Molecular mechanisms of microbial pathways for environmental contaminant remediation
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Toporek, Yael Jordan
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Abstract
This thesis examines the molecular mechanism of alternate strategies for remediation of contaminated environments. Radioiodine, perfluoroalkyl substances (PFAS), and 1,4-dioxane represent emerging contaminants of national concern. For example, microbially catalyzed reductive methylation of 129IO3- has received recent attention as an alternate strategy for remediation of radioiodine-contaminated environments. This thesis identified enzymes required for IO3- reduction coupled to organic acid oxidation in the facultative anaerobe Shewanella oneidensis: cytoplasmic electron donors are oxidized, and the electrons are transferred through the periplasm via cytochromes of the metal-reducing pathway to extracellular dimethylsulfoxide (DMSO) reductase, which directly reduces IO3- to iodide (I-) as an alternate substrate. Future work aims to investigate the apparent import of I- back to the cytoplasm, where it is putatively methylated and volatilized by a promiscuous thiopurine methyltransferase, presenting a potential strategy for bioremediation of radioiodine.
In the case of PFAS, the industrial surfactant and flame retardant perfluorooctanoic acid (PFOA) has been designated as an emerging contaminant. In the present study, the microbially driven Fenton reaction (MFR) was employed to attempt degradation of PFOA by cycling between aerobic and anaerobic ferric iron (Fe(III))-reducing conditions. Under aerobic conditions, S. oneidensis reduced molecular oxygen (O2) to hydrogen peroxide (H2O2), while under anaerobic conditions, S. oneidensis reduced Fe(III) to Fe(II). During aerobic-to-anaerobic transition periods, Fe(II) and H2O2 interacted chemically via the Fenton reaction to produce contaminant-degrading hydroxyl (HO•) radicals, which in turn interacted with PFOA. PFOA concentrations, however, remained unchanged, which most likely reflects the stability of carbon-fluorine bonds and consequent inability of HO• radicals to oxidatively degrade PFOA.
Finally, the present study aimed to determine the redox conditions of the intracellular environment during oxidative stress in S. oneidensis from aerobic respiration and H2O2 stress. In contrast to S. oneidensis anaerobic respiration, aerobic respiration is understudied, but is a key contributor to MFR in degrading organic and chlorinated environmental contaminants like 1,4-dioxane. This work describes the native and perturbed redox environment of the S. oneidensis cytoplasm, as well as the contribution of individual genes, particularly catalases and peroxidases, to intracellular H2O2 scavenging rates using the genetically-encoded ratiometric fluorescent sensor HyPer-3 as a reporter. As measured by HyPer-3, deletion of one or more catalases and peroxidases resulted in dramatic changes in the redox condition of the cytoplasm, while other H2O2-scavenging enzymes provided overlapping H2O2 scavenging activity to combat H2O2 challenges. Based on cytoplasmic HyPer-3 redox signals, results from the present study indicated that periplasmic PgpD, cytoplasmic KatB, and previously overlooked cytoplasmic KatG1 and KatG2 provide first- and second-line defenses to protect against exogenous H2O2 challenges in minimal growth medium.
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2023-01-13
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Dissertation