Theses and Dissertations at Montana State University (MSU)
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Item The biofilm matrix in sulfate-reducing bacterial biofilms: potential roles for electron mediators and large proteins(Montana State University - Bozeman, College of Letters & Science, 2019) Krantz, Gregory Peter; Chairperson, Graduate Committee: Matthew Fields; Kilean Lucas, Erica L.-Wunderlich, Linh T. Hoang, Recep Avci, Gary Siuzdak and Matthew W. Fields were co-authors of the article, 'Bulk phase resource ratio alters carbon steel corrosion rates and endogenously produced extracellular electron transfer mediators in a sulfate-reducing biofilm' in the journal 'Biofouling' which is contained within this dissertation.; Peter J. Walian, Marty Boyl-Davis, Kara De Leon, Judy D. Wall and Matthew W. Fields were co-authors of the article, 'Large extracellular proteins sense hydrodynamic force and drive biofilm formation in Desulfovibrio vulgaris' which is contained within this dissertation.; Marty Boyl-Davis, Kara De Leon, Judy D. Wall and Matthew W. Fields were co-authors of the article, 'Characterization of extracellular biofilm mutants cultivated on 1018 carbon steel in Desulfovibrio vulgaris Hildenborough' which is contained within this dissertation.Sulfate-reducing bacteria grow and form biofilms in soil and benthic environments across much of the Earth's surface. Formation of these prevalent biofilms requires the secretion of an extracellular polymeric substance (EPS) to allow the cells to stick together, as well as adhere to a surface. The specific interactions that occur between EPS components of an SRB biofilm are poorly understood. The data presented in this dissertation suggest the presence of two extracellular mechanisms utilized in these communities. The first mechanism was observed in a study altering the lactate (electron donor) and sulfate (electron acceptor) ratios to create limiting nutrient conditions in Desulfovibrio alaskensis G20 (G20) biofilms. G20 was grown under two conditions: electron donor limited (EDL) and electron acceptor limited (EAL) conditions. When grown on a 1018 carbon steel substrate, the G20 consumes all of the available lactate, and once limited, it turns to the high energy electrons in the Fe 0 for growth. Corrosion rates in the steel increased two fold compared to the EAL condition. Global metabolomic analysis revealed increased lumichrome levels under the EDL condition, which suggested higher flux through the riboflavin/FAD biosynthetic pathway. Previous research showed that synthetically adding riboflavin and FAD increases the corrosion rate of a SRB biofilm on 1018 carbon steel, and paired with these results, suggest G20 produces a flavin-based extracellular electron transfer molecule endogenously, and uses it to harvest high energy electrons from Fe 0 when limited for electron donor. The second mechanism was observed in Desulfovibrio vulgaris Hildenborough (DvH) biofilms grown on glass. Two proteins, DVU1012 and DVU1545 were found to be the most abundant extracellular peptides in a DvH biofilm. Single deletion strains for these proteins grew biofilm similar to the wild type strain, but a double deletion strain had decreased ability to form biofilm, demonstrating that at least one of the peptides must be present in order to form a biofilm. Exposure to increased shear force caused an large increase in wild-type biofilm biomass, yet eliminated the double mutant biofilm. These proteins are required for a DvH biofilm to respond to shear force.Item Understanding physiological adaptations, metabolic potential and ecology in a novel photoautotrophic alga for biofuel production(Montana State University - Bozeman, College of Letters & Science, 2019) Corredor Arias, Luisa Fernanda; Chairperson, Graduate Committee: Matthew Fields; Elliot B. Barnhart, Al Parker, Robin Gerlach and Matthew W. Fields were co-authors of the article, 'Impact of temperature, nitrate concentration, PH and bicarbonate addition on biomass and lipid accumulation of a sporulating green alga' which is contained within this dissertation.; Thiru Ramaraj, Huyen Bui, Mensur Dlakic, Robin Gerlach and Matthew W. Fields were co-authors of the article, 'Genomic insights into a sporulating, non-motile, oligotrophic green microalga (PW95)' which is contained within this dissertation.; Huyen Bui, Thiru Ramaraj, Robin Gerlach and Matthew W. Fields were co-authors of the article, 'Transcriptomic profiling of Chlamydomonas-like PW95 cultivated in coal bed methane production water with the native microbial community' which is contained within this dissertation.; Anna J. Zelaya, Robin Gerlach and Matthew W. Fields were co-authors of the article, 'Associations between sympatric bacterial groups and a novel green alga cultivated in coal bed methane production water' which is contained within this dissertation.Commercial implementation