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Item Sulfolobus as a model organism for the study of diverse biological interests : forays into thermal virology and oxidative stress(Montana State University - Bozeman, College of Letters & Science, 2006) Wiedenheft, Blake Alan; Chairperson, Graduate Committee: Mark Young; Trevor Douglas (co-chair)My research interests have focused on two distinct aspects of Sulfolobus biology: virology and oxidative stress. My major contribution to the emerging field of thermal virology has been the isolation, characterization and comparative genomic analysis of a spindle-shaped virus partical (SSV RH) infecting the thermoacidophilic archaeal host Sulfolobus solfataricus (18). Insights from this comparative genomic analysis have served as a platform for targeted structural studies, as well as providing molecular tools used to follow the viral life cycle in culture and for assessing the ecological significance of these viruses in the environment (9, 19-24). My research endeavors in oxidative stress arose from an early interest in iron metabolism and protective mechanisms that allow life to cope with the paradoxical role that iron plays in biological systems. Pursuit of this interest has lead to the discovery of a new class of proteins termed, "DPS-Like" (7, 17, 24, 25). These previously unrecognized proteins function as antioxidants and are widely distributed across both prokaryotic domains of life.Item DNA binding proteins of archaeal viruses(Montana State University - Bozeman, College of Letters & Science, 2008) Geary, Joella Suzanne; Chairperson, Graduate Committee: Mensur DlakicArchaea are single-celled organisms comprising the third domain of life. The Achaeal species Sulfolobus are infected by the Fuselloviridae virus family: SSV1, SSV2, SSV-RH, and SSV-K. The genomes of these viruses have been annotated and contain putative DNA-binding proteins. The purpose of this work is to identify DNA sequences bound by the SSV1 putative DNA-binding protein C43. C43 protein was cloned, expressed, purified, and assayed at various temperatures for interaction with three SSV1 DNA sequences. C43 binds the T5-promoter, T6-promoter, and C43-promoter sequentially and consistently. Additionally, C43 protein is functional at temperatures of 50°C and 65°C. Thus, C43 appears to be an important regulator of the Fuselloviridae SSV1 viral genome.Item The search for Archaeal viruses in high temperature acidic environments and characterization of Sulfolobus turreted icosahedral virus (STIV)(Montana State University - Bozeman, College of Letters & Science, 2006) Rice, George Ernest; Chairperson, Graduate Committee: Mark YoungViruses of extreme thermophiles are of great interest because they can serve as model systems for understanding biochemical molecular nuances required for life at high temperatures. This two part body of work first reports the discovery and isolation of viruses and virus-like particles from extreme thermal acidic environments (70-92°C, pH 1.0-4.5) in Yellowstone National Park (YNP), and secondly details the characterization of one of these viruses that possesses a capsid structural motif that is found in at least two other families of viruses inhabiting the other two domains of life (Bacteria and Eukarya). This is of particular interest because it is the first example of a predicted but previously undocumented structural relationship between any entities (living or non) that span all three domains of life. The implications of this reported connection lend credence to the theory that there was a common viral ancestor, or ancestors, that predate the division of life into the three separate currently accepted domains.Item Virus dynamics, archaeal populations, and water chemistry of three acidic hot springs in Yellowstone National Park(Montana State University - Bozeman, College of Letters & Science, 2005) Snyder, Jamie Christine; Chairperson, Graduate Committee: Mark J. YoungFew viruses from Archaea have been described and only a limited number have been characterized in molecular detail. Most hyperthermophilic viruses isolated from the archaeal sub-domain Crenarchaeota belong to the Sulfolobales family. We have focused on the isolation and analysis of viruses replicating in Sulfolobus. Sulfolobus is an acidophilic hyperthermophile that lives optimally at 80°C and pH 3. The SSVs (Sulfolobus spindle-shaped viruses) are the viruses most commonly isolated from thermal acidic areas around the world. SSVs are dsDNA spindle-shaped viruses with genomes of approximately 15kb. They are also the predominant virus isolated from samples collected in Yellowstone National Park (YNP). The SIRVs (Sulfolobus islandicus rodshaped viruses) are also commonly isolated from YNP. These viruses have linear dsDNA genomes of approximately 33kb. I have used these two viruses to investigate the interaction of time and viral population dynamics. For a comparison between geographically isolated acidic hot springs, we chose to study three sites in YNP.Item Characterization and isolation of archael thermophilic hosts and viruses from Yellowstone National Park(Montana State University - Bozeman, College of Letters & Science, 2009) Spuhler, Joshua Lupine; Chairperson, Graduate Committee: Mark J. YoungMy research is focused on the identification and characterization of new archaeal viruses that inhabit the thermal features of Yellowstone National Park (YNP). I have undertaken the systematic survey of more than 90 different thermal features found in Yellowstone through a variety of means including culturing of hosts, MDA amplification, qPCR for known archaeal viruses, 16S rRNA gene analysis of potential resident archaeal hosts, tangential flow and end point filtration approaches to sample new viruses, and general water geochemical analysis. From this work a new host has been isolated from YNP. rDNA analysis has shown a 98% similarity to Thermocladium modestius. Routine culturing of this host has lead to the discovery of multiple viruses. Some of these associated viruses have similar morphology to other known archaeal viruses. The first is a 90x60 nm spindle shaped virus that was originally isolated from Rabbit Creek thermal feature (Temp78, pH3.5). Our initial genomic analysis shows that there is no obvious similarity to other known archaeal viruses, included the SSV spindled shaped viruses for Sulfolobus. The second sequencing effort has come from Nymph Lake thermal feature (Temp 85, pH 2.5). This virus population was gathered from a primary enrichment culture. This culture had two dominate virus morphologies present. The first is a 15nmx210nm rod-shaped virus with tapered ends. The second morphology seen is a 90nm spherical virus. Both of these viruses are hoped to be new additions to the archaeal virus families providing a more in depth view of the necessities of life at high temperatures.Item Archaeal host virus interactions(Montana State University - Bozeman, College of Letters & Science, 2011) Wirth, Jennifer Fulton; Chairperson, Graduate Committee: Mark J. YoungViruses are the most abundant biological entity on earth, and virus-host interactions are one of the most important factors shaping microbial populations (Suttle, 2007b). The study of both the cellular and viral members of the domain Archaea is a relatively new field. Thus, the viruses (and their cellular hosts) of Archaea are poorly understood as compared to viruses of Bacteria and Eukarya. This work has sought to expand our understanding of archaeal viruses by two general approaches. The first is by developing and implementing the use of a genetic system for a crenarchaeal virus, Sulfolobus turreted icosahedral virus (STIV), isolated from a hot (82°C) acidic (pH 2.2) pool in Yellowstone National Park, USA. The second approach has been to look at viral communities and their interactions with their cellular hosts in natural environments. We have developed a genetic tool, an infectious clone for STIV, which has allowed for genetic analysis of this virus. A number of viral genes have been knocked out, and their functions investigated using this tool. We have determined that at least three viral genes, A197, B345 and C381, are required for viral replication, while one gene, B116, is not essential. Work continues investigating function for other STIV genes as well as specific interactions with its host, Sulfolobus solfataricus. We have performed total community sequencing (metagenomics) for both the cellular and viral populations of several hot springs in Yellowstone National Park. We have been able to assemble a near full-length putative novel viral genome from one of these sites. We have also performed an in depth analysis of the function of a newly described bacterial and archaeal adaptive immune system (CRISPR/Cas) in a natural environment. This study has provided insights into the function of this immune system in a complex nutrient limited environment, which would not have been observed by studying cultured isolates in a laboratory.