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Isolation, Characterization, and Genomic Comparison of Bacteriophages of Enterobacteriales Order

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Isolation, Characterization, and Genomic Comparison of Bacteriophages of Enterobacteriales Order Brigham Young University BYU ScholarsArchive Theses and Dissertations 2019-07-01 Isolation, Characterization, and Genomic Comparison of Bacteriophages of Enterobacteriales Order Ruchira Sharma Brigham Young University Follow this and additional works at: https://scholarsarchive.byu.edu/etd BYU ScholarsArchive Citation Sharma, Ruchira, "Isolation, Characterization, and Genomic Comparison of Bacteriophages of Enterobacteriales Order" (2019). Theses and Dissertations. 8577. https://scholarsarchive.byu.edu/etd/8577 This Dissertation is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. TITLE PAGE Isolation, Characterization, and Genomic Comparison of Bacteriophages of Enterobacteriales Order Ruchira Sharma A dissertation submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Julianne H. Grose, Chair Bradley D. Geary Steven M. Johnson Perry G. Ridge K. Scott Weber Department of Microbiology and Molecular Biology Brigham Young University Copyright © 2019 Ruchira Sharma All Rights Reserved ABSTRACT Isolation, Characterization and Genomic Comparison of Bacteriophages of Enterobacteriales Order Ruchira Sharma Department of Microbiology and Molecular Biology, BYU Doctor of Philosophy According to CDC, every year at least 2 million people are affected and 23,000 dies as a result of antibiotic resistance in U.S. It is considered one of the biggest threats to global health. More and more bacterial infections are becoming harder to treat. One such infection is fire blight, one of the most destructive disease of apple and pear trees. It is caused by bacteria Erwinia amylovora and its outbreaks have been known to destroy entire orchards in a single season. The conventional method of treatments includes use of antibiotics like streptomycin and oxytetracycline but the incidences like presence of multi-drug resistant bacteria in the mammals grazing in the fields have raised concerns. Phage therapy is considered one of the few ways available to combat bacterial resistance and prevent fire blight. In this method, a cocktail of highly lytic bacteriophages is prepared and sprayed on the trees at different time intervals. Bacteriophages are an “intelligent” drug. They multiply at the site of the infection until there are no more bacteria and then they are excreted back into the nature. These phenomena make them more efficient than an antibiotic, which kills all kind of bacteria including good bacteria and can be maintained in the environment for long periods of time. These qualities of bacteriophage have resulted in many commercially available phage therapies. The initial part of this research focuses on isolation, characterization and genomic comparison of bacteriophages that infect a plant pathogen E.amylovora of Erwiniaceae family of Enterobacteriales order. In this study, 28 novel bacteriophages were isolated, fully sequenced, characterized and grouped into seven families based on phage homology. To take this further, we characterized a novel jumbo family of bacteriophages that has a small burst size of 4.6-4.9 and are most similar to bacteriophages that infect Pseudomonas and Ralstonia rather than Enterobacteriales bacteria by protein similarity. These bacteriophages are shown to infect Erwinia and Pantoea bacterial strains, but no infection of 9 other bacterial strains tested, was seen, under laboratory conditions. The results of this work provide an insight on special characteristics that makes bacteriophage so unique and adaptable. The final part of this research explores the enormous diversity of bacteriophages. In 2014 Grose and Casjens grouped 337 fully sequenced tailed phages into 56 diverse clusters (32 lytic and 24 temperate). We further expanded our current understanding of these clusters by performing the comprehensive analysis of genomes and proteomes of 1037 tailed bacteriophages, posted on GenBank. The results of this work provide insights into diversity and relatedness of bacteriophages and the data is posted on GenBank. Keywords: E. amylovora, bacteriophage, Enterobacteriaceae, phage clusters, fire blight, Pantoea, phage therapy ACKNOWLEDGEMENTS This work is dedicated to my husband Yomesh, and kids Avani and Aniruddh. It would have been impossible without their sacrifice and support. I am indebted to my parents, siblings and in-laws for their trust and unconditional love. I would like to begin with thanking my pillar of support and phabulous mentor Dr. Julianne Grose. Her creative thinking, caring attitude and prompt replies to my dumb questions even at 2 am in the morning, made this a fruitful journey. I would also like to thank my committee Dr. Weber, Dr. Johnson, Dr. Geary and Dr. Ridge for their valuable advice and encouragement. I am grateful to the graduate committee and Dr. Griffitts for admitting me into the program at the last minute and for enhancing the quality of my research with his wonderful insight. A big thank you goes to our academic advisors Marcia and Kevin, business managers Amanda, Becky and Heidi and graduate secretaries for their hard work, time and support. I would also like to thank Sue Pratley for fulfilling my last-minute room-scheduling requests and for reviewing this dissertation. I am lucky to have built everlasting friendships in and out of our lab and greatly appreciate their affection and much needed constructive criticism. Thank you, Jenny, KaiLi, Trever, Elise, Daniel T, Daniel A, Claudia, Deborah, Khin, Israel, Anna, Nidhi, Jeralyn, Evita, Desi, Ian, Jordan and Garrett for mentoring and guiding me and lending your shoulders to cry on. I would also like to thank all the phage hunters and high school interns, who I had the honor of training, for making me a better scientist. I appreciate the patience and kindness of my friends outside of school and everyone who has helped in making this possible. TABLE OF CONTENTS TITLE PAGE ................................................................................................................................... i ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................... iii TABLE OF CONTENTS ............................................................................................................... iv LIST OF FIGURES ........................................................................................................................ x LIST OF TABLES ....................................................................................................................... xiii CHAPTER 1: Isolation, Characterization and Genomic Comparison of Bacteriophages of Enterobacteriales Order ........................................................................................................ 1 1.1 Background ........................................................................................................................... 1 1.2 Introduction to bacteriophages .............................................................................................. 1 1.3 Structure and morphology of Caudovirales bacteriophages ................................................. 2 1.4 The Enterobacteriales order ................................................................................................. 3 1.5 Erwinia amylovora ............................................................................................................... 3 1.6 Fire blight .............................................................................................................................. 4 1.7 The interplay between bacteria-bacteriophage ..................................................................... 6 1.8 Bacteriophage isolation and basic characterization .............................................................. 7 1.9 Summary of research chapters .............................................................................................. 7 1.10 Summary of appendices ...................................................................................................... 9 REFERENCES ............................................................................................................................. 10 CHAPTER 2: Genome Sequences of Nine Erwinia amylovora Bacteriophages ......................... 14 2.1 Abstract ............................................................................................................................... 14 iv 2.2 Introduction ......................................................................................................................... 14 2.3 Materials and methods ........................................................................................................ 15 2.3.1 Bacteriophage isolation and genome sequencing ........................................................ 15 2.3.2 Electron microscopy .................................................................................................... 15 2.3.3 Computational analysis ................................................................................................ 16 2.4 Results and discussion .......................................................................................................
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