of microalgal biomass as bio-oil/chemical feedstocks has been difficult to achieve, and challenges include water/nutrient sources, CO 2 delivery, and community dynamics of mixed cultures. We employed an integrated approach to the study of microalgal production systems to advance towards sustainable implementation of industrial microalgal biofuel production using a native alga (Chlamydomonas-like alga, PW95) isolated from Coal Bed Methane (CBM) production water. Our approach was based on the evaluation of PW95 physiological responses to combinations of growth constraints, the determination of its genomic and functional potential, phylogenetic relations and the implementation of an ecosystem view to algal biomass production. PW95 growth and lipid accumulation (biofuel potential) were ascertained in standardized media and CBM water through the evaluation of mixed effects of temperatures, nitrate levels, pH, and bicarbonate to elucidate interactions between multiple environmental variables and nutritional levels. The biofuel potential of PW95 ranges between 20-32% depending on culture conditions and our results suggest an important interaction between low nitrate levels, high temperature, and elevated pH for trade-offs between biomass and lipid production in the alga. Whole genome sequence was employed to predict biological and metabolic capacity in PW95, and the expression of these capabilities during growth in CBM water with the native microbial consortia was evaluated using RNA sequencing. genome determination and assembly resulted in a draft genome size of 92 Mbp with 14,000 genes predicted and 402 pathways mapped in the KEGG database. The gene complement of PW95 provided a glance into life in an oligotrophic environment (CBM water) and evidence of essential metabolic pathways for cell growth, survival and maintenance, also relevant for cultivation and value-added products generation. Microbial composition and shifts during growth were identified, as well as the algal phycosome. During growth in CBM water, PW95 appeared to be supported by a native microbial consortium and differential expression analysis showed basic metabolic functions and adaptive physiological responses. Our findings build on previous knowledge for improved algal culturing for biomass and industry-valued products while exploring the biology of an organism with relevant impact in energy and water resource management.Item Initiation and pathogenesis of Staphylococcus aureus Pneumonia following influenza A infection(Montana State University - Bozeman, College of Letters & Science, 2019) Borgogna, Timothy Ryan; Chairperson, Graduate Committee: Jovanka Voyich-Kane; Adrian Sanchez-Gonzalez, Kelly Gorham and Jovanka M. Voyich were co-authors of the article, 'A precise pathogen delivery and recovery system for murine models of secondary bacterial pneumonia' in the journal 'JOVE Journal of visualized experiments' which is contained within this dissertation.; Bennett Hisey, Emily Heitman, Joshua J. Obar, Nicole Meissner and Jovanka M. Voyich were co-authors of the article, 'Secondary bacterial pneumonia by Staphylococcus aureus following influenza A infection is saeR/S dependent' in the journal 'Journal of infectious diseases' which is contained within this dissertation.; Madison M. Collins, Kyle A. Glose, Kyler B. Pallister, Tyler K. Pallister and Jovanka M. Voyich were co-authors of the article, 'Uncovering the executioner: disruption of pulmonary surfactant by influenza A triggers Staphylococcus aureus Pneumonia' which is contained within this dissertation.Infection influenza A virus (IAV) leads to increased host susceptibility to secondary bacterial pneumonia. In cases such as these, Staphylococcus aureus (S. aureus) has emerged as the dominant bacterial pathogen associated with severe infection outcomes. S. aureus is a common commensal of the anterior nares and is frequently trafficked into the lower respiratory tract through inhalations, micro-aspirations, and direct mucosal dispersion. Despite recurrent exposure to the lungs and the capacity to cause severe disease, cases of S. aureus pneumonia are rare in immunocompetent hosts. Previous efforts interrogating S. aureus secondary bacterial pneumonia have largely focused on the immunomodulation that occurs during the antecedent influenza infection and have ignored the virulence contributions of the bacterial pathogen. To that end, we developed a murine model of secondary pneumonia to investigate S. aureus pathogenesis following influenza A infection. We identify that secondary bacterial pneumonia by S. aureus is dependent on the activation of the two-component regulatory system (TCS) SaeR/S. Further, studies demonstrated that in the absence of IAV infection the healthy lung environment suppresses virulence gene expression. Characterization of the lung environment revealed that the lipid constituents of