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Item The influence of an iron deficient diet on the murine gut microbiome(Montana State University - Bozeman, College of Letters & Science, 2019) Coe, Genevieve Lea; Chairperson, Graduate Committee: Jennifer DuBoisIron is an essential nutrient for mammals 1. It is involved in multiple redox reactions that are essential for the survival of most organisms 2. There are two main types of iron that are absorbed from the diet: inorganic iron and heme 3. Dietary iron ingested by mammals is mostly absorbed in the small intestine; however, it is unclear whether the gut microbiome is involved in iron homeostasis or whether iron in the diet influences the microbiome. The goal of this project is to characterize the change in microbial composition in response to iron deficiency and iron repletion in conventional mice and define a baseline model for future studies involving the more complex human gut microbiome.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 Ultrafast photochemistry of aqueous iron(III) complexes(Montana State University - Bozeman, College of Letters & Science, 2017) Danforth, Rebecca Ann; Chairperson, Graduate Committee: Erik Grumstrup; Bern Kohler was a co-author of the article, 'Ultrafast photochemical dynamics of hexaaqua iron(III) ion' in the journal 'Chemical physics letters' which is contained within this thesis.The ultrafast photochemical dynamics of aqueous iron(III) solutions were measured utilizing ultrafast pump probe spectroscopy. Aqueous solutions of iron(III) were prepared at low pH (<4.5) and low iron(III) concentration (<5 mM) to allow for small aquairon(III) complexes and ferrihydrite to be studied. Small monomeric and dimeric aquairon(III) complexes were studied to elucidate the mechanisms involved in the formation of OH ° after UV excitation which were previously known to generate OH ° in vastly different quantities. Upon excitation of Fe 3+, a proton is released from a coordinated water molecule to generate FeOH 2+ in less than 200 fs. The newly generated FeOH 2+ can then undergo numerous recombination pathways to regenerate the Fe 3+. Approximately 10% of the excited Fe 3+ undergoes photoreduction and subsequent release of OH ° and Fe 2+ within 20 ps. Exciting FeOH 2+, results in homolysis to form Fe 2+ and OH ° with a wavelength dependent yield with a lifetime of 20 ps. Fe 2(OH) 2 4+ does not appear to generate significant quantities of OH ° however, the dimer is photostable in comparison to Fe 3+ and FeOH 2+. To further the understanding of the primary kinetics of iron(III) in aqueous solutions, ferrihydrite nanoparticles were studied. Ferrihydrite exhibits similar dynamics to hematite in which electrons are excited into the conduction band of ferrihydrite. The electrons can then relax to the bottom of the conduction band within 390 fs before undergoing various recombination process. This limits the amount of iron(III) converted into iron(II) in ferrihydrite. All iron(III) systems studied show unique kinetics after excitation that elucidate the mechanisms behind the generation of OH °.Item Analysis of the role of iron uptake mechanisms and addition of iron-doped apatite nanoparticles in phage infections in Staphylococcus aureus and Mycobacterium smegmatis(Montana State University - Bozeman, College of Letters & Science, 2018) Rost, Linda Christina; Chairperson, Graduate Committee: Greg FrancisAntibiotic resistance has become a significant global public health issue, and phage therapy could serve as an adjuvant to traditional antibiotics. Phages are viruses that kill bacteria and produce more phages. Iron-doped apatite nanoparticles (IDANPs) have been shown to increase phage killing of bacteria. However, the mechanism of JB and Yodasoda infection of bacteria, and mechanism by which IDANPs increase phage infections, is unknown. Based on the iron composition of the IDANP, as well as extensive literature describing Staphylococcus aureus having aggressive iron uptake systems, it was hypothesized that IDANPs may affect such systems, and thereby be involved in the subsequent increase of phage-mediated bacterial death. In this work, the relationship between bacterial exposure to iron and subsequent phage infectivity was established, and IDANP effect on plaque size was determined. S. aureus cells were grown in various iron treatments, plaque assays were performed. There was a strong, positive relationship between iron treatments and plaque counts. The plaque counts were 29% higher in the 0.0004g/L iron treatment, 34% higher in 0.0008g/L, 60% higher in 0.0016 g/L and 82% higher in 0.0032g/L. When S. aureus and M. smegmatis cells