The Role of Plant-Derived Quaternary Ammonium Compounds, Including Carnitine and Choline-O-Sulfate, on the Biology of the Plant
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Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2015 The oler of plant-derived quaternary ammonium compounds, including carnitine and choline-O- sulfate, on the biology of the plant pathogen Pseudomonas syringae and its interactions with the host species Phaseolus vulgaris Michael David Millican Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Agriculture Commons, Microbiology Commons, and the Plant Pathology Commons Recommended Citation Millican, Michael David, "The or le of plant-derived quaternary ammonium compounds, including carnitine and choline-O-sulfate, on the biology of the plant pathogen Pseudomonas syringae and its interactions with the host species Phaseolus vulgaris" (2015). Graduate Theses and Dissertations. 14948. https://lib.dr.iastate.edu/etd/14948 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 Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. The role of plant-derived quaternary ammonium compounds, including carnitine and choline-O-sulfate, on the biology of the plant pathogen Pseudomonas syringae and its interactions with the host species Phaseolus vulgaris by Michael D. Millican A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Microbiology Program of Study Committee: Gwyn Beattie, Major Professor Thomas Bobik Qijing Zhang Byron Brehm-Stecher Larry Halverson Iowa State University Ames, Iowa 2015 Copyright © Michael D. Millican, 2015. All rights reserved. ii DEDICATION To my parents, Doug and Mary Millican. You have selflessly provided me the opportunity to pursue my dreams and no words could express my admiration and appreciation for you and all that you have done for me. And to my wife, Sarani Rangarajan, for your unwavering support I would like to thank you from the bottom of my heart, but for you my heart has no bottom. iii TABLE OF CONTENTS Page ACKNOWLEDGMENTS ........................................................................................ v ABSTRACT………………………………. ............................................................. vi CHAPTER 1 GENERAL INTRODUCTION ...................................................... 1 Introduction ........................................................................................................ 1 Thesis Organization ............................................................................................ 2 Literature Review ................................................................................................ 3 References ........................................................................................................ 13 CHAPTER 2 CARNITINE RELEASED FROM GERMINATING PHASEOLUS VULGARIS SEEDS PROMOTES THE GROWTH OF PSEUDOMONAS SYRINGAE AND REQUIRES DHCAB FOR REMOVAL OF THE INTERMEDIATE ACETOACETATE FOR CATABOLISM .................. 26 Abstract to ........................................................................................................ 26 Introduction ........................................................................................................ 27 Methods and Materials ....................................................................................... 30 Results of the ....................................................................................................... 37 Discussion ........................................................................................................ 50 References ........................................................................................................ 55 CHAPTER 3 CHOLINE-O-SULFATE CATABOLISM IN PSEUDOMONAS SYRINGAE REQUIRES A SULFATE EXPORTER THAT MAY FUNCTION TO RELIEVE SULFATE TOXICITY AND IS ENCODED IN A LOCUS WITH CHOLINE-O-SULFATE SULFATASE ..................................................................... 60 Abstract to ........................................................................................................ 60 Introduction ........................................................................................................ 61 Methods and Materials ....................................................................................... 64 Results of the ....................................................................................................... 66 Discussion ........................................................................................................ 75 References ........................................................................................................ 84 iv CHAPTER 4 PLANT TISSUE-SPECIFIC BENEFITS OF QUATERNARY AMMONIUM COMPOUNDS TO PSEUDOMONAS SYRINGAE DURING LEAF AND SEED COLONIZATION ..................................................................... 89 Abstract to ........................................................................................................ 89 Introduction ........................................................................................................ 90 Methods and Materials ....................................................................................... 95 Results of the ....................................................................................................... 98 Discussion ........................................................................................................ 105 References ........................................................................................................ 113 CHAPTER 5 CONCLUSIONS AND FUTURE DIRECTIONS ......................... 118 References ........................................................................................................... 121 APPENDIX A SUPPLEMENTARY TABLES AND FIGURES FOR CHAPTER II ........................................................................................................... 123 APPENDIX B SUPPLEMENTARY TABLES AND FIGURES FOR CHAPTER III ........................................................................................................... 131 APPENDIX C SUPPLEMENTARY FIGURES FOR CHAPTER IV .................. 140 v ACKNOWLEDGMENTS I would like to thank my advisor, Gwyn A. Beattie, for her endless patience and thoughtful guidance over these years. I would also like to thank my committee members, Thomas Bobik, Qijing Zhang, Byron Brehm-Stecher, and Larry Halverson for their guidance and support throughout the course of this research. In addition, I would also like to thank my wife, without whom this dissertation would be an jarrbelled un reedabel dizazter. I want to thank my friends and family who have supported me all these years. Lastly, but certainly not least, I would like to thank Regina McGrane for her thoughtful input and countless discussions. vi ABSTRACT Knowledge of the chemicals available during plant-microbe interactions is useful for promoting the growth of beneficial bacteria and predicting pathogen populations and thus the probability of disease. Pseudomonas syringae B728a is able to utilize plant-derived quaternary ammonium compounds (QACs), such as L-carnitine, choline, and choline derivatives, as osmoprotectants and nutrient sources. We explored in detail the biological roles of carnitine and choline-O-sulfate in P. syringae cells, including in culture and during host colonization. Although genes involved in carnitine catabolism were identified in other organisms, the roles of these genes have not been fully elucidated. We analyzed conflicting reports of the carnitine to glycine betaine degradation pathway and provided evidence for a modified pathway based on the growth and accumulation of metabolic intermediates in catabolic mutants. Although carnitine is converted to glycine betaine for accumulation as a compatible solute in B728a, we found that carnitine itself can function as a compatible solute in P. syringae. This was based on both the ability of a carnitine catabolic mutant to derive osmoprotection from carnitine, and the accumulation of carnitine in this mutant as detected using MALDI-MSI. Although we did not detect evidence for carnitine production by bean leaves, we discovered that carnitine is released during bean seed germination, specifically during radicle emergence and elongation, and this carnitine strongly contributed to the growth of B728a on germinating seeds. In exploring the genes involved in the catabolism of choline-O-sulfate (COS), we confirmed that a predicted COS sulfatase was required for catabolizing COS as a nutrient and osmoprotectant, but did not impact P. vulgaris leaf or vii seed colonization, indicating little to no available COS in bean plants. However, we found unexpected evidence that a sulfate exporter is critical to the use of COS as a nutrient or osmoprotectant; this supports a model in which this exporter removes sulfate released from the cleavage of COS and prevents accumulation to toxic levels. Although COS was not a significant source of sulfur during colonization of the plant host, we used transcriptome data to elaborate a likely metabolic network highlighting multiple organosulfur sources that supply leaf surface bacteria with sulfur. Lastly, we systematically evaluated the roles of a broad set of QACs, including choline, phosphorylcholine,