pulmonary surfactant suppress S. aureus virulence production. Our data provide a model of secondary bacterial pneumonia wherein infection with IAV significantly reduces surfactant lipid concentrations within the lungs. The reduction of pulmonary surfactant lipids leads to a loss of S. aureus virulence suppression and rapid activation of the major virulence regulator saeR/S. Taken together, these data provide a strong rational for the low incidence of primary S. aureus pneumonia and the increased severity of S. aureus pneumonia following antecedent influenza A infection. Furthermore, these data highlight possible pulmonary surfactant replacement therapies that may significantly alleviate secondary bacterial pneumonia morbidity and mortality.Item Bacterial and archaeal community diversity in relation to organic carbon consumption and sulfate gradients in the Powder River Basin(Montana State University - Bozeman, College of Letters & Science, 2019) Schweitzer, Hannah Doris; Chairperson, Graduate Committee: Matthew Fields and Sara Branco (co-chair); Elliott Barnhart, Al Cunningham and Matthew Fields were co-authors of the article, 'Comparison of attached and planktonic microbial assemblages across geochemically distinct coal seam habitats' submitted to the journal 'International journal of coal geology' which is contained within this dissertation.; Daniel Ritter, Jennifer McIntosh, Elliott Barnhart, Al B. Cunningham, David Vinson, William Orem and Matthew Fields were co-authors of the article, 'Changes in microbial communities and associated water and geochemistry across a sulfate gradient in coal beds: Powder River Basin, USA' submitted to the journal 'Geochimica et cosmochimica acta' which is contained within this dissertation.; Heidi J. Smith was an author and Elliott P. Barnhart, William Orem, Robin Gerlach and Matthew W. Fields were co-authors of the article, 'Linking organic matter degradation and microbial assemblage composition to subsurface methane production in the Powder River Basin' submitted to the journal 'Applied and environmental microbiology' which is contained within this dissertation.; Heidi J. Smith, Elliott P. Barnhart, Boris Wawrik, Amy Callaghan, Luke McKay, Robin Gerlach and Matthew W. Fields were co-authors of the article, 'Metagenomic analysis of recalcitrant rich coal seams from coal seams with varying sulfate concentrations' submitted to the journal 'Applied and environmental microbiology' which is contained within this dissertation.The rate limiting step in biogenic coal bed methane production has been attributed to the predominantly recalcitrant composition of coal, making it difficult for bacteria to anaerobically break down into methanogenic substrates. The significance of different carbon (C) cycling pathways involved in the turnover of recalcitrant, terrestrial C under various redox conditions is still a topic of debate, and in fact, unknown C cycling metabolic pathways are still being discovered in sub-oxic and anoxic environments. Redox transitions exist along gradients of increasingly recalcitrant C in many environments, and subsurface environments represent a large reservoir of C. The Powder River Basin in southeastern Montana is a model environment for studying in situ redox gradients for terrestrial subsurface C and were selected to investigate i) the temporal and spatial variation in the microbial assemblage from four different coal seams with varying depth profiles, ii) the physicochemical controls that impact the turnover of recalcitrant coal to methane, and iii) the functional potential for hydrocarbon degradation under different sulfate concentrations. Similar to the methane-sulfate critical zone in marine habitats, the presented work highlights the crucial role sulfate has on microbial assemblages, methane production, and C consumption in shallow coal seams. Given the accepted differences between groundwater and surface-associated communities of subsurface porous media, diffusive microbial samplers packed with native coal material were used to enhance the establishment of microbial communities that better re-capitulated in situ communities. The microbial community inhabiting low sulfate coal seams consisted of sequences indicative of syntrophic bacteria such as Syntrophomonas and Hydrogenophaga which have previously demonstrated degradation of polycyclic aromatic hydrocarbons (PAH) and coupled growth with hydrogenotrophic methanogens. The assemblages inhabiting high sulfate coal seams were comprised of methylotrophic methanogens and sulfate reducing bacteria. Methylotrophic methanogens are observed in methane producing coal seams that have intermediate levels of sulfate, suggesting an important transition role in early stage methanogenesis. Low sulfate microcosms experienced an increase in humic-like material and consumed more C compared to high sulfate conditions that demonstrated