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Item An omics-based interrogation of disparate microbial systems: multi-omics analysis of a bio-mining archaeon and the effects of arsenic on the E. coli Lipidome(Montana State University - Bozeman, College of Letters & Science, 2023) Fausset, Hunter Lee; Chairperson, Graduate Committee: Brian Bothner; This is a manuscript style paper that includes co-authored chapters.Systems biology represents the next frontier in the elucidation of biochemical mechanisms, disease states, and microorganisms. Rather than approaching individual parts of an organism, such as a specific protein, molecule, or mRNA, a systems biology or "omics" investigation seeks to characterize all proteins, molecules, or RNA simultaneously. This is crucial, because all macromolecules in a lifeform exist in dynamic equilibria with those around them; no one biological process occurs in a vacuum. Omics investigations have ballooned in usage over the last decades due to scientists realizing their power in characterizing complex biological phenomena. This has also been spurred on by advances in technologies enabling the robust elucidation of thousands of molecules at once, particularly benefitting from the modernization of mass spectrometry. This technique can be used to study any number of biological problems including those presented here; a multi-omics investigation into a mineral- eating methanogen and a lipidomic characterization of arsenic exposure in a key member of the gut microbiome, E.coli. Methanosarcina barkeri, a widespread methanogen found in marine sediments, is able to reductively dissolve minerals such as pyrite (FeS2) to satisfy their iron and sulfur requirements. Presented here are two investigations containing transcriptomic, proteomic, metabolomic, and lipidomic analyses, performed in parallel on the same biomass. Together, these experiments suggest that the organism undergoes a significant phenotypic shift in response to changes in just two elements, Fe and S. Overall inferences are echoed in the small molecule analyses; the metabolomes and lipidome of the organism change similarly in to the proteome. Key sulfur equilibria are implied in the process, as are specific lipids, choline, and dethiobiotin. A similar approach was applied to E.coli treated with arsenic, as a proxy for understanding the detoxifcation that takes place in the gut microbiome after ingestion. Marked lipidomic changes were observed in E.coli resulting from treatment, which were dependent both on species of arsenic as well as presence of the Ars operon. As a foundational study, this work answered some and generated many more hypotheses on the biochemical fate of As in microorganisms in the gut microbiome.Item Omics approaches identify molecular mechanisms of arsenic-microbial interactions(Montana State University - Bozeman, College of Letters & Science, 2019) Rawle, Rachel Anna; Chairperson, Graduate Committee: Timothy R. McDermott and Brian Bothner (co-chair); Yoon-Suk Kang, Brian Bothner, Gejiao Wang and Timothy R. McDermott were co-authors of the article, 'Transcriptomics analysis defines global cellular response of Agrobacterium tumefaciens 5A to arsenite exposure regulated through the histidine kinases phor and aios' in the journal 'Environmental microbiology' which is contained within this dissertation.; Monika Tokmina-Lukaszewska, Zunji Shi, Brian Tripet, Fang Dang, Timothy R. McDermott, Valerie Copie, Gejiao Wang and Brian Bothner were co-authors of the article, 'Metabolic responses to arsenite exposure regulated through histidine kinases phor and aios in Agrobacterium tumefaciens 5A' submitted to the journal 'Environmental microbiology' which is contained within this dissertation.Arsenic is a class I carcinogen and causes various cancers and diseases. Its toxicity, prevalence, and potential for human exposure has classified arsenic as the number one environmental toxin according to the Environmental Protection Agency. Contamination of groundwater and soil leads to over 200 million human exposures above the health limit. In every environment where arsenic and microbes coexist, microbes are the principal drivers of arsenic speciation, which is directly related to bioavailability, toxicity, and bioaccumulation. These speciation events drive arsenic behavior in the soil, water, and as recent data suggests, human-associated microbiomes. This dissertation details arsenic-microbial interactions through an omics platform, utilizing transcriptomics, metabolomics, and proteomics profiling as a way to