Brigham Young University BYU ScholarsArchive Theses and Dissertations 2013-03-20 Modulators of Symbiotic Outcome in Sinorhizobium meliloti Matthew B. Crook Brigham Young University - Provo Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Microbiology Commons BYU ScholarsArchive Citation Crook, Matthew B., "Modulators of Symbiotic Outcome in Sinorhizobium meliloti" (2013). Theses and Dissertations. 3946. https://scholarsarchive.byu.edu/etd/3946 This Dissertation is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Modulators of Symbiotic Outcome in Sinorhizobium meliloti Matthew Ben Crook, Jr. A dissertation submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Joel S. Griffitts, Chair Brent Nielsen William R. McCleary Jeff Maughan David L. Erickson Department of Microbiology and Molecular Biology Brigham Young University March 2013 Copyright © 2013 Matthew Ben Crook, Jr. All Rights Reserved ABSTRACT Modulators of Symbiotic Outcome in Sinorhizobium meliloti Matthew Ben Crook, Jr. Department of Microbiology and Molecular Biology, BYU Doctor of Philosophy Microorganisms interact frequently with each other and with higher organisms. This contact and communication takes place at the molecular level. Microbial interactions with eukaryotes can be pathogenic or mutualistic. One of the best-studied symbioses is the complex interaction between nitrogen-fixing soil bacteria, termed rhizobia, and legumes. This symbiosis culminates in the elaboration of a new organ, the root nodule. Many of the molecular signals exchanged between the host plant and the invading rhizobia have been deduced, but there is still much that remains to be discovered. The molecular determinant of host range at the genus level of the plant host has been determined to be lipochitooligomers called Nod factors. The molecular determinants of host range at the species and cultivar level are less well-defined. Part of my work has been to identify and characterize accessory plasmids that disrupt the normal progression of symbiosis between legumes of the genus Medicago and their rhizobial symbiont, Sinorhizobium meliloti. A cre–loxP-based system capable of making large, defined deletions was developed for the analysis of these plasmids. This system is also being employed to cure the laboratory strain, S. meliloti Rm1021 of its two megaplasmids—a loss of nearly half of its genome. I have also done work to determine whether locally-collected sinorhizobia are native, invasive, or native with symbiosis genes acquired horizontally from invasive sinorhizobia. Finally, I have studied Sinorhizobium meliloti as a host by identifying an outer membrane porin that several bacteriophages use to adsorb to the S. meliloti cell surface. Key words: Sinorhizobium meliloti, Medicago, symbiosis, host range, plasmid, pHRC017, bacteriophage receptor, RopA1 ACKNOWLEDGMENTS First and foremost I would like to thank my wife, Leann, who over the last six years has tolerated my working late, my working weekends, my working holidays, and lately, the long hours spent preparing this dissertation. Her patience and loving support has been integral to my success. I would also like to thank my parents who bought me my first microscope and helped me with my first insect collection when I was in first grade. They always encouraged my curiosity and provided me with access to useful sources of knowledge. When at home I could often be found reading from a set of World Book Encyclopedias they kept on their shelves. My mentor, Dr. Griffitts, has taken a personal interest in my development as a scientist and has helped me to take great strides in thinking analytically and scientifically. His tutelage and expertise have made a deep impact on my approach to scientific inquiry. My association with the other members of my graduate committee has also been exceptional. They always gave good advice during my committee meetings and provided me with new insights into my work. I have also received help, support, and camaraderie from other members of the MMBIO faculty. The office staff of the department, both past and present, has been exactly what they should be: helpful and knowledgeable. I always came away from my interactions with them feeling satisfied and encouraged. I would also like to thank Dr. Kim O’Neill and Dr. Paul Savage, who gave me research opportunities before I began my graduate program. I would like to thank the many BYU undergraduates who have contributed to this work in some way: Joshua Bentley, Michael Bevins, Matthew Biggs, Divyesh Choudhri, Casey Crum, Alicia Draper, Krysta Felix, Jordan Guillory, Clarice Harrison, Daniel Lindsay, Gledi Peco, Shawna Rogers, and Elise Scoggin. I have also received invaluable advice and support from the other graduate students in the lab, Ryan VanYperen and Phil Bennellack, and from Skip Price, a post-doctoral fellow in the lab who will be continuing the study of host range plasmids after I leave and who generated most of the data presented in §2.1.6. I would also like to thank members of the BYU Computer Science Department who helped with the assembly and annotation of pHRC017 and its subsequent submission to GenBank: Jared C. Price, Spencer C. Clement, and Dr. Mark J. Clement. Camille Porter, a graduate student in the Biology Department, helped train me in phylogenetics and gave me access to the software I needed. Finally, many other scientists have provided materials and expertise without which this project would not have been possible: P. Van Berkum of the USDA–ARS, M. Hynes of the University of Calgary, H. Krishnan of the University of Missouri, S. Long of Stanford University, I. Oresnik of the University of Manitoba, M. Standing in the microscopy lab here in the college, and Graham Walker of the Massachusetts Institute of Technology. TABLE OF CONTENTS LIST OF TABLES ...................................................................................................................... vii LIST OF FIGURES ................................................................................................................... viii ABBREVIATIONS ...................................................................................................................... X 1 INTRODUCTION................................................................................................................... 1 2 OPUS ...................................................................................................................................... 14 2.1 RHIZOBIAL PLASMIDS THAT CAUSE IMPAIRED SYMBIOTIC NITROGEN FIXATION AND ENHANCED HOST INVASION ...................................................................................................................... 14 2.1.1 Summary ............................................................................................................... 14 2.1.2 Introduction........................................................................................................... 15 2.1.3 Materials and Methods ......................................................................................... 17 2.1.4 Results ................................................................................................................... 23 2.1.5 Discussion ............................................................................................................. 32 2.1.6 Unpublished Data ................................................................................................. 35 2.1.7 Future Directions .................................................................................................. 41 2.2 DISSECTION OF RHIZOBIAL PLASMIDS USING CRE–LOXP .............................................................. 43 2.2.1 Summary ............................................................................................................... 43 2.2.2 Introduction........................................................................................................... 44 2.2.3 Materials and Methods ......................................................................................... 45 2.2.4 Results ................................................................................................................... 49 2.2.5 Discussion ............................................................................................................. 58 2.2.6 Future Directions .................................................................................................. 61 2.3 CHARACTERIZATION OF SINORHIZOBIA AND OTHER RHIZOBIA COLLECTED PRIMARILY IN THE LOWER INTERMOUNTAIN WEST ....................................................................................................... 63 2.3.1 Summary ............................................................................................................... 63 2.3.2 Introduction........................................................................................................... 64 2.3.3 Materials and Methods ......................................................................................... 65 2.3.4 Results ................................................................................................................... 69 2.3.5 Discussion and Future Directions