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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 Insights into the molecular mechanisms of sensing and responding to the host by Staphylococcus aureus(Montana State University - Bozeman, College of Agriculture, 2019) Meishery-Patel, Delisha; Chairperson, Graduate Committee: Jovanka Voyich-Kane; K. B. Pallister and Jovanka Voyich were co-authors of the article, 'Role of SaeR phosphorylation in regulation of Staphylococcus aureus virulence genes' which is contained within this thesis.; Dissertation contains an article of which Delisha Meishery-Patel is not the main author.Two-component systems (TCSs) are highly conserved across bacteria and are used to rapidly sense and respond to changing environmental conditions. The human pathogen Staphylococcus aureus uses the S. aureus exoprotein expression (sae) TCS to sense host signals and activate transcription of virulence factors essential to pathogenesis. Despite its importance, the mechanism by which the sensor kinase (SaeS) recognizes a stimulus and activates its cognate response regulator (SaeR) to regulate transcription of virulence genes is incompletely defined. However, findings from our lab suggest that SaeR/S mediated transcription is unique-to and dependent-on specific host stimuli. Studies outlined in this dissertation suggest that residues in the extracellular loop may be involved in refinement of the sae regulated targets at the single amino acid level. By generating single amino acid replacement mutants in the response regulator SaeR, we identified a key aspartate residue at position 46 (D46) on SaeR to be important in SaeR mediated signaling as mutation D46A prevented the recombinant protein from binding promoter recognition sequence and subsequently influenced virulence regulation. Current studies are aimed to define the phosphorylation patterns in SaeR using SDS-PAGE analysis and mass spectrometry. Overall, these structure-function studies provide insight into the Sae- signal transduction mechanism and raise some new questions regarding the role the Sae system in the larger regulatory network S. aureus uses to control expression of its secreted virulence factors.Item Genetics of laccase in Gaeumannomyces graminis, the take-all fungus(Montana State University - Bozeman, College of Agriculture, 2002) Litvintseva, Anastasia PavlovnaItem Cyanidiales ecology : biodiversity and transcriptomics in Yellowstone National Park(Montana State University - Bozeman, College of Letters & Science, 2013) Skorupa, Dana Jean; Chairperson, Graduate Committee: Timothy R. McDermottThe work presented in this thesis investigated the ecology of a unicellular, and asexual red algae belonging to the order Cyanidiales which are the only phototrophs occurring in low pH (0.5-3.5) and high temperature (38-56°C) geothermal environments. These algae exhibit a dynamic seasonal biomass fluctuation referred to as 'mat decline,' where viability drastically decreases as seasonal ultra-violet (UV) and visible (VIS) irradiance intensify. Temporal experiments coupling UV irradiance filtering with whole-community transcription profiling revealed significant cyanidial gene expression changes occurring as a result of exposure to UV, and that patterns of response adjusted across low and high irradiance time periods. Separate analyses examined genes responding to either increasing seasonal UV or VIS intensity, or by the combined effects of both irradiance wavelengths (UV and VIS). Results not only corroborated known physiological changes to solar irradiance, but also suggested the strategies employed to deal with excess VIS and UV intensity may be highly integrated. Comparative analysis determined that environmental microarrays are highly sensitive in their detection of transcript diversity, and in situ gene expression changes. Proteomic work identified several dominant cyanidial proteins which were also abundantly transcribed, suggesting there is good correspondence between highly abundant proteins and gene transcriptional activity for these algae. Additionally, a study is presented which examined the biodiversity and distribution of the Cyanidiales in Yellowstone National Park (YNP). Phylogenetic reconstruction using the rbcL gene sequence identified two well-supported YNP lineages: Cyanidioschyzon and Galdieria-A, and showed the separation of taxa based on ecophysiological conditions. Galdieria-A phylotypes were found exclusively in soil and endolithic habitats. Cyanidioschyzon was the sole phylotype found in aqueous environments, but was also detected in all soil and endolithic habitats investigated. Culturing efforts demonstrated that moisture availability controls cyanidial viability and distribution in soil habitats, while alternative environmental factors influence endolithic populations. In addition, autotrophic and heterotrophic viable cell counts in combination with rbcL gene sequencing determined that Galdieria dominates the species composition in soil environments, while the endolithic habitat contains both Cyanidioschyzon and Galdieria.Item Quantitative assessment of localized growth rates and gene expression patterns in Pseudomonas aeruginosa biofilms(Montana State University - Bozeman, College of Letters & Science, 2009) Perez-Osorio, Ailyn Cecilia; Chairperson, Graduate Committee: Michael FranklinThis dissertation work provides evidence of heterogeneity in the distribution of gene expression and growth rates among surface associated cells of Pseudomonas aeruginosa. Currently, methodologies used for characterizing biofilm heterogeneity are constrained by the need of in vitro biofilm growth and by the need to genetically manipulate bacteria. This dissertation describes findings obtained by using LCMM, qRT-PCR, qPCR and microarrays. Through combining LCMM with qRT-PCR a housekeeping gene and two quorum sensing induced genes were found to be differentially expressed at the periphery of P. aeruginosa biofilms. qPCR also enabled the growth rate of cells in discrete locations of biofilms to be determined. Cells localized to the deep layers of biofilms were found in a growth state analogous to stationary phase in planktonic cultures, while cells localized to the biofilm periphery were slightly more active with growth rates that approached cells growing exponentially in planktonic cultures. By elucidating the growth rates of subpopulations within the biofilm it was subsequently possible to determine that the most active cells had approximately 7 copies of the mRNA of housekeeping and stationary phase associated genes. Each of the least active cells, those found in the deeper sections of the biofilms, had less than one copy of any of the mRNAs measured. No significant differences in the distribution of 16S rRNA were found along the sections analyzed. The microarray studies revealed several genes, known to be involved in the pathogenesis of P. aeruginosa, to be undergoing active transcription in young biofilms under conditions of low calcium concentrations. This is significant because calcium homeostasis is known to be out of balance in the lungs of cystic fibrosis patients, where P. aeruginosa biofilms grow causing life threatening infections. These results suggest that spatial and temporal heterogeneity within biofilms underscores their ability to not only survive in diverse and sometimes harsh environmental conditions, but to exploit those environments. The methods described in this work are suitable for characterizing heterogeneity of gene expression and growth rate in biofilms collected from their natural environment. These also represent an alternative method for assessing the distribution of populations in multispecies biofilms.