THE SEARCH FOR ARCHAEAL VIRUSES IN HIGH TEMPERATURE ACIDIC ENVIRONMENTS AND CHARACTERIZATION OF SULFOLOBUS TURRETED ICOSAHEDRAL VIRUS (STIV) by George Ernest Rice III A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology MONTANA STATE UNIVERSITY Bozeman, Montana August 2006 © COPYRIGHT by George Ernest Rice III 2006 All Rights Reserved ii APPROVAL of a dissertation submitted by George Ernest Rice III 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 Approved for the Department of Microbiology Dr. Tim Ford Approved for the College of 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, 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 form in whole or in part.” George Ernest Rice III 23 August, 2006 iv TABLE OF CONTENTS 1. THERMOACIDOPHILLIC ARCHAEAL VIRUSES: BACKGROUND AND SCOPE OF THIS RESEARCH ...............................................................................1 Major Questions and Hypothesis.............................................................................2 Thermophiles ...........................................................................................................2 Domain Archaea ..........................................................................................4 Sulfolobus and Archaeal Metabolism ..........................................................8 Biotechnical Applications of Extremophiles .............................................11 Viruses .......................................................................................................13 Evolutionary Significance..........................................................................15 Crenarchaeal dsDNA Viruses....................................................................18 SSV ............................................................................................................19 SIRV ..........................................................................................................22 Other Thermal Viruses...............................................................................27 Viral Morphologies and Diversity from Marine Environments ................34 Summary................................................................................................................37 2. THE SEARCH FOR ARCHAEAL VIRUSES IN HIGH TEMPERATURE, ACIDIC ENVIRONMENTS .................................................................................38 Collecting Virus in Marine Environments.............................................................39 Culture Dependent vs. Culture Independent Approaches to Virus Studies...........42 Host Independent Survey (Filter Concentration) Method ........................43 Host Dependent (Culturing Sulfolobus) Method .......................................43 Materials and Methods ..........................................................................................46 Sampling Locations ...................................................................................46 Establishing Enrichment Cultures..............................................................48 16S rDNA Analysis (performed by Jamie Snyder Doctoral Student in the Young Lab) ..........................................................................................51 Virus Detection Methods ...........................................................................51 Transforming Sulfolobus with Viral Nucleic Acid ....................................52 Viral Genomic Library Construction.........................................................53 Results....................................................................................................................54 Enrichment Cultures ..................................................................................54 16S rDNA Analysis ..................................................................................55 Detection of Viruses and Virus-like Particles............................................56 SSV – Morphologies that Appear Similar .................................................58 SIRV – Morphologies that Appear Similar ...............................................59 SIFV – Morphologies that Appear Similar................................................60 32 nm Spheres – Morphologies Not Previously Identified........................61 Double – Tailers – Morphologies Not Previously Identified.....................63 v TABLE OF CONTENTS - CONTINUED STIV – Morphologies Not Previously Identified.......................................65 Discussion..............................................................................................................66 3. CHARACTERIZATION OF SULFOLOBUS TURRETED ICOSAHEDRAL VIRUS (STIV).......................................................................................................72 Materials and Methods...........................................................................................73 Sampling Locations ...................................................................................74 Virus Isolation............................................................................................75 Virus Purification (Small Batch) ...............................................................75 Virus Purification (Large Batch) ...............................................................77 Isolation, Cloning, and Sequencing the Viral Genome..............................79 Infectivity Assays.......................................................................................81 Identification of Major Virion Protein (performed in Cooperation with Eric Gillitzer, Postdoctoral Fellow in the Young Lab) .............................81 CryoTEM and Image Reconstruction (performed in Cooperation with Liang Tang, Postdoctoral Fellow in the Johnson Lab, Scripps Research Institute) .....................................................................................................82 Results....................................................................................................................83 Virus Isolation.............................................................................................84 Infectivity...................................................................................................85 Genome Analysis (performed in Cooperation with Frank Roberto at Idaho National Lab and Lucigen Corp.) ....................................................87 Identification of the Major Structural Protein ..........................................91 CryoTEM and Image Reconstruction (performed in Cooperation with Liang Tang, Postdoctoral Fellow in the Johnson Lab) ......................94 Discussion...............................................................................................................98 4. CONCLUSIONS TO QUESTIONS AND HYPOTHESIS.................................102 Question 1 ............................................................................................................102 Question 2 ............................................................................................................102 Question 3 ............................................................................................................102 Question 4 ............................................................................................................103 Hypothesis............................................................................................................104 REFERENCES CITED...............................................................................................105 APPENDIX A: Culture Independent Virus Survey in YNP.......................................118 References Cited .........................................................................................................132 vi LIST OF TABLES Table Page 1.1. Viruses of Crenarchaeaota ..........................................................................................36 2.1. Locations of thermal features selected for sampling ..................................................48 2.2. Location, temperature and pH of sampling sites .......................................................54 2.3. Characteristics of YNP viruses ...................................................................................57 3.1. Recipe for DSM media 88 ..........................................................................................76 3.2. Predicted ORFs in STIV genome that are most similar to ORFs in the database.............................................................................................................91