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Structure and Function in Bacteriophage Phi6 City University of New York (CUNY) CUNY Academic Works All Dissertations, Theses, and Capstone Projects Dissertations, Theses, and Capstone Projects 6-2014 Structure and Function in Bacteriophage Phi6 James Carpino 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/183 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] STRUCTURE AND FUNCTION IN BACTERIOPHAGE PHI6 JAMES CARPINO 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 2014 ©2014 James Carpino 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 Dennehy Date Executive Officer Dr. Laurel Eckhardt Dr. John Dennehy, Queens College Dr. Cathy Savage-Dunn, Queens College Dr. Stéphane Boissinot, Queens College Dr. Paul Gottlieb, City College of New York Dr. Dan Dykhuizen, Stony Brook University, SUNY Supervisory Committee THE CITY UNIVERSITY OF NEW YORK iii ABSTRACT STRUCTURE AND FUNCTION IN BACTERIOPHAGE Φ6 by JAMES CARPINO Advisor: Professor John Dennehy The present study of bacteriophage φ6 has been preceded by a great number of exploratory studies of its structure and function, and these studies have formed a basis for φ6’s development into a model organism. In this study, two aspects of the model organism have been examined. 1. There are several uncharacterized and presumed untranslated regions (UTRs) in φ6's 13.3 kilobase-pair dsRNA genome. I examined the impact of specific modification to the 3' UTR of the small segment of bacteriophage φ6. I determined that modification to the purported UTR of the small segment resulted in severe fitness costs, supporting a functional role for unidentified gene products, secondary RNA structure, or both. 2. Bacteriophage φ6 packages its dsRNA genomic segments selectively and sequentially through the function of the packaging motor P4 which occupies fivefold vertices of the φ6 procapsid, and studies support the functioning of one and only one P4 during packaging. The mechanism of this specific phenomenon is not known. I used computational reconstruction of cryoelectron microscopy and examined the occupancy of P4 on the φ6 procapsid, and acquired insight into the mechanism of assembly and packaging. iv Acknowledgement and Dedication I owe my sincerest thanks to a long list of people without whose help and support, my success would have been highly improbable. The list is too long to present here; it includes many advisors, professors, friends, classmates, students, teammates, and one true love, all of whom know who they are. This work is dedicated to the loving memory of my father, Dr. Joseph James Carpino, October 15, 1930 – August 4, 1998. May he rest in peace, and watch over us all with his beloved son, John. My father was the first to teach me to embrace knowledge, truth, and justice, and to seek them through patience, perseverance, and personal sacrifice. This lesson was one of many which he imparted passively, by example, by allowance, and never by force or imposition. He was a teacher, a philosopher, a soldier, a carpenter, and a Christian, and a father more deeply in his heart than anything else. He advised me never to be stingy with ideas, lest the river from which they flow run dry. v TABLE OF CONTENTS ABSTRACT........................................................................................................................... IV ACKNOWLEDGEMENT AND DEDICATION.................................................................V CHAPTER 1: INTRODUCTION..........................................................................................1 1.1 BACTERIOPHAGE Φ6 AS A MODEL ORGANISM ........................................................ 1 1.2 BACTERIOPHAGE Φ6 DESCRIPTION ......................................................................... 4 1.3 BACTERIOPHAGE Φ6 LIFE HISTORY ........................................................................ 5 1.4 GENETIC EXCHANGE , CYSTOVIRIDAE AND REASSORTMENT OF SEGMENTED GENOMES .............................................................................................................................. 7 CHAPTER 2: EVIDENCE FOR A FUNCTIONAL ROLE IN THE 3’ UTR OF THE SMALL SEGMENT OF BACTERIOPHAGE Φ6....................................................11 ABSTRACT : ......................................................................................................................... 11 2.1 INTRODUCTION ....................................................................................................... 12 2.1.1 φ6 Tagging Project .............................................................................................12 2.1.2 The 3’ UTR of the Small Segment of Bacteriophage φ6 ................................14 2.1.3 The Case for Functional Secondary Structure in the 3’ UTR .......................15 2.1.4 The Case for Genes ............................................................................................15 2.1.5 The Curious Precedent of the Bullseye Plaque ...............................................20 2.2 EXPERIMENTAL METHODOLOGY .......................................................................... 25 2.3 MATERIALS AND METHODS ................................................................................... 28 2.3.1 Bacterial and Viral Strains and Plasmids ........................................................28 2.3.2 Culture Conditions .............................................................................................29 2.3.3 Preparation of Plasmids ....................................................................................29 vi 2.3.4 Genetic Modification of RNA Segments of φ6 ................................................29 2.3.4.1 Mutagenesis Methods ........................................................................................29 2.3.4.2 Mutant Classes and Segment Variants ............................................................31 2.3.5 Recombinant Bacteriophage Rescue ................................................................33 2.3.5.1 Competent Cell Preparation .............................................................................33 2.3.5.2 Electroporation and Plating ..............................................................................34 2.3.6 Plaque Isolation and Purification .....................................................................35 2.3.7 RNA Extraction ..................................................................................................36 2.3.8 cDNA Synthesis ..................................................................................................37 2.3.9 PCR Detection of Inserts ...................................................................................37 2.3.10 Sequencing ..........................................................................................................37 2.3.11 Fitness Assay .......................................................................................................37 2.3.12 OD Depletion Assay ...........................................................................................38 2.3.13 Growth Curve of JC1917 ..................................................................................39 2.3.14 RNA Secondary Structure Prediction ..............................................................39 2.3.15 Protein 3D Structure Prediction .......................................................................39 2.4 RESULTS ................................................................................................................. 40 2.4.1 Fitness Impact of Modification to the 3’ Region of φ6 Small Segment .........40 2.4.2 Fitness of Small Tag Mutant .............................................................................40 2.4.3 Identification of ORFs .......................................................................................40 2.4.4 Prediction of Secondary Structure; Impact of Tagging on Secondary Structure .........................................................................................................................40 2.4.5 Construction of New UTR Mutants .................................................................43 vii 2.4.6 DNA Synthesis and Sequence Verification ......................................................43 2.4.7 Fitness of Newly constructed Strains MUT1 through MUT6 ........................43 2.4.8 Relationship between Fitness and Optical Density .........................................45 2.4.9 Growth Curve of JC1917 ..................................................................................47 2.5 DISCUSSION ............................................................................................................. 49 2.5.1 Comparisons .......................................................................................................50 2.5.2 MUT2 vs. MUT5 Comparison Reflects Altered ssRNA Stability .................50 2.5.3 MUT1 vs. MUT3: ORF J Impacts Plaque Morphology but not Fitness ......51 2.5.4 MUT2 and MUT3 demonstrate Different Impacts on Fitness, Supporting Function of One or Two
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