were treated with IDANPs, plaque sizes were significantly larger, which may indicate increased infection in adjacent cells. Plaque sizes from IDANP-treated cells continued to increase in size as plates were incubated over 24, 48 and 96 hours. Plaque sizes also increased in size in the control cells in some time frames. S. aureus cells were also grown in 0.0016g/L iron treatment and treated with IDANPs, and there was a 65% increase in plaque counts. In higher iron treatments, it was difficult to achieve a lawn of cells to perform plaque assays. Cell growth was measured by performing serial dilutions and determining CFU/mL. There was no significant difference between cells grown in M9 minimal media or treated with IDANPs. Cells were also grown in the different iron treatments over three hours, with or without IDANPs. Less growth was observed in high iron treatments, but the differences were not significant. Cell growth was relatively slower in high iron levels in overnight treatments, and the results were significantly different. These data can be used to elucidate the relationship of iron uptake and phage killing, and therefore allow conjectures as to whether or not iron uptake mechanisms may be involved in the IDANP effect. Further research in this field can provide opportunities to develop reliable alternatives to antibiotics to treat bacterial infections.Item Role of the P-cluster and FeMo-cofactors in nitrogenase catalysis(Montana State University - Bozeman, College of Letters & Science, 2017) Keable, Stephen Michael Keable; Chairperson, Graduate Committee: John W. Peters; Andrew J. Rasmussen, Karamatullah Danyal, Brian J. Eilers, Gregory A. Prussia, Axl X. LeVan, Lance C. Seefeldt and John W. Peters were co-authors of the article, 'Three structural states of the nitrogenase P-cluster revealed in MOFE protein structures at poised potentials' submitted to the journal 'Biochemistry' which is contained within this thesis.; Jacopo Vertemara, Karamatullah Danyal, Andrew J. Rasmussen, Brian J. Eilers, Oleg A. Zadvornyy, Luca De Gioia, Giuseppe Zampella, Lance C. Seefeldt and John W. Peters were co-authors of the article, 'Acetylene interaction with the nitrogenase femo-cofactor investigated by structural and computational analysis' submitted to the journal 'Biochemistry' which is contained within this thesis.; Dissertation contains two articles of which Stephen Michael Keable is not the main author.Biological nitrogen fixation has been extensively researched for over four decades, yet due to the complex nature of this process, numerous questions still remain regarding the catalytic mechanism, and investigation of this system has relevance across a number of disciplines. Nitrogen is a fundamental element to all biological systems, primarily occurring in proteins and nucleic acids. However, most nitrogen on Earth is found in the form of nitrogen gas, a form that is biologically unavailable to most organisms owing to the strength of the triple bond between the two nitrogen atoms. The limited abundance of biologically accessible (or fixed) nitrogen has driven an anthropomorphic thrust to supplement the nitrogen cycle with nitrogenous fertilizers, thereby boosting agricultural output. The primary industrial method to produce these fertilizers, derived from the Haber-Bosch synthesis, is an energy intensive process that consumes approximately 1- 2% of the world's energy portfolio. This process utilizes metal iron catalysis, high temperatures and high pressures, along with hydrogen usually obtained from reformed fossil fuels, to reduce atmospheric nitrogen gas to ammonia. Aside from the environmental consequences that arise from the production of nitrogenous fertilizers, long-term agricultural application may also have disastrous ecological ramifications, such as eutrophication. Additionally, biological nitrogen fixation supports more than half the human population, and having a more complete understanding of this complex process has the potential to displace some of the demand for fertilizer production. The aforementioned reasons are clearly enough to warrant serious investigation into biological nitrogen fixation, however, the fascinating intricacies and comparative relevance to other biochemical systems further motivates the study of this system. The enzyme committed to this task, nitrogenase, orchestrates an elegant unidirectional multiple electron reduction and activation of the nitrogen triple bond. Historically, mechanistic characterization of this enzyme has been difficult for a number of reasons; however, studies to date have revealed many aspects of the process as biochemical techniques have improved. Nitrogenase