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Sulfolobus As a Model Organism for the Study of Diverse SULFOLOBUS AS A MODEL ORGANISM FOR THE STUDY OF DIVERSE BIOLOGICAL INTERESTS; FORAYS INTO THERMAL VIROLOGY AND OXIDATIVE STRESS by Blake Alan Wiedenheft A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Microbiology MONTANA STATE UNIVERSITY Bozeman, Montana November 2006 © COPYRIGHT by Blake Alan Wiedenheft 2006 All Rights Reserved ii APPROVAL of a dissertation submitted by Blake Alan Wiedenheft This dissertation has been read by each member of the dissertation committee and has been found to be satisfactory regarding content, English usage, format, citations, bibliographic style, and consistency, and is ready for submission to the Division of Graduate Education. Dr. Mark Young and Dr. Trevor Douglas Approved for the Department of Microbiology Dr.Tim Ford Approved for the Division of Graduate Education Dr. Carl A. Fox iii STATEMENT OF PERMISSION TO USE In presenting this dissertation in partial fulfillment of the requirements for a doctoral degree at Montana State University – Bozeman, I agree that the Library shall make it available to borrowers under rules of the Library. I further agree that copying of this dissertation is allowable only for scholarly purposes, consistent with “fair use” as prescribed in the U.S. Copyright Law. Requests for extensive copying or reproduction of this dissertation should be referred to ProQuest Information and Learning, 300 North Zeeb Road, Ann Arbor, Michigan 48106, to whom I have granted “the exclusive right to reproduce and distribute my dissertation in and from microfilm along with the non-exclusive right to reproduce and distribute my abstract in any format in whole or in part.” Blake Alan Wiedenheft November, 2006 iv DEDICATION This work was funded in part through grants from the National Aeronautics and Space Administration Program (NAG5-8807) in support of Montana State University’s Center for Life in Extreme Environments (MCB-0132156), and the National Institutes of Health (R01 EB00432 and DK57776). Invaluable technical assistance was provided by D. Willits, M. Allen, S. Brumfield, L. Liepold, Z. Varpness, D. Nielsen, A. Ortmann, B. Widener and nearly every other current or previous members of the Mark Young/Trevor Douglas group. Interdisciplinary collaborations with Professors C. Martin Lawrence, Matthew Lavin, Brian Bothner and John Van der Oost have substantially broadened my scientific scope and challenged me to learn the scientific vernacular that comes with each of their respective disciplines. Special thanks to my mentors (Drs. Mark Young and Trevor Douglas) whom have demonstrated a contagious zeal for both a life of science and for life “outside” of science. These two individuals have managed to merge the appeal of Yellowstone National Park with existing scientific interests in virology, biomedicine and material science. I hope to use this example of creativity and selfless collaboration as a template for my future scientific endeavors. Thanks to friends and family, whom frequently tolerated unsolicited lectures on any number of ‘burning’ scientific topics. The patience of these people is a lesson I would do well to learn. Enduring patience has been a reoccurring theme for one individual in particular. Michelle Flenniken has not only been an invaluable scientific reference, but also a partner in diverse adventures on several continents, more then a few of which have been arguably ridiculous. v TABLE OF CONTENTS 1. AN ARCHAEAL PERSPECTIVE ................................................................... 1 Abstract .................................................................................................. 1 Archaea: Life of the Third Kind.................................................................. 2 Current State of Culture Based Archaea ..................................................... 8 Archaeal Genomics: Defining a Domain...................................................... 9 Developing Genetic Systems ................................................................... 16 Arriving at Sulfolobus as the Model System of Choice................................ 22 Overview............................................................................................... 25 2. HOT ARCHAEAL VIRUSES REVEAL DEEP EVOUTIONARY CONNECTIONS ...................................................................................... 31 Abstract ................................................................................................ 31 Viral Prospecting.................................................................................... 31 Crenarchaeal Viruses.............................................................................. 34 Genomic Structure and Content............................................................... 41 Viral Replication Cycles........................................................................... 46 Entry and Uptake .............................................................................. 46 Transcription and Genome Replication ................................................ 49 Assembly and Release ....................................................................... 51 Biogeographic Isolation of Crenarchaeal Viruses ....................................... 52 Future Directions and Questions.............................................................. 54 3. COMPARATIVE GENOMIC ANALYSIS OF THE THERMOACIDOPHILIC ARCHAEAL FUSELLOVIRIDAE VIRUSES .................................................... 57 Abstract ................................................................................................ 57 Introduction .......................................................................................... 58 Material and Methods ............................................................................. 63 Environmental Sampling .................................................................... 63 Enrichment Cultures of Environmental Samples ................................... 64 Virus Purification and Nucleic Acid Isolation......................................... 65 Construction of Genomic Libraries and Sequencing .............................. 65 Sequence Analysis............................................................................. 66 SSV Genomics Comparisons ............................................................... 67 Phylogenetic Comparison of Common ORFs......................................... 70 Analysis of Targeted SSV Integration .................................................. 70 vi TABLE OF CONTENTS - CONTINUED Structural Modeling of tRNAs.............................................................. 71 Results and Discussion ........................................................................... 72 4. NO BOUNDARIES: VIRAL MIGRATION MAINTAINS LOCAL DIVERSITY........ 84 Abstract ................................................................................................ 84 Linking Phylogeny and Ecology of Viruses in Yellowstone’s Hot Springs ........................................................................................... 85 Materials and Methods............................................................................ 96 Sample Collection and DNA Isolation .................................................. 96 Water chemistry................................................................................ 96 PCR Amplification, Cloning and Sequencing ......................................... 96 Phylogenetic Analysis ........................................................................ 97 Dissimilarity Index, Rank Abundance Modeling and Estimations of Migration and Metacommunity ....................................................... 98 5. KILLING THE MESSENGER-– A NOVEL PROKARYOTIC IMMUNE SYSTEM BASED ON RNA INTERFERENCE.............................................................. 99 Abstract ................................................................................................ 99 Introduction .......................................................................................... 99 Preliminary Results................................................................................103 Archaea: A Hot Model for Assessing the Prokaryotic Immune Response ..........................................................................103 A Collaborative Strategy; Microarray, Proteomics and Biochemistry .............................................................................109 Materials and Methods...........................................................................110 Culturing .........................................................................................110 RNA Isolation and Quality Assessment ...............................................110 Northern Analysis.............................................................................110 6. PERSPECTIVES AND FUTURE DIRECTIONS IN THE FIELD OF THERMAL VIROLOGY ............................................................................112 Advancing the Field of Thermal Virology .................................................112 RNA Mediated Gene Regulation..............................................................120 Viral Evolution and Ecology....................................................................121 Brief on Topics Outside the Lab..............................................................122 Outlook................................................................................................124 vii TABLE OF CONTENTS
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