globally assess the impacts of arsenic exposure. This work followed two main aims: (1) characterize cell metabolism during arsenic exposure in soil bacterium Agrobacterium tumefaciens 5A, a model organism for arsenite oxidation, and (2) assess the impacts of specific arsenic-processing bacteria within the gut microbiome of mammals. The results of this work provide a foundational understanding for how arsenic speciation events are regulated and how they affect nutrient cycling in environmental systems, which is necessary for bioremediation and health initiatives.Item Investigating arsenic-microbiome interactions in the gut using murine models(Montana State University - Bozeman, College of Letters & Science, 2019) Coryell, Michael Philip; Chairperson, Graduate Committee: Seth Walk; B. A. Roggenbeck and Seth T. Walk were co-authors of the article, 'The human gut microbiome's influence on arsenic toxicity' submitted to the journal 'Current pharmacology reports' which is contained within this thesis.; M. McAlpine, N.V. Pinkham, T.R. McDermott and Seth T. Walk were co-authors of the article, 'The gut microbiome is required for full protection against acute arsenic toxicity in mouse models' in the journal 'Nature communications' which is contained within this thesis.; M. Yoshinaga, T.R. McDermott and Seth T. Walk were co-authors of the article, 'Speciation of excreted arsenicals from germ free and conventional AS3MT knockout mice exposed to inorganic arsenate' which is contained within this thesis.Drinking water contamination with arsenic is a wide-spread public health concern, potentially affecting over 140 million people across at least 40 different countries. Current understanding of biological and behavioral factors influencing clinical outcomes is insufficient to explain the variation observed in arsenic-related disease prevalence and severity. The intestinal microbiome in humans is a dynamic and active ecosystem with demonstrated potential to mediate arsenic metabolism in vitro and distinct variability between individuals. This dissertation investigates arsenic-microbiome interactions, with a focus on determining how microbiome activity influences host-response and toxicity from arsenic exposures. Chapter 2 overviews common exposure routes, important metabolic pathways, and current evidence of arsenic-microbiome interactions in humans or experimental animal models. Chapter 3, the initial approach was to experimentally perturb the microbiome of common laboratory mice during arsenic exposure, measuring arsenic excretion in the stool and accumulation in host tissues. Arsenic sensitive gene-knockout mice were used to determine the microbiome's influence on subacute arsenic-induced mortality. Disrupting microbiome function--first by antibiotic treatment, then by deriving mice germ free--dramatically reduced survival times during severe arsenic exposures. Transplantation of human fecal communities into germ free mice effectively complemented the loss of function from microbiome disruption in these mice. Chapter 4 examines microbiome's impact on arsenic metabolism in germ free and conventional mice from this same arsenic-sensitive genetic background. These mice are deficient for the primary metabolic pathway involved in arsenic detoxification in both humans and mice, facilitating a more complete experimental isolation of microbiome and host metabolisms. This study provides evidence of microbiome-dependent changes in the elimination routes and metabolic transformation of ingested arsenic and provides a new experimental model for studying arsenic metabolism in the gut.Item Introducing the ArsR regulated arsenic stimulon(Montana State University - Bozeman, College of Agriculture, 2017) Saley, Tara Carolyne; Chairperson, Graduate Committee: Timothy McDermottThe United States EPA ranks arsenic as the number one environmental toxin. Since microorganisms are significant drivers of arsenic toxicity and mobility in nature, it is important to understand how microbes detect and react to arsenic. The microbial arsenic resistance operon (ars) is critical for sensing arsenic in the environment and controlling the cellular response to this toxin. The ars operon is minimally comprised of arsRBC, which codes for an ArsR transcriptional repressor, arsenite effluxer, and an arsenate reductase, respectively, with the operon negatively regulated by the transcriptional repressor, ArsR. Our model organism Agrobacterium tumefaciens 5A carries two ars operons, with each containing two arsR genes. We conducted an RNASeq study to examine