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Causes and Consequences of Diversity Within Experimental Biofilms of Pseudomonas Aeruginosa

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Causes and Consequences of Diversity Within Experimental Biofilms of Pseudomonas Aeruginosa University of New Hampshire University of New Hampshire Scholars' Repository Doctoral Dissertations Student Scholarship Winter 2014 Causes and Consequences of Diversity within Experimental Biofilms of Pseudomonas aeruginosa Kenneth Mark Flynn University of New Hampshire, Durham Follow this and additional works at: https://scholars.unh.edu/dissertation Recommended Citation Flynn, Kenneth Mark, "Causes and Consequences of Diversity within Experimental Biofilms of Pseudomonas aeruginosa" (2014). Doctoral Dissertations. 2165. https://scholars.unh.edu/dissertation/2165 This Dissertation is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. CAUSES AND CONSEQUENCES OF DIVERSITY WITHIN EXPERIMENTAL BIOFILMS OF PSEUDOMONAS AERUGINOSA BY KENNETH MARK FLYNN DISSERTATION Submitted to the University of New Hampshire in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Microbiology December, 2014 i This dissertation has been examined and approved in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology by: Dissertation Director, Cooper, Vaughn S., Associate Professor of Microbiology and Genetics Sullivan, Elise R., Clinical Assistant Professor Bolker, Jessica A., Associate Professor of Biological Sciences Cooper, Timothy F., Associate Professor of Ecology and Evolution, University of Houston O’Toole, George A., Associate Professor of Microbiology and Immunology, Dartmouth College On September 12th 2014 Original approval signatures are on file with the University of New Hampshire Graduate School. ii ACKNOWLEDGEMENTS Over the past six years I have received support and encouragement from many individuals that deserve mention here. First, I would like to thank Dr. Vaughn Cooper for his mentorship and willingness to support me in all my endeavors both in an out of the laboratory. I would also like to thank my dissertation committee of Elise Sullivan, Jessica Bolker, George O’Toole and Tim Cooper for their support and invaluable input on all aspects of my research from PowerPoint presentations to manuscript drafts. In addition, Dr. Joe Harrison provided countless hours of his time to teach me how to perform genetic manipulations with Pseudomonas strains. During data collection and writing, Gabrielle Dowell spent months of her time keeping me on task and listening to me talk about my research. I can say with confidence that this document would not have existed today without her input and encouragement. Wendy Carlson, a laboratory technician when I first started, carried out the original experimental evolution experiment that gave rise to this body of work. Megan McLaughlin, an undergraduate researcher, helped me with numerous day-to-day activities and laboratory experiments. I have also learned so much from the diverse group of graduate students that helped me start Feed the Data Monster. I would also like to thank my parents, Karen Sealund and Peter Flynn, for their love and support. Finally, thank you to Dr. Frank Fekete and Dr. Brooke Jude for giving me the initial push to get started during my time at Colby College. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................ III LIST OF TABLES ......................................................................................... VI LIST OF FIGURES……………………………………………………………….. VII ABSTRACT ................................................................................................... X CHAPTER PAGE I. INTRODUCTION ........................................................................................ 1 II. THE EVOLUTION OF RARE, SUBDOMINANT SPECIALISTS DRIVES COMMUNITY COMPOSITION AND FUNCTION IN EXPERIMENTAL PSEUDOMONAS AERUGINOSA BIOFILMS ........................................................................... 12 INTRODUCTION ..................................................................................... 13 RESULTS ................................................................................................ 15 DISCUSSION .......................................................................................... 29 MATERIALS AND METHODS ................................................................. 34 III. FROM GENOMICS TO ECOLOGY AND BACK: THE EVOLUTION AND MAINTENANCE OF VAST DIVERSITY IN PSEUDOMONAS AERUGINOSA BIOFILMS ......................................... 39 INTRODUCTION ..................................................................................... 40 RESULTS ................................................................................................ 41 DISCUSSION .......................................................................................... 59 MATERIALS AND METHODS ................................................................. 62 IV. EVOLUTION OF HYPERMUTATION IN EXPERIMENTAL PSEUDOMONAS BIOFILMS AS AN ESCAPE FROM CLONAL INTERFERENCE ........................................................................................ 69 INTRODUCTION .................................................................................... 70 iv RESULTS ............................................................................................... 71 DISCUSSION ......................................................................................... 84 MATERIALS AND METHODS ................................................................ 87 V. BIOFILM ADAPTATION THROUGH MODIFYING THE TIMING OF DISPERSAL THROUGH FINE-TUNING OF POLYPHOSPHATE PRODUCTION............................................................ 91 INTRODUCTION ..................................................................................... 92 RESULTS ................................................................................................ 93 DISCUSSION ........................................................................................ 101 MATERIALS AND METHODS ............................................................... 104 LIST OF REFERENCES ........................................................................... 110 APPENDIX A CHAPTER 2 SUPPLEMENTAL MATERIAL ....................... 123 APPENDIX B CHAPTER 3 SUPPLEMENTAL MATERIAL ....................... 134 APPENDIX C CHAPTER 4 SUPPLEMENTAL MATERIAL ....................... 144 v LIST OF TABLES CHAPTER II: Table 1. Characteristics of representative morphotypes from the B1 population isolated following 540 generations of biofilm adaptation .................................................................................... 21 CHAPTER III: Table 1. Low frequency mutations correspond with specific isolates following selection ............................................................. 52 Table 2. Convergence suggests mutational targets resulting in ecological separation ................................................................ 53 CHAPTER IV: Table 1. Expected versus observed mutational counts calculated based on the mutation rate and time in generations .................. 76 Table 2. The expected minimum number of generations needed to accumulate the observed number of mutational calls for individual isolates ........................................................................... 79 Table 3. Comparison of mutational targets identified through convergence ................................................................................................ 82 Table 4. Hierarchical clustering of the variance associated with functional mutations among B1 540 generation isolates ...................... 82 vi LIST OF FIGURES CHAPTER II: Figure 1. Evolution of morphological diversity among three replicate biofilm populations....................................................................................... 17 Figure 2. Replicate biofilm-evolved populations respond differently to diversity. ...................................................................................................... 20 Figure 3. Community fitness requires diversity. ........................................... 23 Figure 4. The D morphotype facilitates the attachment of other types partly defined by variation in c-di-GMP pools. ............................................. 25 Figure 5. Altering c-di-GMP levels in individual B1 morphotypes affects fitness as a whole. ....................................................................................... 28 CHAPTER III: Figure 1. Mutation frequencies in metagenomes and sequenced isolates ....................................................................................................... 42 Figure 2. Biofilm structure preserves immense genetic variation. ............... 46 Figure S1. Methods defining key differences across sequencing approaches. ................................................................................................ 48 Figure 3. Frequencies of mutational calls in different samples of the B1 population and associated cohorts......................................................... 50 Figure 4. Characterization of mutants containing mutations in the anaerobic respiration transcriptional regulator, anr. .................................... 54 Figure 5. Population genetic structure of a biofilm population over long-term and short-term evolution.............................................................
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