Theses and Dissertations at Montana State University (MSU)
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Item Geomicrobiology of hydrogen in Yellowstone Hot Springs(Montana State University - Bozeman, College of Letters & Science, 2019) Lindsay, Melody Rose; Chairperson, Graduate Committee: Eric Boyd; Daniel R. Colman, Maximiliano J. Amenabar, Kirsten E. Fristad, Kristopher M. Fecteau, Randall V. Debes, John R. Spear, Everett L. Shock, Tori M. Hoehler and Eric S. Boyd were co-authors of the article, 'Geological source and biological fate of hydrogen in Yellowstone hot springs' which is contained within this dissertation.; Maximiliano J. Amenabar, Kristopher M. Fecteau, R. Vincent Debes II, Maria Clara Fernandes, Kirsten E. Fristad, Huifang Xu, Tori M. Hoehler, Everett L. Shock and Eric S. Boyd were co-authors of the article, 'Subsurface processes influence oxidant availability and chemoautotrophic hydrogen metabolism in Yellowstone hot springs' in the journal 'Geobiology' which is contained within this dissertation.Hydrogen (H 2) connects the geosphere and biosphere in rock-hosted ecosystems and has likely done so since early in Earth's history. High temperature hydrothermal environments, such as hot springs, can be enriched in H 2 and were likely widespread on early Earth. As such, linking the geological processes that supply H 2 to contemporary hot springs and the distribution of extant thermophilic organisms that can utilize H 2 as a component of their energy metabolism can provide insights into the environment types that supported early H 2 dependent life. Using a series of geochemical proxies, I developed a model to describe variable H 2 concentrations in Yellowstone National Park (YNP) hot springs. The model invokes interaction between water and crustal minerals that generates H 2 that can partition into the vapor phase during decompressional boiling of ascending hydrothermal waters. Fractures and faults in bedrock, combined with topographic features such as high elevation, allow for vapor to migrate and concentrate in certain areas of YNP leading to elevated concentrations of H 2. Metagenomes from chemosynthetic communities in YNP springs sourced with vapor-phase gas are enriched in genes coding for enzymes predicted to be involved in H 2-oxidation. A spring in an area of YNP (Smokejumper, SJ3) sourced with vapor-phase gas, that has the highest concentration of H 2 measured in YNP, and that is enriched in hydrogenase encoding genes was chosen to further examine the biological fate of H 2. SJ3 harbors a hyperdiverse community that is supported by mixing of oxidized meteoric fluids and volcanic gases. Transcripts coding for genes involved in H 2 uptake and CO 2 fixation were detected. The processes that control the availability of oxidants and their effect on the activity and abundance of H 2 dependent organisms was also investigated in two paired hot springs. H 2-oxidizing chemoautotrophs utilized different oxidants in the two springs and this underpinned differences in H2 oxidation activity and their identity. Together, these observations indicate that the subsurface geological processes of decompressional boiling and phase separation influence the distribution, identity, and activity of hydrogenotrophs through their combined effects on the availability of H 2 and oxidants.Item Flexibility in the mineral dependent metabolism of a thermoacidophilic crenarchaeote(Montana State University - Bozeman, College of Letters & Science, 2017) Amenabar Barriuso, Maximiliano Jose; Chairperson, Graduate Committee: Eric Boyd; John W. Peters (co-chair); Everett L. Shock, Eric E. Roden, John W. Peters and Eric S. Boyd were co-authors of the article, 'Microbial substrate preference dictated by energy demand rather than supply' in the journal 'Nature geoscience' which is contained within this thesis.; Daniel R. Colman, Saroj Poudel, Eric E. Roden and Eric S. Boyd were co-authors of the article, 'Expanding anaerobic heterotrophic metabolism with hydrogen' submitted to the journal 'Environmental microbiology' which is contained within this thesis.; Eric S. Boyd was a co-author of the article, 'Flexibility in mineral dependent energy metabolism broadens the ecological niche of a thermoacidophile' submitted to the journal 'Environmental microbiology' which is contained within this thesis.This dissertation focuses on understanding the metabolic flexibility of a thermoacidophilic crenarchaeote, the mechanisms underlying its physiology, and the consequences for its ecology. Acidianus strain DS80 was isolated from an acidic spring in Yellowstone National Park (YNP) and displays versatility in energy metabolism, using soluble and insoluble substrates during chemolithoautotrophic, chemoheterotrophic, and/or chemolithoheterotrophic