The Roles of Moron Genes in the Escherichia Coli Enterobacteria Phage Phi-80
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THE ROLES OF MORON GENES IN THE ESCHERICHIA COLI ENTEROBACTERIA PHAGE PHI-80 Yury V. Ivanov A Dissertation Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY December 2012 Committee: Ray A. Larsen, Advisor Craig L. Zirbel Graduate Faculty Representative Vipa Phuntumart Scott O. Rogers George S. Bullerjahn © 2012 Yury Ivanov All Rights Reserved iii ABSTRACT Ray Larsen, Advisor The TonB system couples cytoplasmic membrane-derived proton motive force energy to drive ferric siderophore transport across the outer membrane of Gram-negative bacteria. While much effort has focused on this process, how energy is harnessed to provide for transport of ligands remains unknown. Several bacterial viruses (“phage”) are known to require the TonB system to irreversibly adsorb (i.e., establish infection) in the model organism Escherichia coli. One such phage is φ80, a “cousin” of the model temperate phage λ. Determining how φ80 is using the TonB system for infection should provide novel insights to the mechanisms of TonB-dependent processes. It had long been known that recombination between λ and φ80 results in a λ-like phage for whom TonB is now required; and this recombination involved the λ J gene, which encodes the tail-spike protein required for irreversible adsorption of λ to E. coli. Thus, we suspected that a φ80 homologue of the λ J gene product was responsible for the TonB dependence of φ80. While φ80 has long served as a tool for assaying TonB activity, it has not received the scrutiny afforded λ. Indeed, only a small portion of its genome (not including a J gene homolog) was known. To facilitate the use of φ80 as a molecular tool, we determined and annotated the full genome of the φ80vir strain commonly used for TonB studies. The 46,285 bp φ80vir genome contained 71 predicted open reading frames, in which the structural genes showed strong synteny with other known lambdoid viruses, with an overall degree of mosaicism indicative of multiple genetic exchanges with other viruses. iv There was also evidence of unique gene acquisition in the form of morons, one of which, termed cor, when cloned and expressed in E. coli, blocked TonB-dependent transport of ferric siderophore and irreversible adsorption of φ80vir to the TonB-dependent outer membrane protein FhuA. These and other findings reported in this dissertation provide a foundation for the use of φ80vir as a tool to dissect the mechanisms of TonB-dependent energy transduction. v Dedicated to my grandfather, Yury Ivanov, who wanted me to become a student, right after I was born. Unfortunately, I cannot tell you that I decided to go even beyond vi ACKNOWLEDGMENTS My career as a scientist would have been not possible without many individuals who played very important roles. But before giving them my regards, I would like to acknowledge Bowling Green State University for opening me new horizons. Next, I would like to acknowledge members of my committee: Drs. Ray Larsen, Scott Rogers, George Bullerjahn, Vipa Phuntumart, and Craig Zirbel. Dr. Larsen: I admire all your input, patience, and guidance that you gave me; I was honored to be your Ph.D. student and I will not let you down; even now, I am still learning from you. Dr. Rogers: you are the reason why I enjoy doing bioinformatics, genomics, phylogeny, and evolution; I will never forget our discussions during the fermentation seminar. Dr. Bullerjahn: you was in my admission committee and I thank you for giving me this opportunity to become a Doctor of Philosophy. Dr. Phuntumart: you combine both a great scientist and a charming person; thank you for all your advice. Dr. Zirbel: I wish I could know you better; I am inspired by the research that you, as a mathematician, are doing in collaboration with Dr. Leontis. Last but not least, I would like to thank my loving family for all their support and patience. To my parents, Victor Ivanov and Alla Ivanova: I owe you everything in my life; I am fortunate to be your son. Maria Ivanova: I cannot wait to see you again, grandmother; thank you for everything you have done for me. Special thanks to my brother, Evgeniy Ivanov: we are so different and yet are very good friends. My aunt, Valentina Ivanova: I always feel your support I would like to acknowledge my support team of friends and colleagues: Andrew Fedorov, Maria Kozelkova, Alex Orlov, Maribeth Spangler, Vadim Solovyov, Alex Goryaynov, Adam Boulton, Tim South, Pavel Borisov, Emil Khisamutdinov, Kate Butler, Matthew Moreau, and many others. Thanks vii to Yury Shtarkman and Alex Bludin who contributed directly to my work and are my very best friends. Finally, special thanks to BGSU faculty: Drs Tami Steveson, Paul Morris, Carmen Fioravanti –you all helped me in various ways and I do remember it. “Never say never” – I refused to believe that I will ever become a Ph.D when I was in high school; you all proved me wrong. viii TABLE OF CONTENTS Page CHAPTER I. A REVIEW OF THE LITERATURE ............................................................ 1 Tailed Bacteriophages ................................................................................................ 1 Membrane Energetics and Iron Transport Systems in Bacteria................................. 3 Iron and Iron Transport in Gram-Negative Bacteria .................................................. 5 Proton Motive Force and TonB-dependent Transporters .......................................... 7 CHAPTER II. ENTEROBACTERIOPHAGE PHI-80 ......................................................... 13 Introduction … ........................................................................................................... 13 Materials and Methods ............................................................................................... 16 Phage Phi-80 Growth ..................................................................................... 16 DNA Extraction ............................................................................................. 17 Restriction Digestion Map ............................................................................. 18 Genome Cloning and DNA Library Construction ......................................... 19 Annotation of Genes ...................................................................................... 21 Comparative Genomics .................................................................................. 21 Electron Microscopy ...................................................................................... 21 Atomic Force Microscopy ............................................................................. 22 Results and Discussion ……… ................................................................................. 22 Overall Features of φ80vir Chromosome ....................................................... 22 The Left Arm ................................................................................................. 23 Head Genes ........................................................................................ 26 Tail Genes .......................................................................................... 26 ix Conserved -1 Translational Frameshift .............................................. 27 The Central Region ........................................................................................ 28 Phage Attachment Site ....................................................................... 28 The Right Arm ............................................................................................... 29 Immunity Region ............................................................................... 29 Phage Morphology ............................................................................. 31 CHAPTER III. GENES WITH MORON FEATURES ........................................................ 40 Introduction … ........................................................................................................... 40 Materials and Methods ............................................................................................... 41 Identification of Transcription Signals .......................................................... 41 G+C Skew ...................................................................................................... 42 Results and Discussion ……… ................................................................................. 42 Genes with Moron Features, or Morons ........................................................ 42 CHAPTER IV. CONVERSION RESISTANT LIPOPROTEIN COR ................................. 51 Introduction … ........................................................................................................... 51 Bacterial Lipoproteins .................................................................................... 51 Materials and Methods ............................................................................................... 54 Bacterial Strains and Construction of Plasmids ............................................. 54 Colicin and Phage Spot-Titer Assays ............................................................ 54 [55Fe3+]ferrichrome Transport ........................................................................ 55 Multiple Sequence Alignment and Phylogenetic Analysis of Cor Protein .... 55 Results and Discussion ……… ................................................................................. 57 Lytic and Lysogenic Conversion: