Central dissimiliatory pathways of the thermophilic acetogen Thermacetogenium phaeum

Thermacetogenium phaeum, the bacterium investigated in this work, can synthesize acetate from hydrogen and carbon dioxide and can as well degrade acetate with a syntrophic partner to hydrogen and carbon dioxide. The aim of this work is to investigate the central dissimilatory pathways of this organism and to characterize their key enzymes. Therefore, Thermacetogenium phaeum was grown axenically either with methanol, formate, hydrogen/CO2 , ethanol or ethanolamine or in co-culture with Methanothermobacter thermautotrophicus with ethanol, ethanolamine or acetate. The genome of Thermacetogenium phaeum was sequenced in 2012, which enabled a proteomic comparison of the different growth conditions. The thus gained results were confirmed by enzyme assays. It was demonstrated that the Wood-Ljungdahl pathway plays a central role under all investigated growth conditions. This raises the question which enzyme systems are responsible for energy conservation. For syntrophic growth with acetate, an alternative activation of acetate via an aldehyde:ferredoxin oxidoreductase was proposed, which would not need ATP investment. As a result, the Wood-Ljungdahl pathway produces one net ATP which, however, is partly consumed in the endergonic oxidation of methyl-tetrahydrofolate with NAD+. The methylene-tetrahydrofolate reductase poses an additional barrier during growth with acetate, as this enzyme releases electrons at an electron potential of -200 mV. These electrons cannot be easily transferred to NAD+ (-320 mV). Therefore, a system was proposed in which the electrons are transferred via a heterodisulfide reductase to a quinone pool. As an electronaccepting system a putatively periplasmic formate dehydrogenase was suggested. This system needs energy input in the form of a proton gradient created by ATPase. Furthermore, degradation pathways for methanol and ethanol were proposed. Microcompartments were formed exclusively during growth with ethanolamine, which was confirmed by transmission electron microscopy. A purification protocol for microcompartments was established and the growth with ethanolamine was investigated in detail. Ethanolamine is deaminated in the microcompartments to acetaldehyde which is then disproportionated to ethanol and acetate. The degradation of ethanol occurs in the cytoplasm as during axenic growth with ethanol as substrate.publishe