Theses and Dissertations at Montana State University (MSU)
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Item Identification of novel ssDNA and RNA coliphage in wastewater(Montana State University - Bozeman, College of Agriculture, 2024) Little, Agusta Rio; Chairperson, Graduate Committee: Blake WiedenheftBacteriophages (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.Item An omics-based interrogation of disparate microbial systems: multi-omics analysis of a bio-mining archaeon and the effects of arsenic on the E. coli Lipidome(Montana State University - Bozeman, College of Letters & Science, 2023) Fausset, Hunter Lee; Chairperson, Graduate Committee: Brian Bothner; This is a manuscript style paper that includes co-authored chapters.Systems biology represents the next frontier in the elucidation of biochemical mechanisms, disease states, and microorganisms. Rather than approaching individual parts of an organism, such as a specific protein, molecule, or mRNA, a systems biology or "omics" investigation seeks to characterize all proteins, molecules, or RNA simultaneously. This is crucial, because all macromolecules in a lifeform exist in dynamic equilibria with those around them; no one biological process occurs in a vacuum. Omics investigations have ballooned in usage over the last decades due to scientists realizing their power in characterizing complex biological phenomena. This has also been spurred on by advances in technologies enabling the robust elucidation of thousands of molecules at once, particularly benefitting from the modernization of mass spectrometry. This technique can be used to study any number of biological problems including those presented here; a multi-omics investigation into a mineral- eating methanogen and a lipidomic characterization of arsenic exposure in a key member of the gut microbiome, E.coli. Methanosarcina barkeri, a widespread methanogen found in marine sediments, is able to reductively dissolve minerals such as pyrite (FeS2) to satisfy their iron and sulfur requirements. Presented here are two investigations containing transcriptomic, proteomic, metabolomic, and lipidomic analyses, performed in parallel on the same biomass. Together, these experiments suggest that the organism undergoes a significant phenotypic shift in response to changes in just two elements, Fe and S. Overall inferences are echoed in the small molecule analyses; the metabolomes and lipidome of the organism change similarly in to the proteome. Key sulfur equilibria are implied in the process, as are specific lipids, choline, and dethiobiotin. A similar approach was applied to E.coli treated with arsenic, as a proxy for understanding the detoxifcation that takes place in the gut microbiome after ingestion. Marked lipidomic changes were observed in E.coli resulting from treatment, which were dependent both on species of arsenic as well as presence of the Ars operon. As a foundational study, this work answered some and generated many more hypotheses on the biochemical fate of As in microorganisms in the gut microbiome.Item Proteomics analysis of the metabolic transition between aerobic and anaerobic conditions in Escherichia coli(Montana State University - Bozeman, College of Letters & Science, 2019) Refai, Mohammed Yahya; Chairperson, Graduate Committee: Brian Bothner; Nina Paris, Hunter Fausset, Monika Tokmina Lukaszewska were co-authors of the article, 'Proteomics analysis of the transition between aerobic and anaerobic growth conditions in Escherichia coli' submitted to the journal 'Biochimica et biophysica acta' which is contained within this dissertation.As a facultative anaerobe, Escherichia coli has the ability to grow in anaerobic and aerobic environments. Despite detailed characterizations of this model organism in the presence and absence of oxygen, an in-depth understanding of changes to the proteome during transitions from aerobic to anaerobic growth is lacking. This thesis work focuses on elucidating how protein thiol oxidation and reduction change during a facultative anaerobe's transition from aerobic to anaerobic growth conditions, and pathways of thiol-mediated cell signaling. Redox driven changes in cysteine oxidation involved in signaling are referred to as 'thiol switches'. These modulate diverse biological activities ranging from gene expression and protein synthesis to environmental stress response. Surprisingly, little is known about the role of thiol switches during microbial transitions from aerobic and anaerobic growth conditions. To explore this uncharted territory, a mass-spectrometry (MS)-based proteomics workflow was developed and refined. Following extensive protocol optimization for high-throughput MS data processing, normalization, and pattern matching, the analytical pipeline was fine-tuned for the specific proteome-wide analysis of cysteine chemical modifications in E. coli. The approach was based on open-source software and publicly accessible databases, creating a transparent, reproducible, and easily sharable proteomics approach. Herein, the redox state and chemical forms of protein-based thiol switches in E. coli were characterized over time as the bacterium reversibly transitioned between aerobic and anaerobic growth conditions. Unexpectedly, differential alkylation analysis of cysteine-containing E. coli proteins revealed a higher degree of protein thiol oxidation under anaerobic growth conditions, a result not reported for E. coli or any other facultative anaerobe. Our proteome-wide analysis also revealed that cysteine redox potentials vary widely, and several specific E. coli proteins contain highly reactive thiols. These findings provide strong evidence for thiol-based signaling in E. coli in response to environmental changes such as aerobic to anaerobic growth transitions. Characterization of specific redox switches underlying metabolic changes associated with oxygen availability has uncovered a previously unknown E. coli cell signaling mechanism. Since transitioning between aerobic and anaerobic environments is associated with bacterial virulence, this work opens new avenues to target pathogenic facultative anaerobes and to develop novel thiol-based antibacterial therapies.