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GREENWALD-DISSERTATION.Pdf AN IN-DEPTH ANALYSIS OF IRON AND PATHOGENICITY REGULATORY PATHWAYS IN Pseudomonas syringae pv. syringae B728A A Dissertation by JESSICA WILLIAMS GREENWALD Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY August 2011 Major Subject: Plant Pathology An In-Depth Analysis of Iron and Pathogenicity Regulatory Pathways in Pseudomonas syringae pv. syringae B728a Copyright 2011 Jessica Williams Greenwald AN IN-DEPTH ANALYSIS OF IRON AND PATHOGENICITY REGULATORY NETWORKS IN Pseudomonas syringae pv. syringae B728A A Dissertation by JESSICA WILLIAMS GREENWALD Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved by: Chair of Committee, Dennis C. Gross Committee Members, Carlos F. Gonzalez Paul de Figueiredo Helene Andrews-Polymenis Head of Department, Leland S. Pierson III August 2011 Major Subject: Plant Pathology iii ABSTRACT An In-Depth Analysis of Iron and Pathogenicity Regulatory Pathways in Pseudomonas syringae pv. syringae B728a. (August 2011) Jessica Williams Greenwald, B.S., The College of William & Mary Chair of Advisory Committee: Dr. Dennis C. Gross Pseudomonas syringae pv. syringae strain B728a (P.s.s. B728a) is an economically significant plant pathogen that is capable of successful epiphytic colonization of leaf surfaces. Although the virulence factors associated with this pathogen‟s ability to cause disease have been well studied, the transition from epiphyte to pathogen is not well understood. The research described in this dissertation utilizes high throughput sequencing transcriptome analyses to define an iron regulatory network that is predicted to be utilized during the epiphytic portion of the P.s.s. B728a lifecycle. This dissertation also describes a collaborative microarray analysis that analyzes the P.s.s. B728a transcriptome at a global level. An iron associated sigma factor, AcsS, encoded within a peptide synthesis rich region of the P.s.s. B728a genome is shown to regulate the citrate siderophore achromobactin. RNA-seq transcriptome analysis reveals that this sigma factor regulates expression of genes predicted to be involved in functions that are important during the epiphytic stage of P.s.s. B728a, including genes involved in iron response, secretion, extracellular polysaccharide production, and cell motility. iv As part of a collaboration, the transcriptomes of the P.s.s. B728a genome and nine deletion mutants in regulatory genes were analyzed by microarray analayses using seven treatment conditions, including epiphytic and in planta conditions. As part of these microarray analyses, results are described for the global regulator, GacS, and a downstream transcription factor, SalA. This study confirms the role of GacS and SalA in the regulation of major virulence components of P.s.s. B728a such as phytotoxin production and Type III secretion. This study also elucidates a role for GacS and SalA regulation of genes important for epiphytic survival and function, including the Type VI secretion system, iron acquisition, and EPS production. v I dedicate this work to my mother and father, who instilled in me the desire to achieve greatness and an appreciation for the greatness of others. And to my loving husband, who complements my mutations. vi ACKNOWLEDGEMENTS I would like to express my sincere gratitiude to my major advisor, Dr. Dennis Gross. As my advisor you have granted me the freedom to express myself and grow, as an individual and a scientist. Thank you for your guidance, support, and patience. I would like to thank my committee members Dr. Carlos Gonzalez, Dr. Paul de Figueiredo, and Dr. Helene Andrews-Polymenis for their constructive criticisms and support of my scientific research. I would like to thank my collaborators: Dr. Tadhg Begley (Texas A&M University), Dr. Benjamin Philmus (Texas A&M University), Dr. Gwyn Beattie (Iowa State University), Dr. Steven Lindow (University of California Berkley), Dr. Daniel Nettleton (Iowa State University), Steven P. Lund (Iowa State University; Russell A. Scott (University of California, Berkley), and Xilan Yu (Iowa State University). I offer a special thanks to Dr. Benjamin Philmus for his perseverance and words of encouragement. I offer my sincerest gratitude to the present and former members of my research laboratory, who have offered scientific and moral support. I offer my thanks to Dr. Brenda Schroeder, Dr. Shien Lu, Dr. Nian Wang, Dr. Angela Records, Amber Lorge, Poulami Basu Thakur, Dr. Aravind Ravindran, and Vanessa Vaughn. Special thanks are given to my undergraduate research assistant and friend, Ryan Cieker. I would like to acknowledge those members of the Texas A&M community who have offered special assistance with or