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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.
Microbial adaptation to cultivation stress using storage compounds
(Montana State University - Bozeman, College of Agriculture, 2022) Arnold, Adrienne Dale; Chairperson, Graduate Committee: Ross Carlson; This is a manuscript style paper that includes co-authored chapters.
Methanotrophs and green algae are microorganisms that grow on single carbon substrates. Methanotrophs are bacteria that use methane as their carbon source, and green algae are eukaryotic phototrophs that grow on CO 2. They are of interest both as primary producers in the environment and as biological catalysts for the conversion of greenhouse gases into value-added compounds. Understanding how methanotrophs and green algae adapt to cultivation stresses is key to understanding carbon cycling in the environment and in industrial settings. This work uses stoichiometric metabolic modeling to investigate the role of carbon storage compounds in the metabolism of C1-utilizing organisms. Storage compounds are accumulated as intracellular reserves of polysaccharides or lipids, which can be catabolized under stress conditions to provide carbon and energy to the cell. Catabolism of carbon storage compounds often results in the excretion of multi-carbon organic compounds that can be utilized as carbon substrates by other members of the microbial community. In silico metabolic models were developed for methanotroph and algal systems and used to examine the breakdown of storage compounds in response to common cultivation stresses. For the aerobic methanotrophs, predictions focused on the use of polyhydroxybutyrate and glycogen in adaptation to O 2 limitation. For the green algae, starch and triacylglycerol reserves are analyzed as sources for compatible solutes, which are produced by cells in response to high salinity conditions. Metabolic modeling of storage compound utilization by methanotrophs and algae helps elucidate the role of these organisms as primary producers and presents an opportunity for industrial production of multi-carbon compounds from single carbon substrates.
Microbiomes and zoonoses: dynamics of the black flying fox (Pteropus alecto) gastrointestinal microbiome
(Montana State University - Bozeman, College of Agriculture, 2022) Jones, Devin Nicole; Chairperson, Graduate Committee: Raina K. Plowright; This is a manuscript style paper that includes co-authored chapters.
Land-use change is increasingly recognized as a driver of spillover of zoonotic pathogens. Australian black flying foxes (Pteropus alecto) are experiencing extensive loss of habitat which reduces available food, particularly in winter. Hendra virus (HeV, family: Paramyxoviridae) was isolated from horses and humans in 1994 and P. alecto was later identified as the reservoir host. As habitat loss threatens these bat populations, and Hendra virus continues to spill over to horses annually, it is important to understand factors that influence bat health and viral shedding. Because gastrointestinal tract (GIT) microbiomes are important for host health and are understudied in flying foxes, the goal of this research was to understand the natural dynamics of the P. alecto GIT microbiome and its associations with diet, body composition, markers of inflammation, and viral shedding. We sampled Pteropus alecto near Brisbane from 2018-2020. We captured bats returning from foraging and collected rectal swabs to determine the GIT microbiome using 16S rRNA amplicon sequencing. In addition to feces for dietary analysis, we also collected samples to measure health and infection, including blood to measure neutrophil-to-lymphocyte ratios, urine to detect Hendra virus, and bioelectrical impedance analysis to measure body fat. These data enabled us to determine how the P. alecto GIT microbiome varied within individuals over time and in the context of physiological, ecological, and dietary shifts. Lastly, we asked if we could predict health outcomes using the GIT microbiome. We found that P. alecto GIT microbiomes are highly dynamic over time, through different life stages, between foraging strategies, and that the type of diet is associated with GIT microbiome diversity. Bats consuming native foods had lower GIT microbiome diversity compared to those consuming introduced and cultivated foods. Despite associations between body fat and HeV infection, the GIT microbiome was not able to predict these health outcomes. These results suggest that P. alecto GIT microbiomes are highly dynamic and may not contribute significantly to host health. Future research should incorporate more health metrics or other approaches to microbiome profiling to determine if the GIT microbiome could be used as a biomarker of health.

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