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dc.contributor.advisorCook, Gregory M.
dc.contributor.advisorBerney, Michael
dc.contributor.advisorSimmonds, Robin
dc.contributor.authorGreening, Christopher Andrew
dc.identifier.citationGreening, C. A. (2014). Physiological roles of the three [NiFe]-hydrogenases in Mycobacterium smegmatis (Thesis, Doctor of Philosophy). University of Otago. Retrieved from
dc.description.abstractMycobacterium is a genus of Actinobacteria that is represented by successful environmental saprophytes and notorious obligate pathogens. Despite primarily deriving their energy from aerobic respiration of organic carbon sources, many mycobacterial species can survive for extended periods during carbon-limitation and oxygen-limitation by entering non-replicative persistent states. There remains an incomplete molecular understanding of how mycobacteria adapt and persist following such environmental changes. In this work, we revealed that hydrogen metabolism has a crucial role in the energetics of the model soil bacterium Mycobacterium smegmatis mc2155 during growth, carbon-limitation, and oxygen-limitation in batch cultures. This metabolism is mediated by three phylogenetically distinct, differentially regulated [NiFe]-hydrogenases, Hyd1 (MSMEG_2262-2263), Hyd2 (MSMEG_2720-2719), and Hyd3 (MSMEG_3932-3928). Gas chromatography revealed that Hyd1 and Hyd2 are both oxygen-dependent uptake hydrogenases. These enzymes have unusually high affinities for H2 (Km Hyd1 = 130 nM; Km Hyd2 = 50 nM), and hence can scavenge the trace concentrations of H2 (0.40 nM) found in the troposphere. While M. smegmatis mc2155 could not sustain chemolithoautotrophy, Hyd1 and Hyd2 enabled mixotrophic growth on organic carbon sources and tropospheric H2; the deletion of these enzymes resulted in severe growth phenotypes, possibly due to loss of reductant required for CO2 fixation. Though contitutively expressed at low levels, Hyd1 and Hyd2 are more active during carbon-limitation; in this condition, they maintain flux through the respiratory chain through coupling oxidation of tropospheric H2 to reduction of O2. In contrast, Hyd3 was activated upon oxygen-limitation by the oxygen- and redox-responsive DosS/DosT-DosR two-component system. This enzyme directly couples the oxidation of reduced coenzymes to the evolution of H2 in a fermentation process; this is essential for intracellular redox homeostasis and long-term survival during oxygen-limitation. The study reveals some crucial roles of lithotrophy and fermentation in the genus Mycobacterium. In addition, it resolves the classes of enzyme that are responsible for the oxidation of tropospheric H2 in soil ecosystems; this process is predicted to be the main global sink in the biogeochemical cycle of molecular hydrogen.
dc.publisherUniversity of Otago
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dc.titlePhysiological roles of the three [NiFe]-hydrogenases in Mycobacterium smegmatis
dc.language.rfc3066en of Microbiology & Immunology of Philosophy of Otago
otago.openaccessAbstract Only
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