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Gyrb and Rpob Genes DIVERSITY AMONG BACTERIAL SYMBIONTS OF ENTOMOPATHOGENIC NEMATODES By HEATHER SMITH KOPPENHÖFER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2017 © 2017 Heather Koppenhöfer In memory of my mother, Linda Huffman, who always believed in me, and to my husband, Albrecht, and our daughter, Katharina. Your encouragement and faith in me has helped make this possible. ACKNOWLEDGMENTS I am deeply grateful for the generosity shown to me by many people. I express my sincere gratitude and appreciation to Dr. Frank Louws (North Carolina State University) who shared his expertise in bacterial diversity and allowed me to conduct all of the rep-PCR portion of my dissertation in his laboratory; to Dr. Susan Webb, who allowed me to use equipment in her laboratory; to Dr. Oscar Liburd, who helped provide reagents; to Drs. John Capinera and William B. Crow, who provided a source of funding for sequencing reactions; and to Dr. Pauline Lawrence, who provided reagents when I had none and took the time to be a mentor to me even though I was not her student. I thank Dr. Randy Gaugler (Rutgers University) for his advice, for providing funding for meetings and for providing me with a quiet office where I could write. I also thank Dr. Michael Klein (The Ohio State University) who provided funding to attend an important meeting in my field and encouragement to not give up, and Dr. Jessica Ware (Rutgers University), who always had an answer to my phylogenetic questions. I would like to thank Dr. Erica Goss for her help with bacterial recombination, and also I would to thank Dr. Matt Gizendanner and Dr. Mark Miller (San Diego Supercomputer Center) for their assistance with HiPerGator and Cipres, respectively. I am thankful for technical assistance from Dr. Khuong Nguyen, Ms. Ellen Dickstein and Mr. Jerry Minsavage. I am grateful to Drs. James and Alejandra Maruniak who opened their lab to me and helped me with various molecular techniques. I am also grateful to Dr. Jim Maruniak for never giving up on me. It is because of his encouragement that I continued this work. I am grateful to Dr. Jeff Jones who took me on as his student though I was from another department, provided laboratory space, directed me to people with different areas of expertise who could help with this project and guided my work. I would like express my deep appreciation for Mrs. Debbie Hall, who not only stayed on top of my program more than I but became a great friend in the process. I am especially grateful 4 for Dr. Heather McAuslane and Ms. Ruth Brumbaugh and all the work they have done so that I could be readmitted into the program. I would like to thank my committee members Dr. James Preston and Dr. David Reed and past committee members Dr. Steve Forst and Dr. Byron Adams for their time, support and encouragement. The research presented her was started under the direction of Dr. Adams. I am most thankful to my chair and co-chair Dr. Jeffrey B. Jones and Dr. James E. Maruniak. Their guidance has been invaluable. I am very grateful for all my wonderful friends and family. I could not have done this without them. Finally, I am especially thankful for Dr. Albrecht Koppenhöfer (Rutgers University), who provided financial support for much of this research, shared his expertise in entomopathogenic nematodes, discussed ideas with me and helped edit my writing. My husband’s strength, generosity and humility continue to amaze me. This dissertation is an acknowledgement of God’s wondrous creation and the privilege of studying it. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF FIGURES .......................................................................................................................10 ABSTRACT ...................................................................................................................................12 CHAPTER 1 GENERAL INTRODUCTION ..............................................................................................14 Xenorhabdus and Photorhabdus Life Cycle...........................................................................15 Phenotypic Variation ..............................................................................................................17 Taxonomy and Systematics of Xenorhabdus and Photorhabdus ...........................................19 Xenorhabdus and Photorhabdus Taxonomy ...................................................................22 The Genus Xenorhabdus Thomas & Poinar, 1979 ..........................................................24 Characters of Xenorhabdus .............................................................................................25 The Genus Photorhabdus Boemare, Akhurst & Mourant, 1993 .....................................26 Characters of Photorhabdus ............................................................................................27 Nematode/Bacteria Associations ............................................................................................29 Phylogenetic Systematics .......................................................................................................35 Coevolution .............................................................................................................................37 Aims and Objectives ...............................................................................................................38 2 DIVERSITY OF Photorhabdus SPP. ISOLATED IN FLORIDA .........................................46 Introduction .............................................................................................................................46 Materials and Methods ...........................................................................................................50 Collection of Nematode Specimens and Isolation of Bacterial Strains ...........................50 Preparation of Genomic DNA .........................................................................................52 Repetitive Sequence-based Genotyping ..........................................................................52 Amplification, cloning and sequencing of partial gyrB and rpoB genes .........................53 Phylogenetic Analyses .....................................................................................................56 Recombination Analyses .................................................................................................59 Results.....................................................................................................................................60 Isolation of nematodes .....................................................................................................60 Diversity as Assessed by rep-PCR ..................................................................................60 gyrB and rpoB based Phylogenetic Analyses ..................................................................62 Recombination .................................................................................................................64 Discussion ...............................................................................................................................68 3 GENETIC DIVERSITY AMONG Xenorhabdus SPP. AND THEIR PHYLOGENETIC INFERENCE ........................................................................................................................101 Introduction ...........................................................................................................................101 6 Materials and Methods .........................................................................................................103 Collection of Nematode Specimens, Isolation of Bacterial Strains and Growth Conditions ..................................................................................................................103 Preparation of Genomic DNA .......................................................................................104 Repetitive Sequence-based Genotyping ........................................................................105 Gene Selection and Amplification .................................................................................106 The gyrB Sequence ........................................................................................................107 The recA Sequence ........................................................................................................107 The groEL Sequence .....................................................................................................107 The rpoB Sequence ........................................................................................................108 The 16S rDNA Sequence ..............................................................................................108 Cloning and Sequencing ................................................................................................109 Alignments ....................................................................................................................111 Phylogenetic Analyses ...................................................................................................111 Recombination Analyses ...............................................................................................113
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