Theses and Dissertations at Montana State University (MSU)
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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 Microbial ecology of mosquitos and ticks(Montana State University - Bozeman, College of Letters & Science, 2017) Pinkham, Nicholas Vernon; Chairperson, Graduate Committee: Seth WalkHost evolutionary history has been shown to select for distinct host associated microbial communities over large evolutionary time scales. The microbiomes of disease vector have been shown to alter the capacity of their host to vector pathogens. Much remains to be understood about how the microbiome of mosquitos and ticks assemble in situ. We conducted a large-scale investigation of microbiome composition between mosquito species as well as a second investigation of microbiomes of brown dog ticks collected in Iquitos, Peru. Intraspecific and interspecific bacterial community diversity was compared across 26 species of mosquitoes collected in Montana. Previous studies of lab reared mosquitoes report greater variation in microbial communities between species than within. Using 16S rRNA sequencing we observed a large amount of intraspecific variation in microbiomes, as well as different species hosting very similar microbiomes. The tick microbiome was found to be dominated by a few select community members that were seen at an extremely high abundance and resembled intracellular tick-borne pathogens. It is common for ticks to host endosymbionts that closely to human pathogens but are not pathogenic themselves. Negative interactions were seen between the most abundant organism observed in the ticks.Item Microbial interactions and the role of environmental stress in natural and synthetic consortia(Montana State University - Bozeman, College of Letters & Science, 2018) Beck, Ashley Esther; Chairperson, Graduate Committee: Ross Carlson; Kristopher A. Hunt, Hans C. Bernstein and Ross P. Carlson were co-authors of the chapter, 'Interpreting and designing microbial communities for bioprocess applications, from components to interactions to emergent poperties' in the book 'Biotechnology for biofuel production and optimization' which is contained within this thesis.; Kristopher A. Hunt and Ross P. Carlson were co-authors of the article, 'Measuring cellular biomass composition for computational biology applications' submitted to the journal 'Processes, methods in computational biology special issue' which is contained within this thesis.; Hans C. Bernstein, and Ross P. Carlson were co-authors of the article, 'Stoichiometric network analysis of cyanobacterial acclimation to photosynthesis-associated stresses identifies heterotrophic niches' in the journal 'Processes, microbial community modeling: prediction of microbial interactions and community dynamics special issue' which is contained within this thesis.; Kathryn Pintar, Diana Schepens, Ashley Schrammeck, Tim Johnson, Alissa Bleem, Hans C. Bernstein, Tomas Gedeon, Jeffrey J. Heys and Ross P. Carlson were co-authors of the article, 'Escherichia coli co-metabolizes glucose and lactate for enhanced growth' submitted to the journal 'Applied and Environmental Microbiology' which is contained within this thesis.; Ross P. Carlson was a co-author of the article, 'Synthetic consortia engineered for push and pull dynamics show conditional optimality over metabolic generalist' which is contained within this thesis.Microbial communities are critical underpinnings of most natural processes, e.g. biogeochemical cycling, and can also be harnessed and engineered for a variety of industrial applications. Despite the abundance of detailed physiological characterization of many individual microorganisms, as well as large data sets describing microbial community composition, the area of interspecies interactions requires further research to truly appreciate and harness the potential of microbial capabilities. Using a combination of in silico metabolic modeling and in vitro laboratory approaches linked to guiding ecological theories, this dissertation investigates metabolite exchange as a mechanism of interspecies interactions and focuses on the role of environmental stress in mediating interactions. A stoichiometric metabolic network model was constructed for the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 and was analyzed with elementary flux mode analysis to predict metabolic acclimations to light and oxygen, two common environmental stressors in photoautotrophic habitats. High stress levels were predicted to activate organic byproduct secretion pathways, which opens a niche to support growth of heterotrophic partners. To further investigate metabolite exchange in the laboratory, synthetic consortia were designed through genetic engineering and pairing of Escherichia coli strains to form metabolically partitioned organic acid cross-feeding systems. These controlled systems were used to investigate the impact of division of labor as well as the effect of byproduct detoxification. Kinetic data from these systems were also applied to interpret ecological theories regarding microbial community structure. Altogether, these studies demonstrate an integrated approach to studying microbial community interactions by combining in silico metabolic modeling and in vitro