Theses and Dissertations at Montana State University (MSU)

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The paradoxical role of ATP in viral infections and cellular defense
(Montana State University - Bozeman, College of Agriculture, 2024) Zahl, Trevor Ronald; Chairperson, Graduate Committee: Blake Wiedenheft; This is a manuscript style paper that includes co-authored chapters.
Conflict between prokaryotes and viruses has driven the evolution of diverse defense systems. The study of these systems has not only expanded our knowledge of the mechanisms underlying genetic conflict, but has also revolutionized biotechnologies. I joined the Wiedenheft lab in early 2021 during the height of the SARS-CoV-2 pandemic. As such, the lab was focused on developing technologies to aid in the identification and study of SARS-CoV-2. I joined a team that was actively repurposing a type III CRISPR system as a molecular diagnostic. Type III CRISPRs are RNA targeting systems that unleash antiviral signaling cascades upon recognition of a target. Previous work in the lab had shown that this activity could be repurposed as a molecular diagnostic, but the first generation technology was not sensitive enough for clinical use. To address this need, we developed a technology termed Capture and Concentrate, which utilized 'CRISPR magnets' to concentrate target RNAs from complex mixtures. Using this technology, we increased diagnostic sensitivity by 1,000 fold. In addition to my work at the bench, I developed an in silico model of the diagnostic to inform future development efforts. While type III CRISPRs are unique due to their ability to generate antiviral signaling cascades, they also contain intrinsic endonuclease activity. In 2023 we demonstrated that this activity could be repurposed as an RNA editing technology. Using the type III system from Streptococcus thermophilus, we introduced precise genomic modifications in a recombinant sindbis virus. This technology removed the need for reverse genetics systems, which hampered the study of the rapidly evolving SARS-CoV-2 virus. In the final year of my graduate studies, I worked with a team focused on determining how, PARIS, a bacterial innate immune system, induced host death upon viral infection. PARIS is a two-component system comprised of the ATPase, AriA, and a nuclease, AriB. Previous studies had shown that PARIS induced abortive infection in response to viral proteins, but the mechanism was not known. We determined that upon antigen recognition, AriB is activated and cleaves host tRNAs to induce cell death and prevent community spread.
Geobiological feedbacks and the evolution of thermophiles in Yellowstone National Park hot springs
(Montana State University - Bozeman, College of Agriculture, 2024) Fernandes Martins, Maria Clara; Chairperson, Graduate Committee: Eric Boyd; This is a manuscript style paper that includes co-authored chapters.
This dissertation focuses on identifying the geobiological feedbacks that shaped the evolutionary ecology of thermophiles in Yellowstone National Park (YNP) hot springs. Hot springs can generally be grouped as acidic, moderately acidic, and neutral to alkaline. Although the geochemistry and microbiology of YNP hot springs have been studied for over a century, fundamental gaps in the understanding of the feedbacks between them remain. Here, the influence of fluid mixing regime on geochemistry, microbial diversity, and productivity was investigated in three geographically co-localized springs whose communities are supported by chemical energy. The results indicate that a higher degree of disequilibrium in electron donor/acceptor pairs due to mixing of highly reduced volcanic gases and oxidized near surface waters was present in the moderately acidic hot spring, which supported higher biodiversity and primary productivity. In contrast, the acidic hot spring had the lowest biodiversity and productivity. Interestingly, acidic springs are generally dominated by members of the archaeal order Sulfolobales which have been suggested to mediate the acidification of these environments through aerobic elemental sulfur (S 8 0 ) oxidation that produces sulfuric acid (H2 SO4 ). Intriguingly, Sulfolobales encode the protein sulfide:quinone oxidoreductase (SQR), proposed to catalyze the oxidation of sulfide (H2 S). However, this metabolism has yet to be demonstrated. Five novel Sulfolobales strains were isolated under H 2S-oxidizing conditions from YNP. This activity was coupled to growth and H 2SO 4 production, expanding the role of Sulfolobales in the oxidative sulfur cycle. S 8 0 oxidation in these strains was also investigated due to the observation that nearly half of Sulfolobales don't encode sulfur oxidoreductase (SOR), the canonical pathway of S 80 oxidation in Sulfolobales. Two Sulfolobales strains were selected, one of which encoded SOR and the other of which did not. SOR disproportionates S 8 0 , yielding H 2S as a product. Since H 2S can react with S 8 0 , promoting its solubilization, it was hypothesized that the strain encoding SOR could grow via indirect contact to the mineral while the non-SOR encoding would need direct contact. This was confirmed through experiments where S 8 0 was sequestered in dialysis membranes. Interestingly, the non-SOR strain was able to grow via indirect contact when H 2S was added to the culture media to mimic SOR mechanism. The results shown here provide new insight into the geological and biological feedbacks that shaped the evolution, ecology, and physiology of thermophiles.