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.Item Mechanisms of CRISPR-mediated immunity in Escherichia coli(Montana State University - Bozeman, College of Letters & Science, 2019) van Erp, Paul Bertram Geert; Chairperson, Graduate Committee: Blake Wiedenheft; Gary Bloomer, Royce Wilkinson and Blake Wiedenheft were co-authors of the article, 'The history and market impact of CRISPR RNA-guided nucleases' in the journal 'Current opinion in virology' which is contained within this thesis.; Ryan N. Jackson and Joshua Carter were authors and Sarah M. Golden, Scott Bailey and Blake Wiedenheft were co-authors of the article, 'Mechanism of CRISPR-RNA guided recognition of DNA targets in Escherichia coli' in the journal 'Nucleic acids research' which is contained within this thesis.; Angela Patterson was an author and Ravi Kant, Luke Berry, Sarah M. Golden, Brittney L. Forsman, Joshua Carter, Ryan N. Jackson, Brian Bothner, and Blake Wiedenheft were co-authors of the article, 'Conformational dynamics of DNA binding and CAS3 recruitment by the CRISPR RNA-guided cascade complex' in the journal 'ACS chemical biology' which is contained within this thesis.; Tanner Wiegand, Royce A. Wilkinson, Laina Hall, Dominick Faith and Blake Wiedenheft were co-authors of the article, 'Protein overexpression reduces specific phage infectivity in prokaryotic argonaute screen' which is contained within this thesis.; Dissertation contains three articles of which Paul Bertram Geert van Erp is not the main author.Prokaryotes are under constant threat from foreign genetic elements such as viruses and plasmids. To defend themselves against these genetic invaders prokaryotes have evolved extensive defense mechanisms. In this thesis I explore two such defense systems: prokaryotic Argonautes and CRISPR-systems. CRISPR-systems acquire short sequences derived from foreign genetic elements and store them in the CRISPR locus. In subsequent rounds of infection these stored sequences are used as guides by Cas proteins to target the invaders. Escherichia coli K-12 contains a type I-E CRISPR system, consisting of two CRISPR loci and eight cas genes. five of these cas genes, together with and 61-nucleotide CRISPR-RNA guide form the RNA-guided surveillance complex Cascade. This complex finds and binds foreign DNA targets that are complementary to its RNA guide. After target binding the helicase/nuclease Cas3 is recruited to the Cascade-DNA complex for destruction of the target. The goal of this research is to understand the molecular mechanisms that lead to target recognition and destruction in the type I-E CRISPR systems. Atomic resolution structures of the proteins involved in these CRISPR systems provide the blueprints of these proteins machines. Structure guided mutational analysis coupled with in vivo and in vitro biochemical experiments are used to investigate the underlying molecular mechanisms of this CRISPR system. Together, these results explain the rules of target recognition and Cas3 recruitment. Prokaryotic Argonautes have been hypothesized to defend against mobile genetic elements such as plasmids and viruses through guided nuclease activity. To test this hypothesis, we overexpressed 8 phylogenetically diverse prokaryotic Argonautes proteins in Escherichia coli and challenged them with seven bacteriophages. This resulted in robust protection against phage Lambda and phage P1 by four of the tested Argonautes, while little impact on phage infectivity was observed for the other phages tested. However, control experiments with a nuclease inactive Argonaute mutant and expression of an unrelated control protein showed similar protection against phage Lambda and phage P1. Collectively, our data suggest that protein overexpression in general, rather than Argonaute expression in particular, results in protection against 2 specific phages.Item The human intestinal organoid as a model system for enteropathogenic Escherichia coli pathogenesis(Montana State University - Bozeman, College of Letters & Science, 2015) Olshefsky, Stephen Christopher (Skip); Chairperson, Graduate Committee: Seth WalkEscherichia coli are Gram-negative, facultative anaerobic bacteria commonly found in the intestine of humans and warm-blooded animals. E. coli can be mutualistic or pathogenic that cause diarrheal disease (diarrheagenic E. coli, DEC). The colonization of E. coli begins with the successful adherence to intestinal epithelial cell (IECs); which is mediated by a variety of colonization factors on the bacterial cell surface. This is the first and most crucial step for E. coli colonization. Therefore, valuable model systems to study E. coli should recapitulate this adherence. Several model systems have been developed and successful reproduce adherence and other