growth, and is widely distributed among YNP springs. This flexibility suggests that strain DS80 is of utility as a model thermoacidophile that allows investigation towards how metabolically flexible microorganisms select among available substrates and how these traits influence their natural distributions. Moreover, this plasticity allows investigation of the agreement between thermodynamic metabolic predictions and physiological measurements. Here, I showed that strain DS80 prefers growth with redox couples that provide less energy (H2/S°) when compared to other redox couples (H2/Fe3+ or S°/Fe3+). I present a bioenergetic-physiological argument for this preference, suggesting that the preferential use of substrates in metabolically versatile strains, such as strain DS80, is dictated by differences in the energy demand of electron transfer reactions rather than the energy supply. These observations may help explain why thermodynamic approaches alone are often not enough to accurately predict the distribution and activity of microorganisms in environments. I then showed that genome-guided predictions of energy and carbon metabolism of organisms may not agree with physiological observations in the laboratory, and by extension, the environment. Using a suite of physiological experiments, I provide evidence that the availability of electron acceptors influences the spectrum of potential electron donors and carbon sources that can sustain growth. Similarly, the availability of H2 enables the use of organic carbon sources in DS80 cells respiring S°, thereby expanding the ecological niche of this organism by allowing them to compete for a wider array of substrates that are available in dynamic environments. Finally, I showed that strain DS80 can use several minerals for chemolithotrophy and that the use of specific metabolisms dictates the requirement for direct access to these minerals. Taken together, the results shown here provide novel insight into the extent and mechanisms of metabolic flexibility of chemolithotrophs and the consequences for their ecology.Item Linking geochemistry with microbial community structure and function in sulfidic geothermal systems of Yellowstone National Park(Montana State University - Bozeman, College of Agriculture, 2015) Jay, Zackary James; Chairperson, Graduate Committee: William P. Inskeep; Doug B. Rusch, Susannah G. Tringe, Connor Bailey, Ryan M. Jennings and William P. Inskeep were co-authors of the article, 'Predominant acidilobus-like populations from geothermal environments in Yellowstone National Park exhibit similar metabolic potential in different hypoxic microbial communities' in the journal 'Applied and environmental microbiology' which is contained within this thesis.; Jacob P. Beam, Alice Dohnalkova, Regina Lohmayer, Brynna Bodle, Brita Planer-Friedrich, Margaret Romine and William P. Inskeep were co-authors of the article, 'Pyrobaculum yellowstonensis strain WP30 respires on elemental sulfur and/or arsenate in circumneutral sulfidic geothermal sediments of Yellowstone National Park' submitted to the journal 'Applied and environmental microbiology' which is contained within this thesis.; Doug B. Rusch, Jacob P. Beam, Mark A. Kozubal, Ryan M. Jennings and William P. Inskeep were co-authors of the article, 'The distribution, diversity and function of predominant Thermoproteales phylotypes in Yellowstone National Park' submitted to the journal 'ISME J' which is contained within this thesis.Members of the archaeal phylum Crenarchaeota are often associated with microbial communities in high-temperature (> 70 °C) geothermal springs. Environmental genome sequencing (metagenomics) has revealed that populations of Sulfolobales, Desulfurococcales, and Thermoproteales are abundant in hypoxic elemental sulfur sediments of Yellowstone National Park (YNP) and possess enzyme complexes that are implicated in the cycling of carbon, sulfur, and arsenic. Therefore, the primary objectives of this work were to (i) identify the abundant Desulfurococcales and Thermoproteales sequences in these habitats, (ii) characterize the growth and curate the genome of the first Thermoproteales representative isolated from YNP (Pyrobaculum yellowstonensis strain WP30), and (iii) establish a linkage between geochemistry and microbial community structure and function by identifying key proteins that are important to these populations in situ. The primary Desulfurococcales populations were related to Acidilobus spp. and exhibited similar metabolic potential in near-neutral (pH 4 - 6) hypoxic elemental sulfur sediments and acidic (pH ~3) iron oxide mats. These populations are primarily anaerobic heterotrophs that ferment complex organic carbon and are auxotrophic with regards to numerous vitamins and cofactors. These