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Pseudomonas Bacteriophage Phi6 As a Model for Virus Emergence

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Pseudomonas Bacteriophage Phi6 As a Model for Virus Emergence City University of New York (CUNY) CUNY Academic Works All Dissertations, Theses, and Capstone Projects Dissertations, Theses, and Capstone Projects 9-2015 Pseudomonas Bacteriophage Phi6 as a Model for Virus Emergence Brian Elwood Ford Graduate Center, City University of New York How does access to this work benefit ou?y Let us know! More information about this work at: https://academicworks.cuny.edu/gc_etds/924 Discover additional works at: https://academicworks.cuny.edu This work is made publicly available by the City University of New York (CUNY). Contact: [email protected] PSEUDOMONAS BACTERIOPHAGE Φ6 AS A MODEL FOR VIRUS EMERGENCE by BRIAN E. FORD Academic dissertation submitted to the Graduate Faculty in Biology in partial fulfillment of the requirements for the degree of Doctor of Philosophy, The City University of New York 2015 © 2015 BRIAN E. FORD All Rights Reserved. ii This manuscript has been read and accepted for the Graduate Faculty in Biology in satisfaction of the dissertation requirement for the degree of Doctor of Philosophy. Date Chair of Examining Committee Dr. John J. Dennehy Date Executive Officer Dr. Laurel Eckhardt Dr. John J. Dennehy, Queens College Dr. Stéphane Boissinot, NYU Abu Dhabi Dr. Nathalia Glickman Holtzman, Queens College Dr. Paul Gottlieb, City College of New York Dr. José A. Fernández-­‐Romero, Population Council Supervisory Committee THE CITY UNIVERSITY OF NEW YORK iii Abstract Bacteriophage Φ6 a s a Model f or Virus Emergence by Brian E. Ford Mentor: John J. Dennehy The Pseudomonas bacteriophage Φ6 has a long and well-­‐established history as a model organism. Here we describe a set of experiments to extend this model system in to concepts previously unclaimed. Chapter 1 presents a brief background of the ecology of viruses that infect microorganisms. Chapter 2 examines genetic mutations allowing for host range expansion. Chapter 3 presents a novel paired strain assay to study how a non genetic host acquired factor affects fitness of these enveloped viruses on subsequent hosts. Chapter 4 is an extension of this system to include how the bacteria host is affected in virus-­‐host coevolution. iv ABSTRACT ................................ ............................................................................................. IV LIST OF TABLES ................................ ...................................................................................... XI LIST OF FIGURES ................................ ................................................................................... XII INTRODUCTION ................................ ................................................................................... XIV CHAPTER 1: EVOLUTIONARY ECOLOGY OF THE VIRUSES OF MICROORGANISMS ..................... 1 ABSTRACT ................................ ...................................................................................................... 2 INTRODUCTION ................................ ............................................................................................... 3 VOM ABUNDANCE, BIODIVERSITY, AND B IOGEOGRAPHY ................................ ......................................... 3 POPULATION D YNAMICS OF V OMS ................................ ..................................................................... 6 Factors affecting rates of VoM population growth. ................................ ............................. 7 Predator-­‐prey dynamics. ................................ ..................................................................... 11 VOM IMPACT ON HOST ATIONS POPUL AND COMMUNITIES. ................................ .................................... 12 VoM-­‐host coevolution and its limitations. ................................ .......................................... 12 VoMs as drivers of host biodiversity. ................................ .................................................. 15 VOM ECOLOGICAL A DAPTATION ................................ ...................................................................... 16 Generation time as an optimizable trait. ................................ ............................................ 16 Coinfection, competition and cheating. ................................ .............................................. 18 Productivity versus latency. ................................ ................................................................. 19 VOM GENOME E VOLUTION ................................ ............................................................................. 21 VoM genomes. ................................ ..................................................................................... 22 Horizontal gene transfer and genome mosaicism. ................................ ............................. 23 v VOM IMPACT ON H OST G ENETICS ................................ .................................................................... 24 VOMS AS E COSYSTEM M ODIFIERS ................................ .................................................................... 26 CLOSING R EMARKS ................................ ........................................................................................ 28 ACKNOWLEDGEMENTS ................................ .................................................................................... 28 FIGURE L EGENDS ................................ ........................................................................................... 29 REFERENCES ................................ ................................................................................................. 34 CHAPTER 2: FREQUENCY AND FITNESS CONSEQUENCES OF BACTERIOPHAGE Φ6 HOST RANGE MUTATIONS ................................ .......................................................................................... 49 ABSTRACT ................................ .................................................................................................... 50 INTRODUCTION ................................ ............................................................................................. 51 METHODS AND M ATERIALS ................................ ............................................................................. 53 Study Organisms and Culture Conditions ................................ ............................................. 53 Virus Stock Preparation ................................ ........................................................................ 54 Host Range Mutant Frequency ................................ ............................................................. 54 Host Range Mutant Isolation ............................................................................................... 55 RNA Extraction and Sequencing ................................ ........................................................... 55 Mutant Characterization ................................ ...................................................................... 56 Plaque Size Estimates ................................ ........................................................................... 56 Mutant Fitness on Native and Novel Hosts ................................ .......................................... 57 Attachment Rate Assays ................................ ....................................................................... 5 7 RESULTS ................................ ...................................................................................................... 58 Mutation Frequency ................................ ............................................................................. 58 vi Mutation Substitution Frequency ................................ ......................................................... 59 Phenotypic Change Analysis ................................ ................................................................. 60 P3 3D Structure Prediction ................................................................................................... 61 Plaque Size ................................ ............................................................................................ 62 Mutant Fitness on Original and Novel Hosts ................................ ........................................ 63 Attachment Rate ................................ .................................................................................. 64 Data Availability ................................ ................................................................................... 65 DISCUSSION ................................................................................................................................. 65 Φ6 Host Range Mutation Frequency ................................ .................................................... 65 Mutation Hotspots ................................ ............................................................................... 69 Structural Speculations ................................ ......................................................................... 70 Fitness on Native and Novel Hosts ................................ ....................................................... 71 Attachment to Native and Novel Hosts ................................ ................................................ 72 Implications for Disease Emergence ................................ ....................................................
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