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The Isolation and Characterization of Tirotheta9, a Novel A4 Mycobacterium Phage

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The Isolation and Characterization of Tirotheta9, a Novel A4 Mycobacterium Phage Western Kentucky University TopSCHOLAR® Honors College Capstone Experience/Thesis Honors College at WKU Projects Spring 5-16-2014 The solI ation and Characterization of TiroTheta9, a Novel A4 Mycobacterium Phage Sarah Schrader Western Kentucky University, [email protected] Follow this and additional works at: http://digitalcommons.wku.edu/stu_hon_theses Part of the Biology Commons Recommended Citation Schrader, Sarah, "The sI olation and Characterization of TiroTheta9, a Novel A4 Mycobacterium Phage" (2014). Honors College Capstone Experience/Thesis Projects. Paper 483. http://digitalcommons.wku.edu/stu_hon_theses/483 This Thesis is brought to you for free and open access by TopSCHOLAR®. It has been accepted for inclusion in Honors College Capstone Experience/ Thesis Projects by an authorized administrator of TopSCHOLAR®. For more information, please contact [email protected]. THE ISOLATION AND CHARACTERIZATION OF TIROTHETA9, A NOVEL A4 MYCOBACTERIUM PHAGE A Capstone Experience/Thesis Project Presented in Partial Fulfillment of the Requirements for the Degree Bachelor of Science with Honors College Graduate Distinction at Western Kentucky University By Sarah M. Schrader **** Western Kentucky University 2014 CE/T Committee: Approved by Dr. Rodney King, Advisor Dr. Claire Rinehart _____________________ Advisor Dr. Audra Jennings Department of Biology Copyright by Sarah M. Schrader 2014 ABSTRACT Bacteriophages are the most abundant biological entities on earth, yet relatively few have been characterized. In this project, a novel bacteriophage was isolated from the environment, characterized, and compared with others in the databases. Mycobacterium smegmatis, a harmless soil bacterium, served as the host and facilitated the enrichment and recovery of mycobacteriophages. A single phage type was purified to homogeneity and named TiroTheta9 (TT9). Electron microscopy revealed that the phage particles have icosahedral heads 58 ± 2 nm in diameter and tails 174 ± 5 nm in length. The TT9 genomic DNA was purified and sequenced using 454 pyrosequencing technology. The 51,367 bp genome contains 87 genes that were identified using the gene prediction programs Glimmer and GeneMark. However, only 31 genes could be assigned functions based on BLAST analysis. Genome wide comparisons using the Phamerator program and BLAST revealed that TT9 is most closely related to members of the A4 subcluster of mycobacteriophages. Although the A4 subcluster phages have been isolated from geographically distinct locations, their genomic sequences are highly conserved. Comparative analysis of the subcluster reveals evidence of evolution through both vertical sequence divergence and horizontal gene transfer, as evidenced by the existence of three unique groups of integrases. These results have contributed to the rapidly ii expanding database of mycobacteriophage genomes; mining this rich source of genetic information should provide new insights into phage diversity and evolution. Keywords: Bacteriophage, Mycobacteriophage, Mycobacterium smegmatis, Phage diversity, Phage evolution, Bioinformatics iii Dedicated to TiroTheta9 iv ACKNOWLEDGEMENTS This project, an endeavor spanning all of my five years at Western Kentucky University, has benefited from the knowledge and support of many wonderful people. Principal among them is my mentor, Dr. Rodney King, whose guidance and encouragement has been indispensable. I am also grateful to the other members of my committee, Dr. Claire Rinehart and Dr. Audra Jennings, for their insight and support. Financial support for various phases of this project was provided by the Howard Hughes Medical Institute Science Education Alliance, the Gatton Academy of Mathematics and Science in Kentucky through a Research Internship Grant and several Research Supplies grants, and the Western Kentucky University Biology Department through a Biology Summer Undergraduate Research Experience Grant. I thank all of these institutions for their generous support of my project. I am also grateful to Dr. John Andersland for his expertise and assistance in taking TEM micrographs of my phage. Additionally, laboratory support was provided at various points by Prasanna Parthsarathy and Ali Wright, to whom I also extend my thanks. Finally, I would like to thank my wonderful parents Steven and Michelle Schrader for their continuous support of my academic endeavors. From Gatton to China (x5) to South Korea and back, they have always encouraged me to follow my dreams. v VITA September 9, 199 ...........................................Born – Ann Arbor, Michigan 2011................................................................The Carol