important aspects of EPEC pathogenesis, but have significant limitations. However, human intestinal organoids (HIOs) are a 3-dimensional tissue culture composed of a single layer of mature, differentiated, columnar epithelial cells that surround a lumen. When compared to traditional cell cultures, animal models and ligated intestines, HIOs have the potential to be more representative human physiology. Here, we begin to demonstrate the use of HIOs as an in vitro model to study E. coli. We hypothesized that HIOs could be used to study epithelial colonization dynamics of E. coli. In this study, we established working protocols for a novel experimental approach for investigating attachment factors involved in E. coli attachment. HIOs are generated from embryonic or pluripotent stem cells into definitive endoderm that gives rise to 3-dimensional structures. These structures were routinely cultured to a diameter of approximately 3.0 mm and embedding in matrigel. An overnight culture of the prototypic EPEC strain, e2348/69 (O127:H6) was transfected with a plasmid carrying a green fluorescent protein that provided visualization by fluorescent microscopy. Approximately, 2.4 x 10 6 CFU was injected into individual HIOs using a microinjector and incubated for 12 hours at 35°C and 5% CO 2. HIOs were then fixed and either stained for fluorescence imaging or processed in 1% osmium tetroxide for examination by field emission scanning electron microscopy (FE SEM). The observations that EPEC was intimately associated with the intestinal epithelium, supports the use HIO for E. coli colonization investigations.Item Understanding Escherichia coli O157:H7 presence, pervasiveness, and persistence in constructed treatment wetland systems(Montana State University - Bozeman, College of Letters & Science, 2015) VanKempen-Fryling, Rachel Joy; Chairperson, Graduate Committee: Anne Camper; Otto R. Stein and Anne K. Camper were co-authors of the article, 'Presence and persistence of wastewater pathogen Escherichia coli O157:H7 in hydroponic reactors of treatment wetland species' in the journal 'Water science and technology' which is contained within this thesis.; Anne K. Camper was a co-author of the article, 'Escherichia coli O157:H7 attachment and persistence within root biofilm of common treatment wetlands plants' submitted to the journal 'Water research ' which is contained within this thesis.; Anne K. Camper was a co-author of the article, 'Using molecular and microscopic techniques to track the wastewater pathogen Escherichia coli O157:H7 within model treatment wetlands' submitted to the journal 'Applied and environmental microbiology' which is contained within this thesis.Treatment wetlands (TW) are a wastewater remediation technology that relies on the natural ability of wetland plant species and the associated microbial consortia to remove pollutants and improve water quality. Although there is substantial research on chemical pollutant remediation by TW, the removal of bacterial pathogens is much more varied and limited in scope. Escherichia coli O157:H7 is a bacterial pathogen that has caused numerous outbreaks and infections in the United States alone and is closely associated with improper water treatment. Understanding how E. coli O157:H7 could potentially persist and survive through a TW process is important in order to appropriately determine the efficacy of TW for treating water and protecting human health. This work used epifluorescent microscopy and qPCR relative DNA abundance to track E. coli O157:H7 tagged with a fluorescent DsRed protein in various environments pertaining to a TW. Two high performing wetland plant species, Carex utriculata and Schoenoplectus acutus, were used in hydroponic and simulated TW columns to better understand how the bacteria localize and persist. Teflon nylon strings (diameter 0.71-1.02 mm), cleaned and with established biofilm, were run hydroponically as control inert surfaces. Unplanted gravel columns were used as a nonplanted control for column experiments. E. coli O157:H7-DsRed were observed by microscopy on root surfaces both in hydroponic reactors and lab scale TW columns. The organisms persisted, forming microcolonies shortly after initial inoculation on both root and nylon surfaces. In the lab scale columns, cells persisted for three weeks, although strong biofilm formation was not observed. qPCR also provided evidence that E. coli O157:H7 was able to persist on the tested surfaces of plant roots, nylon inert surfaces, and gravel, showing higher abundance S. acutus roots than on the inert surface and gravel, however higher in unplanted gravel overall. For the plant types, C. utriculata was statistically lower for E. coli O157:H7 abundance than S. acutus over time. This work provides evidence that E. coli O157:H7 is able to colonize and persist in a TW environment, and plant surfaces may offer a higher inactivation than an inert matrix.Item Effects of ultraviolet irradiation of host and parasite on attachment of Bdellovibrio bacteriovorus to Escherichia coli(Montana State University - Bozeman, College of Agriculture, 1969) Castric, Kathleen ForsgrenItem Genetic studies on a heat stable surface antigen of Escherichia coli K12(Montana State University - Bozeman, College of Agriculture, 1963) Markusen, Aletha SylviaItem Studies on a mutator gene in Escherichia coli(Montana State University - Bozeman, College of Agriculture, 1967) Warren, Guylyn Rea
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