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Title:Hydrogenase utilization and regulation in species of methanosarcina
Author(s):Mand, Thomas David
Director of Research:Metcalf, William W.
Doctoral Committee Chair(s):Metcalf, William W.
Doctoral Committee Member(s):Cronan, John E.; Cann, Isaac; Olsen, Gary J.
Department / Program:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Abstract:Methane producing archaea have a vital role in the global carbon cycle by facilitating the conversion of organic carbon into CO2 and methane. Species of Methanosarcina are unique among methanogens due to a versatile metabolism that allows the use of a wide range of substrates through four overlapping methanogenic pathways. Two of the pathways require H2 as a substrate, while the others can use H2 as an electron carrier via a hydrogen cycling mechanism, which was previously demonstrated for Methanosarcina barkeri. Both uses of H2 require the activity of three different hydrogenase enzymes, however, M. barkeri can also utilize an electron transport system that is independent of hydrogenase activity. In Chapter 2, the interconnected nature of H2-dependent and H2-independent metabolic pathways was investigated in a series of M. barkeri hydrogenase deletion mutants. Phenotypic analysis of these mutants, including characterization of growth, methanogenesis, and gene regulation, allowed detection of an alternative ferredoxin-dependent electron transport system that does not require the production or consumption of H2. Additionally, we found that H2-dependent inhibition of the oxidative branch of methanogenesis required active hydrogenases, and that M. barkeri was potentially able to regulate gene expression based on the redox state of coenzyme F420. In Chapter 3, I explored the regulation of hydrogenases in Methanosarcina acetivorans, a species that is incapable of metabolizing H2, yet encodes hydrogenases with high similarity to the active enzymes found in M. barkeri. Despite the lack of hydrogenase activity in its native host, I found that the Vht hydrogenase from M. acetivorans was fully active when expressed in M. barkeri. Further assessment of Vht from M. acetivorans revealed that transcript levels were diminished when compared to M. barkeri, and that the hydrogenase protein was not detectable when expressed from the native promoter. In addition to Vht regulation at the transcriptional and translational levels, I found that M. acetivorans has a mechanism of post-translational control that disallows maturation of the hydrogenase into a fully active enzyme. Finally, in Chapter 4 I summarize the significant findings of this research, and outline several projects for continued investigation of energy conservation and gene regulation in Methanosarcina.
Issue Date:2018-10-08
Rights Information:Copyright 2018 Thomas Mand
Date Available in IDEALS:2019-02-06
Date Deposited:2018-12

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