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RELEVANCE of Tolc and EXPRESSION OF RELEVANCE OF tolC AND EXPRESSION OF acrAB AND emrAB IN Erwinia chrysandiemi by RAVI DAMODAR BARABOTE, B.Sc, M.Sc. A DISSERTATION IN BIOLOGY Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for die Degree of DOCTOR OF PHILOSOPHY Approved Chai/person of the Committee I •~A \S i * "». Accepted ^ •-'- • • f ^^ ^ ..I y I Dean of the Graduate School December, 2003 ACKNOWLEDGEMENTS This dissertation is dedicated in the loving memory of my mom, Pratibha Barabote, and to my dad, Damodar Barabote, who epitomize unconditional love. They worked very hard and sacrificed many things in order to help me achieve my goals. My interactions with Dr. Michael San Francisco have tiansformed me into a wiser and more self-confident student. He has served as a constant source of inspiration for me and I have leamt a great deal from him, in explicit as well as imphcit ways. He went out of his way to help me in any way he could, even beyond the frontiers of professional hfe. His faith in my skills always boosted my confidence. I would like to express my smcere gratitude to Dr. San Francisco for all his help and support. He is an extiaordinarily understandmg and a nice individual. I extend my heartfelt thanks to him for his mcredible patience, constant encouragement, and support throughout my doctoral work. I sincerely thank him for fmancially supporting me with a research assistantship during several semesters. This work would not be possible without the sustained support of my doctoral committee members, who have also been a great source of inspiration for me. I would Idee to thank Dr. Randy Allen for his encouragement and for letting me use the Kodak Image Station and the Reporter Microplate Luminometer in his lab. I owe a great deal to Dr. Larry Blanton for his help and for allowing me to use the HHMI facilities such as the Biotek Fluorescence Microplate Reader, BioRad Micropulser Electioporator, Stiatalinker UV Crosslinker, BioRad Gel Doc, etc. I thank him for his confidence in my teaching abilities and for choosing me as a Teaching Assistant for the Cell Biology Laboratory during several semesters. Dr. Joe Fralick has always inspired me with his brilliant scientific ideas. I thank him for providing some ofthe bacterial sfrains used in this study. I enjoyed interacting with Dr. Abdul Hamood. I would Idee to thank him for providmg me the media for the protease assay. I greatly appreciate Dr. Randall Jeter for bemg very 11 understanding, supportive, for all his help, and for permitting me to use the French Pressure Cell Press equipment in his lab. I am grateful to all die past and present members ofthe lab for their friendship, help, and for making laboratory work a pleasant experience. I appreciate the help and guidance of Dr. Venugopal Valmeekam, Dr. Brian Haseloff, Todd Anderson, and David Wheeler. Florencia Meyer helped me with her digital camera to take some ofthe pictures included in this dissertation, and she has been a great friend. Ramani Ravirala has been an incredibly helpful and supportive colleague and a very affectionate friend. I thank the Department of Biological Sciences for Summer Research Awards (1999,2001, and 2003), the Graduate School for Smnmer Dissertation Research Award (2002), and die Association of Biologists for Summer Mini Grants (2002 and 2003). I appreciate the travel grants provided by the Association of Biologists, the Student Chapter of American Society for Microbiology at TTU, and the Graduate and Professional Student Government Association. I thank Dr. Susan San Francisco and Ruwanthi Wettasinghe at the TTU Biotechnology Core Facility for their help with DNA sequencing and Oligo synthesis. I would also like to thank Ms. Barbi Dickensheet for her help and suggestions in preparing this manuscript. Ill TABLE OF CONTENTS ACKNOWLEDGEMENTS 11 ABSTRACT vi LIST OF TABLES vii LIST OF FIGURES viii CHAPTER I. OVERVIEW 1 1.1 Erwinias and Soft Rot 1 1.1.1 The Genus Erwinia 1 1.1.2 Model Systems 2 1.1.3 Soft Rot 3 1.2 Pathogen-Plant Interactions 3 1.2.1 Gene-for-Gene Interactions 3 1.2.2 Plant Defense 4 1.3 Erwinia chrysanthemi 5 1.3.1 The Bacterium 5 1.3.2 E. chrysanthemi Phytopathogenesis 7 1.3.2.1 Viralence Factors 7 1.3.2.2 Other Pathogenicity Factors 9 1.3.2.3 Regulation of Pectmase Genes 9 1.3.2.4 Secretion and Uptake Systems 10 1.3.3 Countering the Host Defense 11 1.3.3.1 Avirulence Proteins 11 1.3.3.2 Protective Mechanisms "11 1.3.3.3 Survival Mechanisms 12 1.4 Rationale for die Stiidy 13 1.4.1 Efflux-Mediated Resistance 13 1.4.2 Objectives 14 n. ROLE OF ERWINIA CHRYSANTHEMI TOLC IN ANTIMICROBIAL CHEMICAL RESISTANCE 15 2.1 Introduction 15 2.2 Materials and Methods 15 2.2.1 Bacterial Stiains and Growth Conditions 15 2.2.2 DNA Manipulations 16 2.2.3 £". c/i/75anf/iew/Transformation 16 2.2.4 