laboratory experiments with ecological theory as a basis for interpretation. This dissertation provides insight into rationale for microbial community structure and highlights the role of environmental stress, particularly byproduct inhibition, in driving microbial consortia interactions.Item Bacteriophage in host associated microbial communities examined with continuous culture systems(Montana State University - Bozeman, College of Letters & Science, 2018) Dills, Michael Stefan; Co-Chairs, Graduate Committee: Mark J. Young and Seth WalkMechanistic understanding of the role of extracellular and parasitic elements in host ecosystems is currently lacking. Extensive surveys have catalogued a large diversity of bacteriophage which associate differentially with definable host states. This work is an attempt to aid in the development of a coherent model for complex symbiosis within mammalian host ecosystems by investigating the role of bacteriophage in microbial community structure. It details an investigation of continuous culture systems as a platform to study bacteriophage within polymicrobial communities of the human GI tract. It then describes an experiment testing an extracellular community's ability to modulate bacterial community structure.Item Microorganisms at the intersection of hydrology and CO 2 efflux in subalpine soils(Montana State University - Bozeman, College of Letters & Science, 2018) Anderson, Erik Charles; Chairperson, Graduate Committee: Eric BoydSubalpine forests are responsible for a substantial fraction of carbon (C) cycling in the western United States, with over 70% of the C sink activity taking place at elevations exceeding 750 meters. Soil microbial communities are key drivers of C cycling in these ecosystems, yet, factors that influence the composition of these communities and their activities across these heterogeneous subalpine landscapes are not well understood. Ten geographically distinct coniferous forest watersheds across western Montana were subjected to characterization of soil properties, carbon dioxide (CO 2) efflux, and community composition to determine the influence of heterogeneity in these watersheds on these properties. Moist, alkaline riparian soils had a higher net CO 2 efflux than drier, more acidic upland soils; soil temperature had no detectable effect on CO 2 efflux. The composition of microbial communities was also significantly correlated to variations in soil moisture content and pH. Dominant bacterial phyla in riparian soils were Proteobacteria while those in upland soils were Acidobacteria, suggesting that these components of these respective soil communities are at least partially responsible for variations in CO 2 efflux. Together, these data suggest that patchiness in subalpine soil properties within a watershed drive variation in the composition of soil microbial communities and their C cycling activities.Item In situ and enhanced coal-bed methane production from the Powder River Basin(Montana State University - Bozeman, College of Letters & Science, 2014) Barnhart, Elliott Paul; Chairperson, Graduate Committee: Matthew Fields; Kara Bowen De León, Bradley D. Ramsay, Alfred B. Cunningham, and Matthew W. Fields were co-authors of the article, 'Investigation of coal-associated bacterial and archaeal populations from a diffusive microbial sampler (DMS)' in the journal 'International journal of coal geology' which is contained within this thesis.; Bradley D. Ramsay, Kara Bowen De León, Kristen A. Brileya, Denise M. Akob, Richard E. Macur, Alfred B. Cunningham, Matthew W. Fields were co-authors of the article, 'Stimulation of coal-dependent methanogenesis with native microbial consortia from the Powder River Basin' submitted to the journal 'Applied and environmental microbiology' which is contained within this thesis.; Kiki Johnson, Kristopher A. Hunt, Sean Cleveland, Marcella A. McClure, Matthew W. Fields were co-authors of the article, 'Genomic insight into the evolution of the acetate switch in archaea' submitted to the journal 'Nature' which is contained within this thesis.The majority of the coal in the Powder River Basin (PRB) is located in formations too deep to be economically mined but microorganisms within some of these deep coal seams generate coal-bed methane (CBM) which can be harvested and utilized as an energy source. However, little is known about the in situ microbial community, the environmental conditions conducive to CBM production, or the microbial community interactions that promote CBM production. Several sampling locations within the PRB were identified as methane-producing sites based on geochemical analysis of groundwater. A diffusive microbial sampler (DMS) was utilized for microbial sampling which was loaded with coal and only opened at the bottom of the wells where the coal seam was exposed. Pyrotag analysis of DMS coal identified the predominant in situ bacterial and archaeal populations, providing insight into microbes generating CBM within the PRB. Changes in the composition and structure of microbial communities that occur under stimulated conditions were investigated by applying molecular methods in combination with cultivation techniques (with and without nutrient supplementation) to identify conditions which maximize methane production in