Louisville's Institutional Repository. This title appears here courtesy of the author, who has retained all other copyrights. For more information, please contact [email protected]. CHARACTERIZATION OF THE SECA-SOD OPERON IN BORRELIA BURGDORFERI By Tonya Lynn Nichols B. S., Livingston University, 1986 M. S., Baylor University, 1991 A Dissertation Submitted to the Faculty of the Graduate School of the University of Louisville In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy Department of Microbiology and Immunology University of Louisville School of Medicine Louisville, Kentucky 2001 ABSTRACT Borrelia burgdorferi, the causative agent of Lyme disease, has been characterized as a microaerophilic spirochete. O2 consumption and utilization potentially yield reactive oxygen intermediates, such as superoxide, hydroxyl radicals, and hydrogen peroxide. This study investigated the expression of the sod gene, which encodes the only, identified oxidative defense mechanism in B. burgdorferi. Using primer extension analysis and RT- PCR, it was found that sod and secA are organized as a single transcriptional unit under the control of σ70-like promoter upstream of the secA open reading frame. Generally, gene expression decreases with increased distance from the promoter; however, secA expression was observed to be relatively lower amounts than sod. Both genes were expressed in all phases of growth, with greatest expression observed in stationary phase. Because transcription of most sod genes is tightly regulated by iron concentration, the secA-sod gene expression was analyzed in borrelial cells grown in varying amounts of metal. Iron- restriction did not significantly affect growth nor did it affect transcription of secA or sod. Likewise, no major differences in transcription were observed with restricting or supplementing media with manganese. To test the possibility that the borrelial SOD may function with either iron or manganese as a cofactor as in cambialistic SODs, SOD activity was assayed and the highest activity was observed in increased manganese concentrations. Protein expression profiles of Sh-2-82 cultured in metal-defined media were investigated by one- and two-dimensional gel electrophoresis. Approximately 15 proteins were differentially expressed in response to metal concentration. iii ACKNOWLEDGMENTS There are many individuals who have played pivotal roles in helping me become the scientist and the person that I am today. As a graduate student at the University of Louisville, I was most influenced by my major professor, Dr. Faye Austin. I thank her for the opportunity to work in her laboratory on B. burgdorferi. Under her guidance, I received a good foundation in the difficult study of RNA genetics. From her, I have learned the value of attention to detail in experimental design, experimentation, and record-keeping, which will follow me throughout my scientific career. Dr. Uldis Streips has also been a key player in my success. I appreciate the time he has devoted to my project and me. I have good memories of our trips to WindRiver where scientific thoughts and ideas were exchanged in an informal, enjoyable manner. I also thank Dr. Robert Stout, Dr. Thomas Geoghegan, and Dr. Ronald Doyle, for their helpful suggestions during this project and their participation as committee members. Additionally, I thank Joan Gagel for her coordination efforts in the preparation of this dissertation. For her kindness and assistance, I would like to thank Jan Powars. There were many others who befriended me and supported me throughout my graduate study, and I am thankful for all of my friends at the University of Louisville. Most of all, I am grateful to my husband, Greg, and our two sons, Matthew and Joshua, for their love, support and understanding. Through a family effort, we have accomplished this goal together. iv TABLE OF CONTENTS ABSTRACT….……………………………………………………………………………. iii ACKNOWLEDGMENTS…..……………………………………………………………... iv TABLE OF CONTENTS………………………………………………………………….. v LIST OF TABLES….……………………………………………………………………… viii LIST OF FIGURES………………………………………………………………………... ix LIST OF ABBREVIATIONS…………………………….…………………...…………... xi INTRODUCTION….……………………………………………………………………… 1 I. Lyme disease….…………………………………………………………………. 1 A. Epidemiology……………………………………………….…………….. 1 B. Clinical features…………………………………………………………... 