Identification of novel ssDNA and RNA coliphage in wastewater
(Montana State University - Bozeman, College of Agriculture, 2024) Little, Agusta Rio; Chairperson, Graduate Committee: Blake Wiedenheft
Bacteriophages (phages) are the most abundant biological entities on Earth. However, our understanding of their diversity is limited, with a vast gap in knowledge regarding single- stranded DNA (ssDNA) and RNA phages. This study addresses this gap by isolating and characterizing ssDNA and RNA coliphages from wastewater, a suspected rich source of these understudied phages. Traditional phage isolation methods favor double-stranded DNA (dsDNA) phages, resulting in the underrepresentation of ssDNA and RNA phages. To overcome this bias, we employed enrichment strategies using small molecules that inhibit dsDNA phage replication. Additionally, we utilized an RNase-A assay to identify potential RNA phage candidates. These enrichment techniques led to the isolation of a circular ssDNA phage (POI 1) and a ssRNA phage (POI 8). A combination of biochemical assays, sequencing, and microscopy techniques were utilized to characterize these phages. Overall, this work demonstrates the effectiveness of enrichment strategies for isolating ssDNA and RNA phages and underscores the importance of developing optimized techniques to unlock the true diversity of these understudied phage populations.
An interrogation of herpes simplex virus type-1 gene expression during neuronal infection
(Montana State University - Bozeman, College of Agriculture, 2024) Domanico, Luke Frank; Chairperson, Graduate Committee: Matt Taylor; This is a manuscript style paper that includes co-authored chapters.
Herpes Simplex virus-type-1 (HSV-1) is a ubiquitous human pathogen casually referred to as "the gift that keeps on giving". The seemingly benign recurring herpetic lesions caused by acute HSV-1 infection are an obnoxious reminder of an incurable infection. HSV-1 maintains lifelong persistence in the infected host through a unique form of infection in peripheral neurons, conventionally termed latency. The latently infected neuron acts as a viral reservoir and is the focal point of herpetic disease. The latent HSV-1 infection represents a brilliant orchestration of viral gene regulation, manipulation of highly polarized cells, and seamless evasion of immunological clearance. Though, the viral mechanisms and cellular factors that govern the establishment, maintenance and reactivation from latency are elusive and challenging to study. The work included here aims to uncover the cryptic factors involved in and supporting the latent HSV-1 infection. Authored publications include the demonstration of a recombinant HSV-1 that enables temporal discretion of viral gene expression, and the revelation of a stunning, yet obscure phenotype of neuronal infection. Next is the implementation of a single-cell culturing method using drop-based microfluidic technology to resolve HSV-1 infection in isolated neurons. Together, this work reveals that the early events of neuronal infection are critical to determining the lytic or latent outcome of infection. Inoculating dose impacts the kinetics of viral replication, and the establishment of lytic or latent HSV-1 infection. Furthermore, evaluation of viral gene expression during latent HSV-1 infection suggests that the distinction between lytic and latent HSV-1 infection is less mutually exclusive than is historically appreciated. Finally, I present preliminary and ongoing research suggesting that a cellular transcription factor called nuclear factor-kappa B (NF-kB) differentially engages in HSV-1 infection. NF-kB supports efficient lytic gene transcription in epithelial cells, while promoting the establishment of latent HSV-1 infection of neurons.
The interaction of heavy metals with the mammalian gut microbiome
(Montana State University - Bozeman, College of Agriculture, 2022) Coe, Genevieve Lea; Chairperson, Graduate Committee: Seth Walk; This is a manuscript style paper that includes co-authored chapters.