batch, bench-scale incubations. Results from these studies indicated the addition of yeast extract resulted in an increase in methane production as well as a shift to a microbial population capable of acetate production and/or acetate utilization. Isolation methods targeting coal utilizing Bacteria and methanogenic Archaea were applied in addition to DNA based methods to infer microbial community members present within coalbeds. The acetoclastic methanogen Methanosarcina was isolated which is the only identified methanogen with the high-efficiency acetate kinase (Ack) / phosphotransacetylase (Pta) methane production pathway. This pathway provides increased growth and methane production when acetate concentrations are high which can result from microbial stimulation with nutrients. Genomic analysis revealed Ack evolved through gene duplication and divergence of acetyl CoA synthetase within the methanogenic genome. This research provided novel insight into the evolution of the high-efficiency Ack/Pta pathway. Collectively, this dissertation presents a novel link between the Ack/Pta pathway, stimulated CBM production and genomic insight into the development of this pathway.Item Physiological heterogeneity and starvation in mature Pseudomonas aeruginosa biofilms(Montana State University - Bozeman, College of Agriculture, 1999) Xu, Dongxin KarenItem Selective cloning of 16S rRNA molecules to describe naturally occurring microorganisms(Montana State University - Bozeman, College of Agriculture, 1990) Weller, RolandItem The fate of fermentation products and glycollate in hot spring microbial mats with emphasis on the role played by Chloroflexus aurantiacus(Montana State University - Bozeman, College of Agriculture, 1983) Tayne, Timothy AldenItem Varied physiological responses of the facultative gamma-proteobacterium, Shewanella oneidensis MR-1, and the delta-proteobacterium Desulfovibrio vulgaris hildenborough to oxygen(Montana State University - Bozeman, College of Letters & Science, 2011) Sundararajan, Anitha; Chairperson, Graduate Committee: Matthew Fields; Jacob Kurowski, TingFen Yan, Dawn. M. Klingeman, Marcin. P. Joachimiak, Jizhong Zhou, Jeff. D. Gralnick, Belen Naranjo and Matthew. W. Fields were co-authors of the article, 'A Shewanella oneidensis MR-1 sensory box 1 protein involved in aerobic and anoxic growth' in the journal 'Applied and environmental microbiology' which is contained within this thesis.Evolution of molecular oxygen and accumulation in Earth's atmosphere is considered to be the one of the most significant changes on Earth that impacted the evolution of life. Over the past 600 million years, there have been fluctuations in atmospheric oxygen concentrations that have driven the evolution of species in all three domains of life. Over the years, microbes and other life acquired different strategies to survive efficiently in the presence of oxygen through mutagenic evolutionary mechanisms. This dissertation demonstrates how a facultative bacterium, Shewanella oneidenisis MR-1, through signaling mechanisms, senses oxygen as an external stimulus and regulates metabolism accordingly. These signaling molecules reside within open reading frames as small domains that sense/transmit signals based on stimulus and subsequently trigger a response within the cell. One such open reading frame, SO3389, containing multiple domains was characterized in the first two chapters of this dissertation. Physiology and genetics based experiments were employed to address hypotheses that a putative sensory-box protein was involved in oxygen sensing. It was elucidated that this protein plays a role in sensing dissolved oxygen (DO) levels that affected both aerobic biofilm formation and transitions to anoxia. While oxygen can be an attractant in aerobic growth mode, it is considered to be toxic to strict anaerobes, such as Desulfovibrio vulgaris Hildenborough, a sulfate-reducing bacterium. Recent studies, however, reveal that even strict anaerobes can tolerate micromolar concentrations of oxygen. These organisms have evolved several protective mechanisms to combat oxidative stress and some may even possess oxygen-utilization machinery. Chapters 3 and 4 address the phenomenon of oxygen tolerance in D.vulgaris planktonic and biofilm cells and the variation in this response based on available carbon and energy sources. Physiology- and genetic-based approaches revealed that D.vulgaris cells grown on pyruvate exhibit increased tolerance towards DO but lactate-grown cells utilized oxygen for energy production at intermediate levels of DO. The substrate-dependent nature of oxygen response in D. vulgaris has not been previously reported, and could impact remediation strategies as well as possible implications for community interactions. The results demonstrated in this dissertation underscore two major findings with respect to oxygen responses: (i) the elucidation of unknown function for a conserved hypothetical protein, and (ii) the substrate-dependent nature of oxygen utilization in a "strict" anaerobe. The elucidation of function for these genes/proteins/organisms will further our fundamental understanding of microbial physiology, of the versatility that allows adaptation to constantly changing environments, and help improve future remediation strategies.