Biochemistry of thermophilic degradation of C1 and C2 compounds in Thermacetogenium phaeum

• Applicant: Dr. Nicolai Müller
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms
• Term: since 2015
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 280732050

Oxidation of acetate to CO2 by the thermophilic bacterium Thermacetogenium phaeum is accomplished in syntrophic cooperation with a methanogenic partner and occurs through the Wood-Ljungdahl pathway of acetogenesis (WLP). Its role in syntrophic acetate oxidation is incompletely understood, as the WLP is originally known from the opposite exergonic reaction, (CO2 to acetate). Thus, syntrophic acetate oxidation cannot be a simple reversal of the WLP. Most genes of the WLP are encoded in the genome of T. phaeum only once, meaning that the same reaction chain has to be used in both operating directions. We have shown that this reaction chain is present in both acetate oxidizing and acetate producing T. phaeum, and that reducing equivalents from acetate oxidation are transferred to the methanogenic partner by membrane bound formate dehydrogenase. The latter enzyme is exclusively present during syntrophic acetate oxidation and thus complements the WLP to allow its operation in the reverse direction. Acetate production occurs through the degradation of methanol, ethanol, H2/CO2 or ethanolamine in pure cultures of T. phaeum. Under all growth conditions, NAD+-independent methylene-THF reductase (MTHFR) and aldehyde:ferredoxin-oxidoreductase (AOR) are abundant in the proteome and also active in enzyme assays. However, the electron carriers of these redox enzymes were not identified yet, which will be accomplished in the research project proposed here. Degradation of ethanolamine occurs in bacterial microcompartments (BMC), which we previously demonstrated by electron microscopy in thin sections of T. phaeum. Our proposed study will focus on the isolation of functional BMC to reveal the function of BMC-associated AOR and acetylating acetaldehyde dehydrogenase (AcAldDH). The genome of T. phaeum contains only one gene for AcAldDH that is located in an ethanolamine utilizing (eut)-gene cluster which harbors genes for the BMC. Apparently, genes of the eut gene cluster in T. phaeum are differentially regulated by the presence or absence of ethanolamine, and AcAldDH, as required for all other growth conditions, seems to be recruited from the eut gene cluster, independently regulated from the ethanolamine stimulus. We will therefore investigate the regulatory mechanisms of this vital gene cluster.In summary, our previous DFG-funded research on T. phaeum, especially during syntrophic growth with acetate, has significantly contributed to the understanding of the energy metabolism of this bacterium. However, many questions on the redox interactions of the participating enzyme complexes are still unanswered. We will combine conventional biochemical methods and enzyme assays together with modern proteomics tools, especially cross-linking proteomics, to reveal the architecture of these enzyme complexes and their interactions during growth of T. phaeum with C1 and C2 compounds.

• DFG Programme: Research Grants