changes in more labile C, including amino acid-like molecules. Moreover, we used a highly curated anaerobic and aerobic hydrocarbon degradation (AnHyDeg and AromaDeg) and redox (nitrogen, sulfur, methane cycle) gene database and pipeline to analyze metagenomic samples that were obtained from three different coal beds that had increasing sulfate levels. While the functional potential for methanogenesis (mcrA) was detected in all metagenomes, the diversity and relative quantity of these genes was greater in the coal beds that contained methane. Of interest was a significantly greater percentage of aerobic hydrocarbon degradation genes (dioxygenases) from one of the methane-containing coal bed samples. These metabolic markers were identified in co-assembled metagenomes. These results provide an enhanced understanding of recalcitrant carbon turnover in the terrestrial subsurface under different redox conditions and the presumptive metabolic capacities involved in subsurface C turnover in relationship to biogenic CH4.Item Metabolic interactions and activity partitioning in a methanogenic, interdomain biofilm(Montana State University - Bozeman, College of Letters & Science, 2019) Camilleri, Laura Beth; Chairperson, Graduate Committee: Matthew Fields; Kristopher A. Hunt, Aurelien Mazurie, Jennifer Kuehl, Alex Michaud, James Connolly, Egan Lohman, Zack Miller, Adam M. Deutschbauer and Matthew W. Fields were co-authors of the article, 'Differential gene expression of a bacterial-archaeal interdomain biofilm producing methane' submitted to the journal 'Biofilms' which is contained within this dissertation.; B.P. Bowen, C.J. Petzold, T.R. Northen and M.W. Fields were co-authors of the article, 'Activity partitioning in an archaeal-bacterial biofilm' submitted to the journal 'Letters in applied microbiology' which is contained within this dissertation.; Matthew W. Fields was a co-author of the article, 'Methanococcus maripaludis factor causes slowed growth in Desulfovibrio vulgaris Hildenborough' submitted to the journal 'Letters in applied microbiology' which is contained within this dissertation.; Matthew W. Fields was a co-author of the article, 'Growth effects of sulfopyruvate and sulfoacetate on the sulfate-reducing bacterium, Desulfovibrio vulgaris Hildenborough, and the methanogenic archaeon Methanococcus maripaludis S2' submitted to the journal 'Scientific reports' which is contained within this dissertation.; Matthew W. Fields was a co-author of the article, 'Methane production in Pelosinus fermentans JBW45' submitted to the journal 'Letters in applied microbiology' which is contained within this dissertation.Biofilms are an ancient survival strategy in which communities of organisms can grow as a cohesive unit, generally attached to a surface and/or at interfaces. Despite the paradigm that 99% of microorganisms grow as a biofilm in the environment, current research methods are largely limited to monoculture planktonic studies. Although more investigations are trying to improve culture complexity by evaluating interactions between two or more populations, experiments are still more readily performed with microorganisms in the planktonic growth mode. The research presented here aims to elucidate the complexity of interactions between two microorganisms from different domains of life that results in enhanced metabolism due to localization of cells in close proximity within an anaerobic biofilm. Desulfovibrio vulgaris Hildenborough (DvH) and Methanococcus maripaludis S2 (Mmp) form a syntrophic mutualism when grown in sulfate-limited media that requires electron flux from DvH to Mmp through what is commonly assumed to be interspecies hydrogen transfer, thereby establishing cross-feeding. The biofilm has been shown to promote a stable and more even carrying capacity for both populations that is likely linked to improved hydrogen transfer (and/or other potential carbon and electron co-metabolites) as compared to planktonic populations. Transcriptomic and proteomic analyses, utilizing RNA-seq and deuterated water respectively, were used to elucidate genes and proteins that contribute to the biofilm growth mode that results in a more efficient metabolism for the syntrophic co-culture (defined by biomass per substrate flux). The results demonstrate the expression of many genes with unknown functions, and others that contribute to cell-cell interactions as well as active proteins in electron processing (e.g., lactate oxidation) in DvH and CO2 reduction (e.g., methanogenesis) in Mmp. A metabolic model of the coculture provided reinforcement for transcriptomic assumptions and aided in the identification of a sulfonate and other amino acids as important syntrophic metabolites. Assessment of biofilm co-culture activity utilizing a new method, Biorthogonal Noncanonical Amino Acid Tagging (BONCAT), showed Mmp was less active in the uptake