One-carbon metabolism in acetogenic and sulfate-reducing bacteria

ABSTRACT One-carbon metabolism in acetogenic and sulfate-reducing bacteria Life on earth is sustained by the constant cycling of six essential elements: oxygen, hydrogen, nitrogen, sulfur, phosphorous, and carbon. The continuous cycling of these elements is due to geo-chemical processes and the combined metabolism of all life on earth. Microorganisms like bacteria and archaea play a major role in this. This is also true for the carbon cycle. In this cycle carbon dioxide and methane are two important C-1 compounds present in the atmosphere. Carbon dioxide is the highest oxidative state of carbon while methane is the highest reduced form of carbon. The art to use light to produce organic compounds and conserve energy from the highest oxidative state of carbon is called photosynthesis and is performed by plants, algae and cyanobacteria. Photosynthesis is not the only system to fix carbon from carbon dioxide. Chemolithotrophs can fix carbon from carbon dioxide using inorganic electron donors, like hydrogen. Subsequently, fixed carbon can be used by other organisms, which also makes life possible for them. Microorganisms play a major role in the degradation of complex organic matter, producing smaller compounds including C-1 compounds. C-1 compounds other than carbon dioxide are e.g. carbon monoxide (CO), methanol and formate. Bacteria and archaea can utilize these relative simple compounds in the presence and absence of oxygen, alone and in cooperation with others (syntrophy). The complex and simple carbon compounds are finally oxidized to carbon dioxide, which closes the carbon cycle. In addition to their importance to the carbon cycle, one carbon compounds like CO, methanol and formate are important for several applications. They are used as a building block for the production of chemicals. They are also used for bioremediation purposes and for wastewater treatment. Therefore, it is important to gain insight in the one carbon metabolism of microorganisms. The research described in this thesis focuses on the proteins and encoding genes involved in anaerobic degradation of C1 compounds by using genome and proteome analysis. In Chapter 2 the genomes of two closely related sulfate-reducing bacteria, Desulfotomaculum nigrificans and D. nigrificans strain CO-1-SRB, are compared including their CO metabolism. Both the D. nigrificans type strain and strain CO-1-SRB can grow with CO. However, there are differences. The type strain can grow with 20% CO coupled to sulfate reduction in the presence of yeast extract, while strain CO-1-SRB can grow with 100% CO in the presence of yeast extract. Moreover, strain CO-1-SRB can grow with CO in the presence and absence of sulfate. It couples the oxidation of CO to carbon dioxide to hydrogen production. This conversion, the protein complex involved, and the genes coding for these proteins have been described before in other microorganisms. The genome of strain CO-1-SRB contains the genes coding for this protein complex while the genome of the D. nigrificans type strain does not. However, the genome of the type strain contains genes encoding two other CO dehydrogenases. This indicates that one or both are necessary for the type strain to grow with 20% CO. Additional research on the different CO dehydrogenases and their regulation is essential to assess if all different CO dehydrogenases can facilitate growth and how they are linked to for example creating a proton motive force for ATP production. The methanol metabolism of anaerobic bacteria seems to differ more from that of methanogens than initially described. Methanogens use a methanol methyltransferase system that consists of two methyltranferases, methyltransferase 1 (subunits MtaB and MtaC) and methyltransferase 2 (MtaA). The methyl group from methanol is transferred to the MtaC subunit by MtaB. Subsequently, MtaA transports the methyl group from MtaC to coenzyme M. A genome and proteome analysis of the acetogenic bacterium Sporomusa strain An4 suggests that instead of MtaA a methyl-tetrahydrofolate methyltransferase is involved in the transport of the methyl bound to MtaC to tetrahydrofolate (Chapter 3). Research done on the methanol metabolism of the sulfate-reducing bacterium Desulfotomaculum kuznetsovii also shows differences with that of methanogens (Chapter 5). The methanol methyltransferase system is vitamin B12 and cobalt dependent. D. kuznetsovii grows with methanol and sulfate, but can do this in presence and absence of vitamin B12 and cobalt. In the absence of vitamin B12 and cobalt D. kuznetsovii grows slower and reaches a lower optical density compared to growth in the presence of vitamin B12 and cobalt. This suggests that D. kuznetsovii can use both a methyltransferase system and a vitamin B12 and cobalt independent system for the degradation of methanol. Proteome results confirm this and suggest that the vitamin B12 and cobalt independent system consists of an alcohol dehydrogenase and an aldehyde ferredoxin oxidoreductase. Moreover, the alcohol dehydrogenase seems to be involved in the oxidation of both methanol and ethanol (Chapter 5). The presence of two methanol degradation pathways give an ecological advantage to D. kuznetsovii in environments containing methanol and sulfate but limiting cobalt and vitamin B12 concentrations. Future research should elucidate if more sulfate-reducing bacteria, or perhaps even acetogenic bacteria, have two methanol degrading pathways. Additional to the genome analysis of D. kuznetsovii to assess the genes coding for the proteins involved in the two methanol degradation pathways, the genome was also analyzed to assess genes encoding other degradation pathways (Chapter 4). This analysis shows many genes present in D. kuznetsovii are also present in Pelotomaculum thermopropionicum. P. thermopropionicum is known to degrade propionate in syntrophic interaction with a methanogen. D. kuznetsovii can also degrade propionate, but only coupled to sulfate reduction and not in syntrophy with methanogens. Moreover, P. thermopropionicum is not able to reduce sulfate. D. kuznetsovii is the only close related, non-syntrophic, propionate degrader of which the genome is available. Therefore, a genome comparison was performed between D. kuznetsovii and P. thermopropionicum to define the differences between a non-syntrophic and a syntrophic lifestyle. D. kuznetsovii misses membrane bound protein complexes like hydrogenases and an extra-cytoplasmic formate dehydrogenase. In order to expand the analysis between non-syntrophs and syntrophs, more genomes of propionate- and butyrate-degrading bacteria were included (Chapter 6). This extended analysis shows that the genomes of non-syntrophs do not contain genes coding for an extra-cytoplasmic formate dehydrogenase, in contrast to all syntrophs included in the analysis. This indicates the importance of this protein complex and the importance of formate as an interspecies electron carrier in syntrophic degradation of propionate and butyrate. Thanks to the extra cytoplasmic formate dehydrogenase the syntrophic bacteria can couple the degradation of propionate and butyrate to formate production. Subsequently, the formate is utilized by methanogens to produce methane. This keeps the formate concentration low, which is necessary for the entire process to be energetically favorable.