organisms are often found together with members of the Thermoproteales, which are widely distributed in elemental sulfur sediments, acidic iron oxide mats, and streamer communities. P. yellowstonensis strain WP30 was obtained from a hypoxic elemental sulfur sediment habitat with high concentrations of arsenic. This organism was shown to reduce elemental sulfur and/or arsenate in the presence of yeast extract. The complete genome of str. WP30 contained numerous dimethylsulfoxide molybopterin (DMSO-MPT) proteins, which are inovolved in redox reactions of inorganic constituents (i.e. sulfur and arsenic), and genomic comparisons revealed that this organism is closely related to native Pyrobaculum populations. The distribution of Thermoproteales populations was correlated with pH, while the presence of respiratory complexes (terminal oxidases, DMSO-MPT, and dissimilatory sulfate reductases) was correlated with the presence of key electron donors and acceptors. Intron sequences identified in Thermoproteales 16S rRNA genes and were shown in silico to prevent the binding of 'universal' primers that are often used in environmental surveys. These metagenomic, microbiological, and geochemical studies have advanced the understanding of Crenarchaeota diversity and function in YNP.Item Chemosynthetic carbon metabolism in thermophiles(Montana State University - Bozeman, College of Letters & Science, 2015) Urschel, Matthew Robert; Chairperson, Graduate Committee: Eric Boyd; Michael D. Kubo, Tori M. Hoehler, John W. Peters and Eric S. Boyd were co-authors of the article, 'Carbon source preference in chemosynthetic hot spring communities' in the journal 'Applied and Environmental Microbiology' which is contained within this thesis.; Matthew R. Urschel, Trinity L. Hamilton, Eric E. Roden and Eric S. Boyd were co-authors of the article, 'Substrate preference and uptake kinetics in a facultatively autotrophic and hyperthermophilic crenarchaeote' submitted to the journal 'Applied and environmental microbiology' which is contained within this thesis.Microbial communities inhabiting high temperature (>73°C) environments are supported by chemical energy, providing a unique opportunity to investigate the processes that supported life prior to the advent of photosynthesis. Previous work has focused on the importance of autotrophy in supporting such communities, and recent reports of organic substrate utilization in several high temperature springs in Yellowstone National Park (YNP), Wyoming, USA suggest that chemosynthetic populations are facultatively autotrophic. Nevertheless, little is known about the factors influencing relative rates of autotrophy and heterotrophy in these systems, and few studies have addressed the potential role of facultative autotrophs in supporting these ecosystems. This work addressed these compelling questions using in situ microcosm assays to directly quantify organic and inorganic substrate transformation rates in 13 geochemically diverse YNP chemosynthetic communities. The results provide the first conclusive evidence that dominant autotrophs in these ecosystems are facultative, and can alter their metabolism over short time spans to preferentially exploit more thermodynamically favorable organic substrates at rates comparable to or exceeding those of inorganic substrate utilization. Multivariate statistical analysis of co-registered substrate transformation rates, geochemical measurements, and phylogenetic data collected from these communities suggests an important relationship between environmental variation, community composition, and the relative importance of autotrophic and heterotrophic metabolisms supporting these communities. Elevated formate utilization rates in crenarchaea-dominated chemosynthetic communities inhabiting acidic, sulfur-rich geothermal springs motivated the isolation of the first hyperthermophilic crenarchaeon (Thermoproteus sp CP80) capable of coupling formate oxidation to elemental sulfur reduction. Physiological characterization demonstrated that CP80 is a facultative autotroph that alters its metabolism to preferentially utilize formate over CO 2. Similar formate utilization characteristics by CP80 and its native population strongly suggests that this and other sulfur reducing crenarchaea may be responsible for high rates of formate utilization in high temperature, sulfur rich YNP systems. Overall, these results indicate an important, previously-underestimated role for organic substrates in supporting chemosynthetic communities inhabiting geochemically diverse YNP hot springs. Future work should focus on identifying additional organic carbon sources, measurement of carbon flux through chemosynthetic communities, and further characterization of the biochemical mechanisms