Heavy metals are for the most part, naturally occurring elements found in the environment. Some are essential, meaning they are involved in critical biochemical pathways, in all branches of life. Other heavy metals are non-essential and disrupt metabolic functions in most organisms rendering them toxic. The following research explored the interactions of the mammalian gut microbiome with an essential heavy metal, iron, and a non-essential heavy metal, methylmercury, acquired through the diet. The overarching goal was to define and characterize the influence of gut microbial interactions with heavy metals on host health. Novel experimental designs using murine models were designed to examine 1) the consequences of low iron challenge on the murine gut microbiome and whether host iron availability was affected and 2) the potential influence of the gut microbiome in methylmercury elimination rate and demethylation in conventional, germ-free, gnotobiotic, and humanized mice. Culturing in vitro and toxicity assays, 16S sequencing, deep metagenomic sequencing of human stool, bioinformatic analysis, transcriptional analysis of iron biomarkers, quantification of iron and mercury by ICP-MS and HPLC-ICP-MS methods were performed as well as the use of mouse models to examine iron and methylmercury interactions with the gut microbiome in vivo. Our results from this project indicate that the gut microbiome is significantly affected by loss of iron from the diet, and does not fully recover post-iron repletion, while the host is relatively unaffected by low-iron challenge to the gut microbiome. Methylmercury elimination and demethylation is significantly faster and higher, respectively, in mice with a gut microbiome, providing novel evidence in support of a role for the gut microbiome in methylmercury demethylation and elimination. However, exact mechanisms of microbial interactions with methylmercury in the gut have yet to be elucidated. Our data also suggests the possibility of host-mediated mechanisms of methylmercury demethylation, by yet unknown mechanisms that warrant further exploration.
Aerobic bacterial methane synthesis in the human gastrointestinal tract
(Montana State University - Bozeman, College of Agriculture, 2023) Jackson, Thomas Robert; Chairperson, Graduate Committee: Seth Walk
Aerobic bacterial methane synthesis constitutes a paradigm-shifting novel metabolism recently described in aquatic environments. It challenges the traditional model of methanogenesis as being a strictly anaerobic process carried out by archaeal methanogens. To date, the presence of aerobic bacterial methane synthesis has not been studied within the context of the human gastrointestinal tract. The goal of this work was to investigate the possibility of the presence of such metabolisms in the human gut microbiome. To investigate this, fecal samples from six individuals were first screened for the ability to produce methane under aerobic conditions. Bacteria from two of those fecal samples were isolated and evaluated for their ability to utilize methylamine, a known substrate involved in aerobic bacterial methane synthesis, as a sole nitrogen source. The ability of those isolates to produce methane under aerobic conditions from methylamine was then evaluated. Additionally, a flask-independent culture-based assay was developed in order to screen larger numbers of future isolates for the ability to utilize methylamine as a sole nitrogen source. This work demonstrates the first evidence of aerobic bacterial methane synthesis from members of the human gastrointestinal tract, finding two isolates capable of producing methane under aerobic conditions. Such findings broaden the understanding of methane-generating pathways that may have implications for the development of dysbiosis and atherosclerosis in human hosts.
Phycosomal dynamics in xenic cultures of the alkalitolerant green Microalga chlorella sp. SLA-04
(Montana State University - Bozeman, College of Agriculture, 2023) Miller, Isaac Robert; Chairperson, Graduate Committee: Matthew Fields; This is a manuscript style paper that includes co-authored chapters.