the regulatory roles of the encoded four ArsR regulatory proteins as a function of +/- arsenite. We report that the regulatory influence of the ArsR proteins extends well beyond the ars operon, with both activation and repression effects. In addition to the expected arsenic resistance response, many cellular functions were impacted, including: phosphate acquisition/metabolism, sugar transport, chemotaxis, copper tolerance, and iron homeostasis. Each of the ArsR proteins uniquely influenced different sets of genes and an arsR regulatory hierarchy was observed, wherein ArsR1 is auto regulatory and negatively regulates arsR4, ArsR4 activates arsR2, and ArsR2 negatively regulates arsR3. ArsR3 is the least active with respect to number of genes regulated. To summarize, this study provides a more complete understanding of how microbial gene expression and biogeochemical cycling may be influenced by arsenic in the environment.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 Arsenite oxidation by a Hydrogenobaculum sp. isolated from Yellowstone National Park(Montana State University - Bozeman, College of Agriculture, 2002) Donahoe-Christiansen, JessicaItem Linking microbial populations and geochemical processes in soils, mine tailings, and geothermal environments(Montana State University - Bozeman, College of Agriculture, 2004) Macur, Richard Eugene; Chairperson, Graduate Committee: William P. Inskeep.The primary goal of this work was to identify and characterize the microbial populations responsible for transformations of As and 2,4-D in soils and waters. Chemical, spectroscopic, and microscopic techniques were used to characterize the aqueous and solid phase geochemistry of soils, mine tailings, and a geothermal spring. The role of specific microbial populations in these systems was examined using cultivation-independent molecular methods [total DNA extraction, 16S rDNA amplification, denaturing gradient gel electrophoresis (DGGE), and sequence analysis] coupled with either characterization of microorganisms isolated from the same systems, or inference of physiological characteristics from (i) closely related (16S rDNA sequence) cultured microorganisms and (ii) the geochemical environments in which they were detected. The microbial reduction of As(V) to As(III) and the subsequent effects on As mobilization in contaminated mine tailings was examined under transport conditions. Enhanced elution of As from mine tailings apparently resulted from the enrichment of aerobic As(V)-reducing Caulobacter leidyi, Sphingomonas yanoikuyae, and Rhizobium loti -like populations after liming. Arsenite was rapidly oxidized to As(V) via microbial activity in unsaturated Madison River Valley soil columns. Eight aerobic heterotrophic bacteria with varying As redox phenotypes were isolated from these columns. Three isolates, identified as Agrobacterium tumefaciens, Pseudomonas fluorescens, and Variovorax paradoxus -like organisms, were As(III) oxidizers and all were apparently important members of the soil microbial community responsible for net As(III) oxidation. Successional changes in microbial communities colonizing an As-rich acid-sulfate-chloride geothermal spring stream channel in Norris Geyser Basin of Yellowstone National Park were examined. Enhanced As(III) oxidation correlated in time and space with the appearance of three Hydrogenobaculum -like populations. The formation of an As(V)-rich hydrous-ferric-oxide mat correlated with the detection of Thiomonas, Acidimicrobium, and Metallosphaera —like populations whose nearest cultivated relatives (based on 16S rDNA sequence) were Fe-oxidizers. Fingerprints of microbial communities (DGGE) established under increasing concentrations of 2,4-D (0 - 500 mg kg'1) in batch soil microcosms showed that at least 100 mg kg'1 2,4-D was required to obtain apparent shifts in community structure. The microbial community selected at high 2,4-D concentrations was predominantly composed of Burkholderia -like populations, which harbored homologs of tfdA genes.Item Photochemical oxidation of arsenic(III) in ferrioxalate solutions and elk exposure to arsenic in Yellowstone's geothermal environments(Montana State University - Bozeman, College of Agriculture, 2002) Kocar, Benjamin DavidItem Uptake and phytotoxicity of arsenic III and V in four grass species(Montana State University - Bozeman, College of Agriculture, 1995) Tice, Stephanie WagnerItem Processes controlling arsenic solubility and mobility in soils(Montana State University - Bozeman, College of Agriculture, 1998) Jones, Clain A.