4 C. Immunopathogenesis……………………………………………………… 6 D. Treatment……………………………………………….………………… 11 E. Vaccine……………………………………………………………………. 11 II. Borrelia burgdorferi….…………….…………………………………………… 13 A. Taxonomy ……………………………………………………………… 13 B. Genetic organization……………………………………………………… 14 C. Morphological features…………………………………………………… 16 D. Metabolism…..…………………………………………………………… 17 E. Virulence Factors….……………………………………………………… 18 III. Transcriptional control in bacteria...…………………………………………… 21 A. Organization of bacterial promoters……………………………………… 21 B. Organization of borrelial promoters……………………………………… 23 C. Processing of bacterial mRNA…..……………………………………….. 25 IV. Bacterial responses to stress and environmental signals….…………………… 27 A. Stationary phase….……………………………………………………….. 27 B. Heat shock response….…………………………………………………… 28 C. Borrelial heat shock response…………………….……………………….. 29 D. Oxidative stress response….……………………………………………… 30 E. Borrelial oxidative stress response……………….………………………. 33 F. Metalloregulation of oxidative stress…..………………………………… 35 V. SecA-dependent protein secretion……………………………………………… 40 A. Overview of secretion mechanisms….…………………………………… 40 B. Transcriptional organization and regulation of SecA…..………………… 42 C. Secretory genes in B. burgdorferi…….…………………………………... 43 SPECIFIC AIMS…...……………………………………………………………………… 45 v MATERIALS AND METHODS…..……………………………………………………… 47 Bacterial strains…..…………………………………………………………… 47 Media and growth conditions………………………………………………… 47 RNA isolation………………………………………………………………… 50 Northern blot analysis….…...………………………………………………… 50 Reverse transcriptase polymerase chain reaction (RT-PCR)….……………… 53 Primer extension (PE) analysis…..…………………………………………… 53 Polymerase chain reaction (PCR)….…………………………………………. 54 DNA sequencing……………………………………………………………… 54 Ribonuclease protection assay (RPA)….……………………………………... 55 Secondary structure analysis…..……………………………………………… 57 Preparation of cell-free extract (CFE)………………………………………... 57 Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE)….……………………………………………………….. 58 Non-denaturing PAGE and SOD activity staining….………………………... 58 Spectrophotometric analysis of SOD activity………….……………………... 59 Two-dimensional gel electrophoresis………………………………………… 59 In-gel tryptic digestion of proteins……………………………………………. 61 Computer imaging……………………………………………………………. 62 Statistical analysis…………………………………….………………………. 62 RESULTS………………………………………………………………………………….. 63 Chapter 1 – Transcriptional analysis of sod in B. burgdorferi….……………. 63 Detection of secA-sod transcript……………………………………… 63 Determination of secA and sod promoter…………………………….. 66 Prediction of secondary structure of secA and sod mRNA transcripts…….…………………………………………………... 79 Chapter 2 – Identifying a possible role for iron in B. burgdorferi……………. 82 Identification of Fur-binding boxes…………………………………... 82 Development of iron-limiting culture conditions…………………….. 82 Chapter 3 – Expression of secA-sod as a function of metal availability……… 101 Development of ribonuclease protection assay………………………. 101 Detection of secA-sod mRNA transcript in different phases of growth….………...…………………………………… 104 Detection of secA-sod mRNA transcript as a function of metal availability…………………………….………………. 107 Chapter 4 – Protein profiling of B. burgdorferi Sh-2-82…..……………….… 114 vi Demonstration of SOD activity…...…………………………………. 114 Effect of metal availability on protein profiles by SDS-PAGE…... 117 Protein profiling by 2D gel electrophoresis….…………………… 120 DISCUSSION….………………………………………………………………………... 133 LITERATURE CITED….……………………………………………………………… 142 VITA….………………………………………………………………………………… 179 vii LIST OF TABLES Table 1. Formulations of Barbour-Stoenner-Kelly (BSK) media….…..….……. 48 Table 2. Oligonucleotide hybridization probe or primer sequences…………….. 52 Table 3. Biotinylated oligonucletide probes used in ribonuclease protection assays…………………………………. 56 Table 4. Summary of growth patterns of Sh-2-82 in metal-restricted media…… 110 Table 5. Protein identification by MALDI-TOF...……………………………… 124 viii LIST OF FIGURES Figure 1. Northern blot analysis of Sh-2-82 mRNA…..……………………………. 65 Figure 2. RT-PCR analysis of the sod transcript…………………………………… 68 Figure 3. Approximate locations of primers used in primer extension analyses….... 70 Figure 4. Lack of σ70-like promoter region upstream of B. burgdorferi sod…..……
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