is an oxygen sensitive, complex two-component enzyme that is mechanistically pertinent to many other biochemical processes. Presented here are studies revealing insight into substrate binding and the unique gated electron transfer mechanism of this fascinating enzyme.Item High temperature chlorosilane corrosion of iron and AISI 316L stainless steel(Montana State University - Bozeman, College of Engineering, 2016) Aller, Joshua Loren; Chairperson, Graduate Committee: Paul E. GannonChlorosilane gas streams are used at high temperatures (>500°C) throughout the semiconductor, polycrystalline silicon, and fumed silica industries, primarily as a way to refine, deposit, and produce silicon and silicon containing materials. The presence of both chlorine and silicon in chlorosilane species creates unique corrosion environments due to the ability of many metals to form both metal-chlorides and metal-silicides, and it is further complicated by the fact that many metal-chlorides are volatile at high-temperatures while metal-silicides are generally stable. To withstand the uniquely corrosive environments, expensive alloys are often utilized, which increases the cost of final products. This work focuses on the corrosion behavior of iron, the primary component of low-cost alloys, and AISI 316L, a common low-cost stainless steel, in environments representative of industrial processes. The experiments were conducted using a customized high temperature chlorosilane corrosion system that exposed samples to an atmospheric pressure, high temperature, chlorosilane environment with variable input amounts of hydrogen, silicon tetrachloride, and hydrogen chloride plus the option of embedding samples in silicon during the exposure. Pre and post exposure sample analysis including scanning electron microscopy, x-ray diffraction, energy dispersive x-ray spectroscopy, and gravimetric analysis showed the surface corrosion products varied depending on the time, temperature, and environment that the samples were exposed to. Most commonly, a volatile chloride product formed first, followed by a stratified metal silicide layer. The chlorine and silicon activities in the corrosion environment were changed independently and were found to significantly alter the corrosion behavior; a phenomenon supported by computational thermodynamic equilibrium simulations. It was found that in comparable environments, the stainless steel corroded significantly less than the pure iron. This is likely due to the alloying elements present in stainless steel that promote formation of other stable silicides. Mechanistic models were developed to describe the formation and evolution of metal silicide and/or metal chloride surface corrosion products in chlorosilane environments. These models will help inform materials selection and/or support process development for next-generation chlorosilane-based production and deposition systems. The implementation of low cost materials of construction in these systems could lower the cost of final products in these industries.Item Biochemical characterization of the six-transmembrane epithelial antigen of the prostate family of metalloreductases(Montana State University - Bozeman, College of Letters & Science, 2015) Kleven, Mark Daniel; Chairperson, Graduate Committee: C. Martin Lawrence; George H. Gauss was the main author, and Mark D. Kleven, Anoop K. Sendamarai, Mark D. Fleming and C. Martin Lawrence were co-authors of the article, 'The crystal structure of six-transmembrane epithelial antigen of the prostate 4 (Steap4), a ferri/cuprireductase, suggests a novel interdomain flavin-binding site' in the journal 'Journal of biological chemistry' which is contained within this thesis.; Mark D. Fleming and C. Martin Lawrence were co-authors of the article, 'Characterization of a single B-type heme, FAD and metal binding sites in the transmembrane domain of six trans-membrane epithelial antigen of the prostate (Steap) family proteins' submitted to the journal 'Journal of biological chemistry' which is contained within this thesis.Iron and copper are the two most abundant transition metals in humans and are mediators of many essential cellular processes. The entry of these metals into cells require controlled processes, including their reduction prior to uptake. A group of integral membrane enzymes, the six-transmembrane epithelial antigen of the prostate (Steap) family, are able to perform this function. Steap3, in particular, functions as the primary ferric reductase in the transferrin cycle, the dominant mode of erythrocyte iron uptake. How these enzymes perform these functions has remained ill-defined. Here, the biochemical