suggestions about my research. These include: vii Dr. Larry Dangott, Dr. Herman Scholthof, Dr. Veria Alvarado, Dr. Joshua Yuan, Dr. Weibing Shi, Dr. Kevin Ong, Dr. Mehdi Kabbage, Dr. Jim Starr, Dr. Daniel Ebbole, Dr. Heather Wilkinson, and Dr. Won Bo Shim. I would like to thank Patsy Bolch, Dora Lee-Haberstroh, Karen Hodges, Chris Court, and Tracy Hurych for their assistance, compassion, and friendship. While I appreciate all of the technical skills that these individuals provided to my research, I am most grateful for their kind words, encouragement, and friendship. I would like to thank the two Karens in my life for being my mentors, my soul sisters, and my friends. I offer my love and gratitude to my husband, Charles, for his scientific knowledge, expertise, love, support, and especially for his sense of humor. I would like to thank Mom, Dad, Granny, and Grandma for their endless support and love. Thank you to my friend Sara, for listening to my endless rants and pointing out the silver lining in every rain cloud. For keeping me grounded, I thank my friend Angela. For stirring the pot and encouraging my fiery nature, I thank Dustin. For providing sound scientific advice and delicious Kosher-friendly meals, I would like to thank Samir. I offer thanks to my dear friend Gabby, whose wicked humor makes me smile. Finally I offer thanks to four little people that I love unconditionally, Sam, Jack, Olivia, and Carter. Thank you for encouraging me to enjoy life and to smile more. viii TABLE OF CONTENTS Page ABSTRACT .............................................................................................................. iii DEDICATION .......................................................................................................... v ACKNOWLEDGEMENTS ...................................................................................... vi TABLE OF CONTENTS .......................................................................................... viii LIST OF FIGURES ................................................................................................... x LIST OF TABLES .................................................................................................... xi CHAPTER I INTRODUCTION ................................................................................ 1 II AN IRON RESPONSIVE SIGMA FACTOR, AcsS, REGULATES ACHROMOBACTIN BIOSYNTHESIS IN Pseudomonas syringae pv. syringae B728a ......................................... 6 Overview ........................................................................................ 6 Introduction and Literature Review ............................................... 7 Materials and Methods ................................................................... 24 Results and Discussion ................................................................... 39 III TRANSCRIPTOME ANALYSIS OF GLOBAL REGULATORS GacS AND SalA .................................................................................. 70 Overview ........................................................................................ 70 Introduction and Literature Review ............................................... 71 Materials and Methods ..................................................................... 83 Results and Discussion .................................................................... 96 IV CONCLUSIONS .................................................................................. 130 ix Page REFERENCES .......................................................................................................... 133 APPENDIX A ........................................................................................................... 146 VITA ......................................................................................................................... 163 x LIST OF FIGURES FIGURE Page 1 Genomic region of Dickeya dadantii strain 3937 containing the NIS synthetase genes responsible for the biosynthesis of the siderophore achromobactin ........................................................................ 20 2 Proposed scheme for the biosynthesis of achromobactin ........................... 22 3 A peptide synthesis region of the P.s.s. B728a genome ............................ 40 4 Sequence homology of the achromobactin gene clusters in P.s.s. B728a, P.s.p.1448a, Dickeya dadantii strain 3937, and Dickeya zeae strain Ech1591 ...................................................................... 42 5 The P.s.s. B728a RND efflux system PseABC .......................................... 57 6 The P.s.s. B728a gene cluster homologous to the mangotoxin biosynthesis cluster found
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