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Regulation of Brucella Abortus Catalase As a Defensive Mechanism Against Oxidative Stress Jeonga Kim Iowa State University

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Regulation of Brucella Abortus Catalase As a Defensive Mechanism Against Oxidative Stress Jeonga Kim Iowa State University Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1997 Regulation of Brucella abortus catalase as a defensive mechanism against oxidative stress Jeonga Kim Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Immunology and Infectious Disease Commons, Medical Immunology Commons, Microbiology Commons, and the Molecular Biology Commons Recommended Citation Kim, Jeonga, "Regulation of Brucella abortus catalase as a defensive mechanism against oxidative stress " (1997). Retrospective Theses and Dissertations. 12219. https://lib.dr.iastate.edu/rtd/12219 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. INFORMATION TO USERS This manuscript has been reproduced fi-om the microfihn master. UMI fihns the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter &ce, \^e others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. ICgher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Ifowell Information Conqnny 300 North Zeeb Road, Ann Aibor NO 48106-1346 USA 313/761-4700 800/521-0600 Regulation of Brucella abortus catalase as a defensive mechanism against oxidative stress by Jeonga Kim A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Genetics Major Professor: John E. Mayfield Iowa State University Ames, Iowa 1997 UMI Number: 9737732 UMI Microform 9737732 Copyright 1997, 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 ii Graduate College Iowa State University This is to certify that the doctoral dissertation of Jeonga Kim has met the dissertation requirements of Iowa State University Signature was redacted for privacy. Signature was redacted for privacy. Signature was redacted for privacy. For the Graduate College iii To my Parents, and Eury iv TABLE OF CONTENTS ACKNOWLEDGEMENTS vii CHAPTER L GENERAL INTRODUCTION 1 Dissertation organization 1 Animal brucellosis 1 Stinictiiral components of Brucella 2 Pathogenesis of Brucella infection 3 Oxidative stress in phagocytes 5 Bacterial defensive mechanisms 7 Catalase and superoxide dismutase 8 Transcription factors of the LysR family 10 Rationale for this study 11 References 12 CHAPTER n. BRUCELLA ABORTUS PERIPLASMIC CATALASE IS REGULATED BY EXTERNAL HYDROGEN PEROXIDE 21 ABSTRACT 21 INTRODUCTION 22 MATERIALS AND METHODS 24 Bacterial strains and medium 24 Construction of the deletion plasmid 24 Transformation of Brucella abortus 25 Halo assay (Hydrogen peroxide sensitivity assay) 25 2-D protein gel analysis 25 Western blot 26 Catalase assay 27 V Dot blot 27 RESUUrS 28 Sensitivity of the catalase deletion mutant to HzCh 28 2-D gel analysis and identification of protein spots 28 Response of Brucella abortus to oxidative stress 29 2-D gel analysis on Cu-Zn SOD deletion mutant 29 2-D gel analysis on catalase deletion mutant 30 Enzyme activity 30 Dot blot of catalase RNA 30 DISCUSSION 31 REFERENCES 34 CHAPTER in. OXYR REGULATES PERIPLASMIC CATALASE IN BRUCELLA ABORTUS 52 ABSTRACT 52 INTRODUCTION 53 MATERIALS AND METHODS 54 Bacterial strains and medium 54 Primer extension 54 Gel mobility shift 55 Southwestem blot 55 Qoning and sequencing of Brucella oxyR 56 Hydrogen peroxide sensitivity assay (Halo assay) 56 RESUUTS 57 Primer extension 57 Gel mobility shift 57 Southwestem blot 58 vi aoning and sequencing 58 Complementation (Hydrogen peroxide sensitivity) 59 Complementation (Gel mobility shift with bOxyR) 59 DISCUSSION 59 REFERENCES 62 CHAPTER IV. GENERAL CONCLUSIONS 79 Catalase is regulated by external hydrogen peroxide 79 OxyR regialates the catalase transcription 80 APPENDIX. BRUCELLA ABORTUS ARGINASE AND ORNITHINE CYCLODEAMINASE GENES ARE SIMILAR TO TI PLASMID ARGINASE AND ORNITHINE CYCLODEAMINASE 81 vii ACKNOWLEDGEMENTS First of all, I thank God to bring me here and to let me perform this work. He always has been with me on both smooth and rough days. I would like to credit all honor to Him, because he is the source of all that I have accomplished. I would like to express my deepest appreciation to my professor, John Mayfield. He gave me advice, constructive criticism, suggestions, comments about the research, and the preparation of this thesis. In addition, he taught me not only as a scientist, but also as a "real" educator, and as a human being. I am indebted to my Graduate Committee: Dr. Allen Atherly, Dr. Drena Dobbs, Dr. Dan Voytas, and Dr. James Thomas for their helpful suggestions. Special thanks goes to Dr. Enger, the chairman of Zoology and Genetics Department, who always has been supportive from adjustments needed in my beginning years at Iowa State University to giving advice about many decision making situations. I gratefully appreciate the supportive and helpful departmental environment which allows many graduate students to concentrate on study and research. I would like to express gratefulness to my parents who always have been patient, and encouraged me throughout all my life. They have always given me opportunities to leam and achieve things, and their warm heart has provided me with courage and a sense of love. I appreciate Yotmgtae Kim's technical support. He helped me when I needed assistance on computer problems, and took care of Eury when I had to go to lab at night, even though he also was busy at his own research. Finally, I want to thank Eury, my daughter, who has sacrificed in many ways for this work. Eury, to you I say, "I love you the most in the world, v^th my whole heart." 1 CHAPTER I. GENERAL INTRODUCTION Dissertation organization This dissertation is organized into four chapters plus appendix. The first chapter describes general introduction to the organism (Brucella abortus), and literature summary for the backgroimd of this study. The second and third chapters include manuscripts which will be submitted to the identified journals. The fourth chapter describes the general conclusions that were drawn from the results of the study. I include a brief report about the arginase operon in the appendix section, since the study is not directly relevant to my thesis project. A cDNA clone was made by reverse transcription of total Brucella RNA using a primer from the middle of the catalase gene. However, 1 foimd a cDNA which contained arginase and ornithine cyclodeaminase genes, not the catalase gene. We concluded that the clone was made by nonspecific binding of the primer to the arginase operon mRNA. In this brief paper, I present the nucleotide sequences of Brucella arginase and ornithine cyclodeaminase genes and discuss the possible physiological importance of these genes to B. abortus. Animal brucellosis Animal brucellosis is a disease that causes fever and reproductive failiire including abortion, epididjonitis, and sterility in males. Different Brucella species cause brucellosis in different animals; B. abortus (cattle), B. melitensis (goats), B. suis (swine), B. canis (dogs), B. ovis (sheep), and B. neotomae (desert rat) (51). Bovine brucellosis has been reported in many countries including the United States of America (20). Although there has been an active vaccination program, it has been 2 difficult to eradicate the disease from this cotintry (51,55). The impact of bovine brucellosis concerns both economic loss and human health (55). Structural components of Brucella Currently, six species of Brucella are included in the genus. The species have been separated into biotypes by colony morphology, metabolic patterns, CO2 and serum requirements, susceptibility to bacteriophage, and biological behavior (51). However, DNA-DNA hybridization studies revealed that four species (B. abortus, B. suis, B. melitensis and B. neotomae) have nearly 100% homology in nucleotide sequences (35,36). These same studies reported that B. cms has 94% homology in nucleotide sequences and proposed that the species is most likely a deletion mutant of one of the other species. Verger (84) proposed Brucella should be a single species. Other molecular genetic studies (46) also
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