underpinnings of Steap metalloreductase activity have been investigated. To elucidate these mechanisms, expression systems for Steap3 and Steap4 have been developed in bacterial, insect, and human cell lines and purified to varying degrees. By analyzing the truncated cytoplasmic oxidoreductase domain of Steap4, it was found that NADPH is oxidized by transferring a pair of electrons to a flavin. With this truncation, however, flavin only binds weakly and the construct shows no ability to preferentially bind one type of flavin. In contrast, when the full length Steap3 was partially purified, it exhibits high-affinity FAD-binding, indicating that the transmembrane region of the protein contains the major structural features of the FAD binding site. Further, it was found that the cytoplasm-oriented loops between transmembrane helices formed the site. The next cofactor in the electron transport chain is a single b-type heme. Two strictly conserved histidines were identified that coordinate the heme and both are required for heme incorporation. The metal binding site at the extracellular face of the membrane was also characterized. Here, it was found that Steap3 and Steap4 share a conserved high-affinity iron binding site. Additionally, iron and copper both bind with similar affinities to Steap4. Two critical residues of the metal binding site were determined and their predicted proximity to the heme cofactor suggests that the electron is transfer is direct between cofactor and metal. Finally, it was found that Steap's are able to dimerize in the cells, forming homo- and heterodimers Together, the enzymatic mechanism has been characterized in-depth for the first time for these physiologically-significant enzymes.Item Iron nutrition of plants and interactions with vascular wilt disease and light(Montana State University - Bozeman, College of Agriculture, 1989) Macur, Richard EugeneThe relationship between iron nutritional status and Verticillium Wilt disease in tomato possessing single gene resistance to Race 1 of Verticillium dahliae was investigated using hydroponic culture media. Iron limiting conditions increased the sensitivity of resistant tomatoes to the pathogen as expressed by wilting and chlorosis. Distance of fungal vascular invasion was approximately the same in both iron replete and iron limited treatments. Comparison of near-isolines revealed that the magnitude of disease expressed in Fe deficient Pixie II (resistant) was considerably less than that expressed by the susceptible Pixie variety. Infection of tomato did not enhance iron stress severity as quantified by root peroxidase activity and chlorophyll content of young leaves. The release of iron from horse spleen ferritin through photochemical reduction of Fe(III) to Fe(II) was studied in vitro. Spectrophotometric measurement of the Fe(ferrozine)3^2+ complex (specific for Fe(II)) was used to quantify rates of Fe mobilization: Cool white fluorescent plus incandescent light effectively promoted the rate of Fe release. Compounds known to be present in plants may provide further regulation of photorelease. Reductive removal from ferritin was inhibited by phosphate, and hydroxide, whereas citrate, oxalate, tartrate, and caffeate enhanced the release. Of the organic acids studied, caffeate was the only compound which induced detectable Fe release in the absence of irradiation. Rate constants ranged from 2.7 x 10^-3 sec^-1 (pH = 4.6) to 2.1 x 10^-3 sec^-1 (pH = 7.1) at 26.5°C. Synthesis of the photosynthetic apparatus is dependent on both light and iron. Thus, the findings provide one possible mechanism coupling chloroplast iron demand with iron release from ferritin. Treatments known to alter either phenolic metabolism or overall enzyme activity were utilized to examine the Fe reductive mechanisms involved in iron stress response at the roots. Although specific compounds caused elevation of internal o-dihydroxyphenol content, the overall root reduction capacity of Fe stressed plants was significantly suppressed. However, plant roots retained significant capacity to reduce Fe after tissues were subjected to severe protein denaturizing treatments. Thus, indications for both secreted reductant and enzymatic reduction mechanisms were observed.Item The effects of iron on the macroinvertebrates of Soda Butte Creek(Montana State University - Bozeman, College of Letters & Science, 1974) Chadwick, James WoodrowItem Large scale synthesis and purification of iron responsive element RNA for crystallographic studies of its interactions with iron regulatory proteins 1 & 2(Montana State University - Bozeman, College of Letters & Science, 2003) Eilers, Brian John