of a methionine analog as compared to DvH. Alternate assessments confirmed that Mmp was in fact active (based upon methane generation) although translational activity was below the detection limit. Further investigation of the system under sulfate stress showed that the metabolic pairing is more stable than previously thought and could indicate survival strategies that drive the seemingly 'mutualistic' relationship as a forced cooperation. The sulfate stress response coincided with observed lags in DvH growth when grown in Mmp spent medium that was associated with a decoupling of lactate-oxidation and sulfate-reduction. Together the results demonstrate metabolic interactions and activity partitioning within a methanogenic archaeal-bacterial biofilm. The dogma of mutualism being synonymous with equal reciprocity is challenged as it pertains to this model biofilm system. Moreover, this unique bacterial-archaeal biofilm represents interdomain interactions that could represent systems that contributed shared metabolic processes that lead to the development of eukaryotic life.Item Virus host interactions at the single cell level in hot springs of Yellowstone National Park(Montana State University - Bozeman, College of Letters & Science, 2019) Munson-McGee, Jacob Hampton; Chairperson, Graduate Committee: Mark J. Young; Jamie C. Snyder and Mark J. Young were co-authors of the article, 'Introduction to archaeal viruses' in the journal 'Genes' which is contained within this dissertation.; Ross Hartman was an author and Mark J. Young were co-authors of the article, 'vFish for the quantification of viral infection in natural environments' submitted to the journal 'Environmental microbiology' which is contained within this dissertation.; Erin K. Field, Mary Bateson, Colleen Rooney, Ramunas Stepanauskas and Mark J. Young were co-authors of the article, 'The identification and characterization of a nanoarchaeota, its cellular host and a nanoarchaeal virus across Yellowstone National Park hot springs' which is contained within this dissertation.; Colleen Rooney and Mark J. Young were co-authors of the article, 'An uncultivated virus infecting a nanoarchaeal parasite in the hot springs of Yellowstone National Park' submitted to the journal 'Virology' which is contained within this dissertation.; Shengyun Peng, Samantha Dewerff, Ramunas Stepanauskas, Rachel J. Whitaker, Joshua Weitz and Mark J. Young were co-authors of the article, 'A virus or more in (nearly) every cell: ubiquitous networks of virus-host interactions in extreme environments' in the journal 'The ISME journal' which is contained within this dissertation.Viruses are the most abundant biological entities on the planet and virus-host interactions are some of the most important factors in shaping microbial community structure and function and global chemical cycling. The high temperature low pH hot spring of Yellowstone National Park contain simplified microbial communities of 8-10 Archaeal species, and comparatively simple viral communities. These idealized communities that contain only viruses and their Archaeal hosts represent a model natural environment for the study of viruses and their hosts. This work presented here builds on previous population level studies of the viral and microbial communities to examine virus-host interactions at the single cell level. The identification of viral infection has long been a scourge of environmental virologist. In order to identify viral infection in natural environments we have adapted Fluorescent in situ hybridization (FISH) techniques to directly identify viral sequences. We further advance this technique to be compatible with flow cytometry analysis for the rapid quantification of viral infection of uncharacterized viruses in natural environments. This technique is used to quantify viral infection of two different viruses, previously only characterized by metagenomic sequencing analysis, in four geographically separate low pH high temperature hot springs of Yellowstone National Park. Finally, we combine viral and cellular metagenomics with cellular transcriptomics and single cell genomics to identify virus host interactions at the single cell level and identify viruses that are replicating in the hot springs. This work suggests that a majority of cells in the hot springs are interacting with viruses and that a majority of the cells are interacting with multiple viruses at any given time. We also identify RNA sequences from a majority of the viral types present in the hot springs suggesting that viral replication is occurring and is an important force in determining the structure and function of the microbial communities in these hot springs. Together these works represent a significant advancement of our understanding of virus host interactions in natural environments as well as new techniques to be used in future studies.