Theses and Dissertations at Montana State University (MSU)
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/733
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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 Hyperthermal reactions of O(p3sP) with hydrogen and methane(Montana State University - Bozeman, College of Letters & Science, 2004) Garton, Donna Joan; Chairperson, Graduate Committee: Lee SpanglerHyperthermal reactions of O(3P) occur at the surfaces and in the exhaust gases of spacecraft that travel through the residual atmosphere of the Earth at high altitudes (200-600 km). These reactions may degrade materials through oxidation and erosion, or they may yield internally excited reaction products which emit radiation and contribute to the “signature” of a rocket plume. Crossed-beams experiments were used to study model reactions of O(3P) with H2, D2, CH4, and CD4 at center-of-mass collision energies in the range 8-75 kcal mol^-1. Interpretation of the experimental results has been strengthened by theoretical calculations carried out by collaborators. A study of the OH scattered flux as a function of collision energy has led to the determination of an experimental excitation function in the threshold region for the O(3P)+H2 → OH+H reaction. The experimental excitation function clearly matched the theoretical prediction, which confirmed that the laser-detonation source produces O(3P) atoms. The excitation function for the O(3P) + H2 reaction and the dynamics of the O(3P) + D2 reaction, observed experimentally for the first time, demonstrate that these reactions proceed mainly on triplet potential energy surfaces, with little or no intersystem crossing. Experiments on the reactions of O(3P) with methane have revealed a previously unobserved reaction pathway, which involves H-atom elimination: O(3P) + CH4 → OCH3 + H. The excitation function for this reaction has been measured, and the reaction barrier has been determined to be ∼46 kcal mol^-1. In addition, the expected H-atom abstraction reaction, O(3P) + CH4 → CH3 + OH, has been observed, and the dynamics have been investigated. Theoretical calculations identify a triplet-singlet curve crossing below the triplet barrier for the H-atom elimination reaction, but the observed dynamics indicate reaction exclusively on the two lowest-lying triplet surfaces. While it remains to be seen whether intersystem crossing will affect the outcome of other reactions involving hyperthermal atomic oxygen, unknown reactions which have high barriers are likely to be common in extreme environments such as low-Earth orbit, where spacecraft surfaces and exhaust gases suffer high-energy collisions with ambient atomic oxygen.Item Studies of the action of molecular singlet oxygen on proteins and amino acids(Montana State University - Bozeman, College of Letters & Science, 1974) Fischer, James RossItem Reactive scattering of oxygen and chlorine atoms on hydrocarbon surfaces(Montana State University - Bozeman, College of Letters & Science, 1998) Garton, Donna Joan