Martin Gatton Academy of Mathematics and Science in Kentucky Greenwood High School- Bowling Green, Kentucky 2011................................................................United States Presidential Scholar 2011................................................................Barry M. Goldwater Scholar 2011................................................................Princeton University Summer Undergraduate Research Program – Princeton, New Jersey 2012................................................................Teach and Learn in Korea Scholar – Gimje, South Korea 2012-13 ..........................................................Chinese Flagship Program Capstone Year – Nanjing andShanghai, China 2013................................................................Pearson Prize for Higher Education 2013................................................................New York University School of Medicine Summer Undergraduate Research Program – New York, New York 2013................................................................Harry S. Truman Scholarship Finalist 2013................................................................Marshall Scholarship Finalist vi 2013................................................................Rhodes Scholarship Finalist 2014................................................................Gates-Cambridge Scholarship Finalist 2014................................................................National Science Foundation Graduate Research Fellowship 2014................................................................National Defense Science and Engineering Graduate Fellowship PUBLICATIONS Schrader SM. 2014. [On the “Right” to Bear Arms]. The Juhe Supplement. 10:53-55. Essay published in Chinese. Schrader SM. 2014. [View of Intercultural Marriage Among Chinese University Students]. The Juhe Supplement. 10:37-38. Essay published in Chinese. Schrader SM. 2013. [A Frustrating Experience]. The Juhe Supplement. 9:17-19. Essay published in Chinese. Schrader SM, Parthasarathy PT, King RA, et al. 2011. Mycobacterium Phage TiroTheta9, complete genome. GenBank. Accession number JN561150.1. Howard CM, Rinehart CA, Parthasarthy PT, King RA, Schrader SM, et al. 2011. Mycobacterium Phage Backyardigan, complete genome. GenBank. Accession number JF704093.1. Rinehart CA, King RA, Schrader SM, et al. 2010. Sequence and annotation of the wizard007 mycobacterium phage genome. BMC Bioinformatics. 11(Suppl 4):P15. FIELDS OF STUDY Major Field 1: Biology Major Field 2: Chemistry Major Field 3: Mandarin Chinese (Self-Designed) Minor Field: Mathematics vii TABLE OF CONTENTS Page Abstract ......................................................................................................................... ii Dedication ..................................................................................................................... iv Acknowledgements ....................................................................................................... v Vita ................................................................................................................................ vi List of Figures ............................................................................................................... xi List of Tables ................................................................................................................ xiii Chapters: 1. Introduction ............................................................................................................. 1 Part I: Introduction to Bacteriophage – Biology, History, and Applications ................ 3 2. Biological Characteristics of Bacteriophage ........................................................... 4 A. Bacteriophage Structure .................................................................................... 4 B. Bacteriophage Lifecycles .................................................................................. 5 C. Bacteriophage Classification ............................................................................ 7 D. Bacteriophage Ecology ..................................................................................... 11 3. History of Bacteriophage Research ........................................................................ 16 A. The Discovery of Bacteriophage ...................................................................... 16 B. Bacteriophage Research from the 1920s to 1940s ............................................ 17 C. Bacteriophage Research since 1950.................................................................. 18 viii 4. Current Applications of Bacteriophage .................................................................. 22 A. Basic Research Applications............................................................................. 22 B. Industrial Applications
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