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 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 Investigation of octopamine-glutamate dual transmission neurons(Montana State University - Bozeman, College of Letters & Science, 2020) McKinney, Hannah Margaret; Chairperson, Graduate Committee: Steven R. Stowers; Lewis Sherer, Jessica L. Williams, Sarah Certel and Steven R. Stowers were co-authors of the article, 'Characterization of drosophila mimic-converted octopamine receptor GAL4 lines' in the journal 'Journal of Comparative Neurology' which is contained within this dissertation.; Dissertation contains a paper of which Hannah Margaret McKinney is not the main author.Dual transmission, or the ability of a neuron to signal with more than one neurotransmitter, is now a well-established phenomenon in the field of neuroscience. However, many questions about this type of signaling process still remain with regards to its mechanisms and its impacts on neural circuitry and organism behavior. In particular, the mode of neurotransmitter release from synaptic vesicles can have significant profoundly affects elements on neural circuitry and, subsequently, on behaviors of an organism. In Drosophila melanogaster, a particular subset of neurons important for the behaviors of courtship and aggression signal with the neuromodulator octopamine and the excitatory neurotransmitter glutamate. Whether these two neurotransmitters are released simultaneously (co-release) or are housed for separate synaptic release (cotransmission) is unknown. The mechanism of release for these neurotransmitters in this population of neurons is investigated here through the development of synaptic vesicle visualization tools, synaptic vesicle isolation, and an examination of the expression of octopamine and glutamate receptors; I explored the hypothesis that receptor expression downstream of dual transmitting neurons will provide information about the co-release or co-transmission of octopamine and glutamate. Results from these experiments demonstrated release of octopamine and glutamate from the same synaptic site, with some variation, and a significant amount of presynaptic receptor expression. The results indicate these dual transmission neurons may release octopamine and glutamate at the same synapse for both post-synaptic signaling as well as pre-synaptic signal modulation.Item Temporal dynamics of Escherichia coli and the microbiome(Montana State University - Bozeman, College of Letters & Science, 2020) Martinson, Jonathan Nathan Vernon; Chairperson, Graduate Committee: Seth Walk; Seth T. Walk was a co-author of the article, 'Escherichia coli residency in the gut of healthy human adults' submitted to the journal 'EcoSal plus' which is contained within this dissertation.; Nicholas V. Pinkham, Garrett W. Peters, Hanybul Cho, Jeremy Heng, Mychiel Rauch, Susan C. Broadaway and Seth T. Walk were co-authors of the article, 'Rethinking gut microbiome residency and the enterobacteriaceae in healthy human adults' in the journal 'The ISME journal' which is contained within this dissertation.; Nicholas V. Pinkham and Seth T. Walk were co-authors of the article, 'Phenotypic predictors of Escherichia coli residency in the gut of healthy human adults' submitted to the journal 'Applied and environmental microbiology' which is contained within this dissertation.Over the past two decades, our understanding of the gut microbiome has increased dramatically. However, most studies involving healthy adults have relied almost exclusively on cross-sectional design, negating the changes occurring within an individual's microbiome through time. With this, we performed a small longitudinal study over a period of ~2 years with a cohort of 8 healthy adults. By sequencing the DNA encoding the 16S ribosomal RNA gene, we assessed the community level change in this cohort through time. Similar to previous findings, we found that using these methods there was remarkable stability through time with nearly 50% of the microbiome remaining the same throughout the study period in the participants. However, analysis of 16S ribosomal RNA sequences limits taxonomic resolution. By cultivating members of the Enterobacteriaceae, we found that turnover at the clone-level (below the species level) was common. Within the Enterobacteriaceae, Escherichia coli was the most numerically dominant species and most often observed as a long-term member of the gut (i.e. resident). Longitudinal analysis of Escherichia coli revealed that some phylogenetic groups within the species are more often long-term residents than other phylogroups. We next assessed the means by which the resident E. coli were capable of establishing and maintaining themselves in the gut. We found that residents were much more likely to produce antagonism (inhibition of other clones) than the E. coli that did not reside in the gut long-term.