The production of microalgal biomass and biofuel is an important component of the transition away from a petroleum-based economy. Industrial scale microalgal cultures are often xenic, meaning they are comprised of microalgae as well as a phycosome (i.e., microbiome). The microalgal field has begun to appreciate the ubiquity and potential influence of the phycosome, but there remains a critical need for comprehensive research to unravel the intricate metabolic and ecological relationships between microalgae and the respective phycosome that can be comprised of mainly bacteria but also other microorganisms (i.e., archaea, fungi, protists, viruses). Phycosome research is essential for potentially using these interactions to enhance the stability, productivity, and cost-efficiency of industrial microalgal cultivation. Chlorella sp. SLA-04 is an oleaginous, alkalitolerant microalga isolated from the alkaline Soap Lake (Washington, USA). Under alkaline conditions, SLA-04 can be grown to high biomass levels without reliance on the delivery of concentrated CO 2, an improvement in producing competitively priced biomass and biofuel. The high pH, high alkalinity systems are able to capture CO 2 directly from the air in open systems (e.g., raceway ponds) but the open systems can be dynamic in terms of stability and productivity. Despite growing knowledge of the importance of phycosomes in open production systems, little is known about how alterations to cultivation conditions can be used to maintain a xenic system with controllable outputs, especially under high pH, high alkalinity conditions. The work outlined in this dissertation employed long term temporal community studies, open outdoor raceway experiments, diel-cycle-resolved temporal sampling coupled with activity-based probing (bioorthogonal non-canonical amino acid tagging (BONCAT)), and quantitative measures of algal physiology to better understand the relationship between microalgal phenotype and the respective phycosomes. SLA-04 phycosome composition and culture physiology were consistent over time when maintained in xenic cultures under low and high alkalinity. When xenic cultures were used in successive open, outdoor raceway experiments, compositional community changes coincided with seasonal temperature and light shifts, providing evidence that abiotic and biological environmental stresses impact directly and indirectly SLA-04 productivity and phycosome composition. By employing temporally resolved sampling and probing the relationship between diel-cycle-dependent metabolism and the phycosome, we identified active bacterial populations that may play a role in culture productivity. Expanding beyond augmenting SLA-04 productivity, aggregation of xenic cultures was assessed as a quantifiable phenotype, uncovering a relationship between aggregation, taxonomic composition and algal growth conditions (i.e., alkalinity level). All together, these results represent an initial description of the ecology (e.g., composition, succession, activity) of alkaline microalgae cultures and provide methodology and perspective for future phycosome studies.
Rhizobiome dynamics in plant growth promotion and abiotic stress response
(Montana State University - Bozeman, College of Agriculture, 2023) Goemann, Hannah Marie; Chairperson, Graduate Committee: Brent M. Peyton
Soil microorganisms play vital roles in global nutrient cycling. Understanding the complex relationships between plants and soil microbes and their implications is one of the greatest challenges facing microbial ecology today. Soil microbes can play beneficial roles in supporting plant growth by increasing access to nutrients, water, and decreasing plant stress signaling under abiotic stresses such as drought and heat. With increasing climate variability due to climate change, it is imperative to make scientifically informed management decisions to best support global biodiversity and plant productivity in natural and agroecosystsms. In this dissertation I summarize four separate investigations of plant-microbe interactions. The first is using nitrogen-fixing cyanobacterial biofertilizers to promote plant growth of perennial second generation bioenergy crops switchgrass (Panicum virgatum) and tall wheatgrass (Agropyrun elongatum). The second and third studies seek to better understand plant-microbe carbon exchange under drought stress in the native North American prairie grass blue grama (Bouteloua gracilis). The final study explores the potential microbial contribution to heat tolerance of panic grass (Dichanthelium lanuginosum) across a natural soil temperature gradient in Yellowstone National Park. Next-generation amplicon sequencing using the Illumina Miseq platform is the primary technique utilized across the three studies to investigate microbial community dynamics. The main results of the biofertilizer study were that tall wheatgrass is better suited to the SW Montana growing season than switchgrass, and similar plant yields were achieved with the cyanobacterial biofertilizer as with urea chemical fertilizer without negatively impacting the microbial community diversity. The first blue grama study found that severe and mild drought had distinct, phylogenetically linked responses within the blue grama rhizobiome with Planctomycetes, Thermoproteota (ammonia-oxidizing archaea), and Glomeromycetes (arbuscular mycorrhizal fungi) exhibiting notably altered relative abundances. The second blue grama study found that climate legacy plays an important role in shaping blue grama drought response. Finally, from the D. lanuginosum study in Yellowstone National Park we learned that pH and temperature both strongly influence community composition, and that D. lanuginosum selects for unique community members in its rhizosphere at higher temperatures. Collectively, these studies contribute to furthering our understanding of the dynamics of plant-associated microbiomes.
Evolutionary consequences of gene flow in the absence or inhibition of dispersal in microbial communities
(Montana State University - Bozeman, College of Agriculture, 2023) Munro-Ehrlich, Robert Mason; Chairperson, Graduate Committee: Jovanka Voyich-Kane; This is a manuscript style paper that includes co-authored chapters.