underlying organic and inorganic substrate metabolismin Thermoproteus sp. CP80.Item Fossil viruses, redox paradigms and predictive metabolism from a systems biology perspective(Montana State University - Bozeman, College of Letters & Science, 2014) Heinemann, Joshua Vance; Chairperson, Graduate Committee: Brian Bothner; Walid S. Maaty, George Gauss, Narahari Akkaladevi, Susan K. Brumfield, Vamseedhar Rayaprolu, Mark Young, C. Martin Lawrence and Brian Bothner were co-authors of the article, 'Fossil record of an HK-97-like provirus' in the journal 'Virology' which is contained within this thesis.; Timothy Hamerly, Walid S. Maaty, Navid Movahed, Joseph D. Steffens, Benjamin D. Reeves, Jonathan K. Hilmer, Jesse Therien, Paul A. Grieco, John W. Peters and Brian Bothner were co-authors of the article, 'Expanding the paradigm of thiol redox in the thermophilic root of life' in the journal 'Biochimica et biophysica acta' which is contained within this thesis.; Aurélien Mazurie, Monika Tokmina-Lukaszewska, Greg J. Beilman and Brian Bothner were co-authors of the article, 'Application of support vector machines to metabolomics experiments with limited replicates' in the journal 'Metabolomics' which is contained within this thesis.; Brigit Noon, Mohammad J. Mohigmi, Aurélien Mazurie, David L. Dickensheets and Brian Bothner were co-authors of the article, 'Real-time digitization of metabolomic patterns from a living system using mass spectrometry' submitted to the journal 'Journal of the American Chemical Society' which is contained within this thesis.One of the goals of systems biology is to develop a model which encapsulates the molecular, structural and temporal complexity of a living organism. While modern omics experiments can deliver a high resolution view of an organism's molecular complexity, methods for correlating the information from multiple biomolecular systems (i.e. genes, proteins and metabolites) and their changes over time remain greatly underdeveloped. Presented in this research are: (1) methods for understanding the inter-relation of multiple biomolecular systems correlating genomics, proteomics and metabolomics experiments; (2) techniques for machine learning based metabolic biomarker selection; (3) robotics technology for real-time measurement of changes in metabolism. The methods for correlating information from multiple biomolecular systems have provided a new perspective of biomolecular adaptation and evolutionary relationships in the thermophilic archaea. The techniques for biomarker selection have provided a method to assess the reliability of biomarkers in experiments where limited samples are available. The new technology has provided an engineered system for automated analysis of metabolic patterns and how they change over time. Together, these results have created a framework for future improvement of our understanding of biology through the use of molecular biology, machine learning and robotics.Item Mineralogical and geochemical characterization of two Sulfolobales harboring hot spring systems : Rabbit Creek and Ragged Hills, Yellowstone National Park, WY, USA(Montana State University - Bozeman, College of Letters & Science, 2003) Hanna, Braden ThomasItem 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 X-ray crystallographic studies of sulfolobus turetted icosahedral virus (STIV) : a hyperthermophilic virus from Yellowstone National Park(Montana State University - Bozeman, College of Letters & Science, 2006) Larson, Eric Thomas; Chairperson, Graduate Committee: C. Martin LawrenceSulfolobus turreted icosahedral virus (STIV) was isolated from acidic hot springs of Yellowstone National Park and was the first hyperthermophilic virus described with icosahedral capsid architecture. Structural analysis of the STIV particle and its major capsid protein suggests that it belongs to a lineage of viruses that predates the division of the three domains of life. Functional predictions of the viral proteins are hindered because they lack similarity to sequences of known function. Protein structure, however, may suggest functional relationships that are not apparent from the sequence. Thus, we have initiated crystallographic studies of STIV and expect to gain functional insight into its proteins while illuminating the viral life cycle. These studies may also provide genetic, biochemical, and evolutionary insight into its thermoacidophilic host and the requirements for life in these harsh environments. The first three proteins studied in structural detail are A197, B116, and F93. As anticipated, these structures suggest possible functions. The structure of A197 reveals a glycosyltransferase GT-A fold.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 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.