Theses and Dissertations at Montana State University (MSU)
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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 Copper injury of Escherichia coli(Montana State University - Bozeman, College of Agriculture, 1984) Domek, Matthew JosephItem A physiological study of the elevated temperature test for fecal coliforms(Montana State University - Bozeman, College of Agriculture, 1977) Dockins, William SchalerItem Expression and mutagenesis of recombinant cholera toxin A subunit(Montana State University - Bozeman, College of Agriculture, 1991) Vadheim, Kirsten Louise; Chairperson, Graduate Committee: Jerry M. Keith; Clifford W. Bond (co-chair)Item The genetic basic of flagellar structure in Escherichia coli(Montana State University - Bozeman, College of Agriculture, 1960) Markusen, Aletha S.Item Physiological studies of chlorine injury in Escherichia coli(Montana State University - Bozeman, College of Agriculture, 1977) Camper, Anne Kosteczko