Marker-Exchange Mutagenesis 17 IV 2.2.5 hnmunoblot Analysis of TolC 17 2.2.6 Zone Inhibition Assays 18 2.2.7 Determination of Minimal Inhibitory Concentiation (MIC) 18 2.3 Results and Discussion 18 2.3.1 Constmction of the E. chrysanthemi tolC Mutation 18 2.3.2 Confirmation ofthe tolC Mutant 21 2.3.3 Characterization ofE. chrysanthemi tolC 21 2.4 Conclusions 26 ni. ROLE OF ERWINIA CHRYSANTHEMIIOLC IN PHYTOPATHOGENESIS 27 3.1 Introduction 27 3.2 Materials and Methods 27 3.2.1 Bacterial Stiauis and Growth Conditions 27 3.2.2 Phytopathogenicity Assay 28 3.2.3 Survival In planta 29 3.2.4 Minimum Inhibitory Concentiation (MIC) Assay 29 3.2.5 Determmation of Berberine Accumulation 29 3.3 Results and Discussion 30 3.3.1 Role ofE. chrysanthemi TolC in Phytopathogenesis 30 3.3.2 Survival of the TolC Mutant In planta 32 3.3.3 Sensitivity of die TolC Mutant to Plant Antimicrobials 33 3.3.4 Accumulation of Berberine in the TolC Mutant 35 3.4 Conclusions 3 6 IV. EXPRESSION 0¥lYfE ERWINIA CHRYSANTHEMI ACRAB AND EMRAB EFFLUX PUMP GENES 37 4.1 Infroduction 37 4.2 Materials and Methods 39 4.2.1 Bacterial Sfrains and Growth Conditions 39 4.2.2 In planta Expression 39 4.2.3 Expression in the Presence of Plant Chemicals 40 4.3 Results and Discussion 41 4.3.1 Constmction of acrA:: lux and emrA:: uidA Fusions 41 4.3.2 Expression ofthe acrAwlux and emrA::uidA In planta 41 4.3.3 In vitro Expression of acrA::lux and emrA::uidA 43 4.4 Conclusion 50 V. SUMMARY 51 REFERENCES 54 ABSTRACT Plants produce a repertoire of antimicrobial chemicals, some of which are produced in response to an infection. Altiiough much is understood about the vimlence capabilities of many bacterial plant patiiogens, little is known about the mechanisms employed by phytopatiiogens to survive die onslaught ofthe plant chemical environment. Here, die first report ofthe role of TolC in phytopathogenesis is presented. TolC is the outer membrane component of several multi-drug resistance (MDR) efflux pumps, such as AcrAB and EmrAB in E. coli, and plays an important role in the survival and virulence of many bacterial animal patiiogens. A tolC mutant ofE. chrysanthemi was found to be exttemely sensitive to antimicrobial agents mcluding several plant-derived chemicals. This mutant was unable to grow in planta and its ability to cause plant tissue maceration was severely compromised. The tolC mutant was shown to be defective m the efflux of berberine, a model antimicrobial plant chemical. These resuUs suggest that the E. chrysanthemi to/C plays an important role in the survival and colonization ofthe pathogen in plant tissue by conferring resistance to the antimicrobial compounds produced by plants. Therefore, in order to assess the plausible implication of MDR during plant disease, the expression ofthe E. chrysanthemi acrAB and emrAB homologs was investigated using their respective promoter-fusions to reporter genes. Both pumps appear to be expressed in planta and in vitro in the presence of several plant-derived molecules. Plant-derived molecules, such as sahcylic acid, hydrogen peroxide, paraquat, and genistein have previously been shown to stimulate antibiotic resistance in other bacteria. Interestingly, jasmonate, which is produced in plants as part ofthe plant defense response to pathogen invasion, was found to stimulate expression of both the pumps in E. chrysanthemi. This is the first report of jasmonate-dependent expression of bacterial efflux pump genes. Avimlence ofthe E. chrysanthemi tolC mutant and expression of acrAB and emrAB pumps in planta as well as in vitro in the presence of plant-defense related molecules suggest that MDR efflux pumps may play an important role in pathogenesis of this bacterium. VI LIST OF TABLES 1.1 Examples of Antibacterial Plant Compounds 6 2.1 Bacterial Strains and Plasmids 16 2.2 Sensitivity to Antimicrobial Agents 24 2.3 MICoftheAntinucrobial Agents 25 3.1 Bacterial Sfrains and Plasmids 28 3.2 MIC of Plant-Derived Chemicals 34 4.1 Bacterial Strains and Plasmids 40 vu LIST OF FIGURES 1.1 Gene-for-Gene friteractions m Pathogen-Plant Interactions 4 1.2 Disease Resistance Pathways 6 1.3 E. chrysanthemi Patiiogeiucity: A Multi-Factorial Process 8 1.4 Schematic Representation an Efflux Pump 14 2.1 Constmction of £". chrysanthemi tolC Mutation 20 2.2 Marker-Exchange Mutagenesis 20 2.3 Southem Blot Analysis of the tolC Mutant 22 2.4 PCR Analysis of die tolC Mutant 22 2.5 Immunoblot Analysis ofthe TolC Mutant 23 2.6 Zone Inhibition Assay 23 2.7 Analysis ofthe Outer Membrane Proteins 25 3.1 Phytopathogenicity of the TolC Mutant 31 3.2 Semi-Quantitative Estimation of Phytopathogenesis 31 3.3 Complementation of the Maceration Defect 3 2 3.4 /n/7/a/ito Survival ofthe TolC Mutant 33 3.5 Berberine Accumulation in TolC Mutant 35 4.1 Regulation of the acrAB and emrAB in E.
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