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Host Range of Mycobacteriophage and Exploring Lysogenic Relationships Between Mycobacteriophage and Their Hosts

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Host Range of Mycobacteriophage and Exploring Lysogenic Relationships Between Mycobacteriophage and Their Hosts The University of Maine DigitalCommons@UMaine Honors College Spring 5-2017 Host Range of Mycobacteriophage and Exploring Lysogenic Relationships Between Mycobacteriophage and Their Hosts Emily Illingworth University of Maine Follow this and additional works at: https://digitalcommons.library.umaine.edu/honors Part of the Biological Engineering Commons Recommended Citation Illingworth, Emily, "Host Range of Mycobacteriophage and Exploring Lysogenic Relationships Between Mycobacteriophage and Their Hosts" (2017). Honors College. 444. https://digitalcommons.library.umaine.edu/honors/444 This Honors Thesis is brought to you for free and open access by DigitalCommons@UMaine. It has been accepted for inclusion in Honors College by an authorized administrator of DigitalCommons@UMaine. For more information, please contact [email protected]. HOST RANGE OF MYCOBACTERIOPHAGE AND EXPLORING LYSOGENIC RELATIONSHIPS BETWEEN MYCOBACTERIOPHAGE AND THEIR HOSTS by Emily Illingworth A Thesis Submitted in Partial Fulfillment of the Requirements for a Degree with Honors (Biochemistry) The Honors College University of Maine May 2017 Advisory Committee: Sally Molloy, Assistant Professor of Genomics, Advisor Keith Hutchison, Professor Emeritus Dorothy Croall, Professor of Biochemistry Richard Powell, Director of the Cohen Institute for Leadership Melissa Ladenheim, Associate Dean of the Honors College ABSTRACT Mycobacteriophage (phage) are abundant viruses that infect bacteria of the genus Mycobacterium. Pathogenic species of Mycobacterium carry prophage that are hypothesized to contribute to virulence. The term “prophage” describes a dormant phage during lysogeny, which occurs when a phage genome integrates itself into the host genome. Understanding the interactions between bacterial host and prophage may improve our ability to treat disease caused by bacteria. Phage are highly diverse and are sorted into clusters based on genome sequence similarity. To determine which phage infect and form lysogens in mycobacterial fish pathogens, we applied viral dosages of phage representative of clusters A–X to lawns of M. chelonae and M. marinum. The ability for phage to infect these alternative hosts is cluster-dependent. Two phage, BPs (cluster G) and Wildcat (cluster V), infected M. chelonae the most efficiently. BPs was able to form lysogens in M. chelonae. The ability for BPs to form lysogens in M. chelonae is not surprising since its genome encodes an integrase and a repressor, and BPs is also able to form stable lysogens in its isolation host, M. smegmatis. However, despite their highly conserved genomes, Wildcat and an additional cluster V phage, EniyanLRS, display different lysogen phenotypes. Future work includes identifying genes in both Wildcat and EniyanLRS that may play a role in lysogenic maintenance and performing RNA sequencing analysis to determine how prophage affect bacterial gene expression. DEDICATION This thesis is dedicated to my younger brother, Brian. I love you and hope you always feel brave enough to follow your dreams. iii ACKNOWLEDGEMENTS I would like to say a special thank you to the following people: • Dr. Sally Molloy and Dr. Keith Hutchison for your constant guidance and support both in and out of the classroom • Members of my advisory committee, Dr. Dorothy Croall, Dr. Melissa Ladenheim, and Dr. Richard Powell, for your flexibility, time, assistance, and support throughout this project • The University of Maine Honors College and Department of Molecular and Biomedical Sciences • Past and current undergraduates in the Molloy Lab for their support and mentorship: Katrina Harris, Gwen Beacham, Emily Whitaker, Robert Soohey, Erica Sewell, Daniel Lesko, and Emma Freeman • The SEA-PHAGES program and Dr. Graham Hatfull • My loving, supportive, dysfunctional family and group of friends. I don’t know what I would do without you. Thank you to the following funding sources: • The INBRE Junior Year Research Award and Comparative Functional Genomics Thesis Fellowship • University of Maine Center for Undergraduate Research • Charlie Slavin Research Fund • Dept. of Molecular and Biomedical Sciences Frederick Radke funds iv TABLE OF CONTENTS LIST OF TABLES ............................................................................................................. iii LIST OF FIGURES ........................................................................................................... iv 1.0 Introduction ....................................................................................................................1 2.0 Literature Review ...........................................................................................................3 2.1 Prophage in pathogens .......................................................................................3 2.2 Mycobacteria ......................................................................................................4 2.3 Mycobacterium tuberculosis ..............................................................................5 2.4 Mycobacterium chelonae and Mycobacterium marinum ...................................6 2.5 Mycobacteriophage abundance and diversity ....................................................6 2.6 Host range of mycobacteriophage .....................................................................8 2.7 Life cycles of mycobacteriophage .....................................................................9 3.0 Materials and Methods .................................................................................................10 3.1 Bacterial growth conditions .............................................................................10 3.2 Viruses .............................................................................................................10 3.3 Host susceptibility testing ................................................................................11 3.4 Full titer assays and plaque recovery of mycobacteriophage on M. chelonae .11 3.5 Lysogen isolation in M. chelonae and M. smegmatis ......................................12 3.6 Lysogen confirmation: checking for phage particles in culture supernatant ...12 3.7 Lysogen confirmation: immunity assays .........................................................12 3.8 DNA isolations of lysogens .............................................................................13 3.9 Polymerase chain reaction of Wildcat and EniyanLRS lysogens ....................13 3.10 Agarose gel electrophoresis .......................................................................13 v 4.0 Results ..........................................................................................................................14 4.1 Optimal optical density of M. chelonae for plaque assays ..............................14 4.2 Host range of M. smegmatis phage isolates .....................................................15 4.3 Wildcat and BPs form lysogens in M. chelonae ..............................................19 4.4 EniyanLRS forms lysogens in M. smegmatis ..................................................22 5.0 Discussion ....................................................................................................................26 5.1 M. marinum and M. chelonae can be used to study relationships between phage and pathogenic hosts .............................................................................27 5.2 The host range of 32 M. smegmatis phage isolates has been determined in M. marinum and M. chelonae ................................................................................27 5.3 BPs and Wildcat mutants demonstrate an increased efficiency of plating ......29 5.4 BPs is confirmed to form lysogens in M. chelonae .........................................29 5.5 Cluster V phage display different lysogen phenotypes in M. smegmatis ........31 5.6 Conclusions and future work ...........................................................................33 REFERENCES ..................................................................................................................34 BIOGRAPHY OF THE AUTHOR ....................................................................................38 LIST OF TABLES Table 1. Mycobacterial susceptibility to mycobacteriophage A–X .................................18 Table 2. Phage efficiencies of plating in M. chelonae compared to M. smegmatis after plaque recovery from M. chelonae ....................................................................................18 vi LIST OF FIGURES Figure 1. Growth curve of M. chelonae .............................................................................15 Figure 2. Host range assays of mycobacteriophage representing clusters A–X ................17 Figure 3. Plaque morphology of BPs and Wildcat on M. smegmatis and M. chelonae .....19 Figure 4. Detection of phage particles in culture supernants of BPs and Wildcat lysogens of M. chelonae on lawns of M. chelonae ...........................................................................21 Figure 5. Immunity assay of BPs M. chelonae lysogen compared to control ...................19 Figure 6. Immunity assay of Wildcat M. chelonae lysogen compared to control .............22 Figure 7. Detection of phage particles in supernatant of EniyanLRS lysogens of M. smegmatis ...........................................................................................................................22 Figure 8. Immunity assay of EniyanLRS M. smegmatis lysogen compared
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