Much of our understanding of the evolutionary dynamics of microbial populations is derived from population level studies which focus on the immediately present populations and ignore the contributions of nearby communities. Microbial ecology studies typically do not distinguish between gene flow, i.e., the movement of genetic material between populations, and dispersal, i.e., the movement of those populations themselves. These two processes are indeed linked, but not identical. We have known for centuries that genetic material can be transferred between physically distant and taxonomically disparate microbial populations; molecular biology tools like cloning are dependent on this capability. In other words, gene flow can occur even without dispersal. However, our ecological and evolutionary studies of microbial populations typically fail to acknowledge the evolutionary impact and genetic contributions of outside populations. Unique evolutionary scenarios arise when dispersal between two or more populations is prevented or limited, but gene flow can still occur between them. We hypothesized that this scenario would impact microbial populations by facilitating speciation, selection, and local adaptation. We aimed to test this hypothesis by studying endemic Meiothermus populations inhabiting serpentinite rocks in the subsurface of the Samail ophiolite in Oman. Samail Ophiolite microbial communities, of which Meiothermus populations are a component, are dispersed across the subsurface and separated by meters of solid rock and by chemical and pH gradients spanning orders of magnitude. Despite barriers to dispersal that are significant enough to shape community structure, we found that gene flow still occurred between nearly all observed populations of Meiothermus. This gene flow is contributing to disruptive selection amongst cohabiting populations, and may also be contributing to local adaptation, both at the genetic and genomic level. We also identified potential mechanisms for this gene flow, including abundant viral elements. The sequence similarity of mobile genetic elements in these Meiothermus populations implies that this gene flow occurred after colonization by a common Meiothermus ancestor and that diversification is likely ongoing. To our knowledge, this is the first demonstration of gene flow across barriers to dispersal in an environmental microbial system. In conclusion, these results suggest that the capacity for microbial populations to undergo gene flow even in the absence or inhibition of dispersal is a natural process, has substantial consequences for the evolution of the effected population, and may also have consequences for the microbial and surrounding environment.
Investigation of the cellular pathology underlying the optic neuropathy in a mouse model of familial Dysautonomia
(Montana State University - Bozeman, College of Agriculture, 2023) Schultz, Anastasia Mardell; Chairperson, Graduate Committee: R. Steven Stowers; This is a manuscript style paper that includes co-authored chapters.
Familial dysautonomia (FD) is a rare, recessive, progressive autosomal disorder that affects the nervous system. This neurological disorder is caused by a splice mutation in the Elongator complex I (ELP1) gene. The mutation results in a tissue-specific reduction of ELP1 protein due to unstable mRNA targeted for nonsense-mediated decay. ELP1 is a highly conserved scaffolding protein and core subunit of the six-subunit Elongator complex required for normal translation, neuronal development, and survival. Insufficient ELP1 leads to the developmental death of neurons in the peripheral and autonomic nervous systems in addition to central and peripheral nervous system neurodegeneration. Patients suffer from congenital and progressive neuropathies, such as cardiovascular dysfunction, reduced peripheral sensory function, poor growth, and digestive and respiratory problems. Outside of the risk of death in early adulthood, one of the most debilitating conditions affecting patients' quality of life is progressive blindness marked by continual loss of retinal ganglion cells (RGCs). Within the FD community, there is a concerted effort to develop treatments to prevent the loss of RGCs, thereby improving patients' quality of life. This study aims (1) to elucidate mechanisms underlying the death of RGCs in the absence of Elp1 and (2) to obtain pre-clinical intervention data that can eventually be translated into therapeutics for rescuing RGCs in FD. Using histology and confocal microscopy in conjunction with biochemistry, this study provides evidence for disrupted cellular homeostasis and inflammation preceding RGC death, and as the disease progresses, the retinal cells fail to mount a correct stress response to restore neuronal homeostasis. Furthermore, this study provides first-of-its-kind pre-clinical data using targeted gene therapies to rescue RGCs. Understanding the biological crosstalk and signaling mechanisms underlying the death of RGCs in the absence of Elp1 will allow for more targeted and effective therapeutics that will benefit not only the FD community but also individuals affected by other retinal diseases and neurological diseases that result from a faulty Elongator complex. This study provides a novel characterization of the FD retina and establishes baseline methods to further investigate rescuing RGCs.

Theses and Dissertations at Montana State University (MSU)

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