Energy Conservation in Syntrophic Acetate Oxidation

Energy conservation in syntrophic acetate oxidation Dissertation submitted for the degree of Doctor of Natural Sciences at the University of Constance Faculty of Sciences Department of Biology Presented by Dirk Oehler Tag der mündlichen Prüfung: 16.12.2014 Referent: Bernhard Schink Referent: Peter Kroth Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-279179 Table of Content Chapter 1 Summary 1 Chapter 2 Zusammenfassung 2 Chapter 3 Introduction 3 Chapter 4 Genome-guided analysis of physiological and morphological traits of the fermentative acetate oxidizer Thermacetogenium phaeum 12 Chapter 5 Cloning, Overexpression and Purification of Ferredoxin of Clostridium pasteurianum using a modified reconstitution protocol 33 Chapter 6 Proteome analysis of the syntrophic acetate oxidizer Thermacetogenium phaeum 43 Chapter 7 Discussion 58 Record of Achievement 63 References 64 Chapter 1 Summary The concept of syntrophic acetate oxidation exists for nearly half a century. However, research focus started recently on this topic after several syntrophic cooperation’s were identified. Most publications deal with the ecological role of this process, and it seems that the syntrophic acetate oxidizers are able to utilize acetate under stress conditions under which their main competitors the acetoclastic methanogens have difficulties to grow. However, less is known about the energy conservation of this pathway. It was already known for T. phaeum that the Wood-Ljungdahl pathway is used. Hence this work focused on sequencing and annotation of the genome and finding potential proton translocation enzymes via proteome analysis and enzyme assays and compare them with well-known systems of other acetogens. Genome analysis revealed a high abundance of different kinds of energy-conserving systems, but none of them could be excluded via proteome analysis or enzyme assays. Hence it is difficult to describe a distinct electron flow scheme. It is reasonable that not all enzymes are involved in energy conservation during syntrophic acetate oxidation and they are more likely involved during growth on other substrate. Though for further analysis of the energy conservation the proteins have to be purified. Due to low growth yield it is recommendable to use a recombinant expression for purification, which was successfully shown in this work. 1 Chapter 2 Zusammenfassung Das Konzept der syntrophen Acetat Oxidation existiert seit etwa einem halben Jahrhundert. Aber die Forschung konzentrierte sich auf das Thema erst, nachdem die ersten syntrophen Kooperationen isoliert wurden. Die meisten Publikationen konzentrierten sich auf die ökologische Rolle der syntrophen Acetat-Oxidierer. Es zeigte sich, dass diese unter Stressbedingungen in der Lage sind Acetat umzusetzen, wenn die acetoklastischen Methanogenen Probleme haben zu wachsen. Jedoch war wenig bekannt über die Energie konservierenden Mechanismen dieses Stoffwechselweges. Es war bisher bekannt, dass T. phaeum den Wood-Ljungdahl Weg nutzt. Daher konzentrierte sich diese Arbeit auf die Sequenzierung und Annotation des Genoms, um potentielle Protonen-translozierende Enzyme mit Hilfe von Proteom Analyse und Enzym Assays zu finden und diese mit gut untersuchten Systemen anderer Acetogenen zu vergleichen. Die Genom Analyse zeigt verschiedene Arten von Energie konservierenden Systemen, jedoch konnte keine von diesen durch Proteom Analyse und Enzym Assays ausgeschlossen werden. Daher ist es schwierig, ein bestimmtes Elektronenflussschema zu beschreiben. Es ist anzunehmen, dass nicht alle Energie konservierenden Systeme für die syntrophe Acetat Oxidation verwendet werden und es wahrscheinlicher ist, dass diese während des Wachstums auf anderen Substraten verwendet werden. Daher ist es notwendig, für eine weitere Analyse die Enzyme zu reinigen. Jedoch aufgrund des geringen Wachstumsertrages ist eine rekombinante Expression für die Reinigung zu empfehlen, diese konnte bereits erfolgreich in dieser Arbeit gezeigt werden. 2 Chapter 3 Introduction Up to 80% of the methane produced by microbes in anoxic environments derives from acetate1,2. Two ways of acetate conversion to methane are known: The aceticlastic reaction which is used by members of the order Methanosarcinales splits acetate into a methyl and carboxyl group. The methyl group is further reduced to methane, the carboxyl group is oxidized to carbon dioxide. The other pathway includes two microorganisms, one that oxidizes acetate completely to carbon dioxide and hydrogen and the second microorganism, a hydrogenotrophic methanogen, which forms methane from these products. This kind of cooperation, where two microorganisms depend on each other for energetic reasons to degrade a certain substrate, is called syntrophy3. The second pathway was first described by Barker4, but it took a long time until the first isolates using this pathway were described. One of the first observations on complete acetate conversion to methane was made by Hoppe-Seyler5. In the following decades the research focus was mainly on the identification of potential substrates for methane production. However, Buswell and Barker generalised the idea that every substrate leads to methane production in anoxic environments4,6. During this time the first pure culture of Methanosarcina strains7,8 and also the first acetogenic bacteria, which are involved in the formation of acetate, were obtained 9. But not until the development of the Hungate technique10 the isolation of anaerobic microorganisms became easier. During that time, despite the hypothesis of Barker, it was believed that the aceticlastic pathway is the only way of decomposition of acetate to methane, because only acetoclastic methanogens were discovered. This changed after the first syntrophic acetate oxidazing (SAO) microorganism were isolated by Zinder et al.11, which do not use the acetoclastic pathway. Using 14C-labeled acetate, these authors discovered that both carbon atoms of acetate are converted to methane and the methanogen isolated in this culture grew only on H2/CO2 or formate, which was direct evidence for the pathway proposed by Barker. Later several other thermophilic and mesophilic acetate-oxidizing cocultures were found12,13. With these findings the question arose through the recent years if both pathways compete for the same substrate in the same environment, and which conditions favour either pathway. Several factors were investigated in recent years such as temperature, pH, and concentration of ammonia or volatile acids to answer this question. Aceticlastic methanogens play a dominant role in conversion of acetate to methane. However, under stress conditions such as high ammonia or fatty acid concentration or high temperatures, the microbial community shifts to hydrogenotrophic 3 methanogens, and acetate is converted via the SAO14-18. These results also explain why it took so long to prove Barkers hypothesis because it appears that those cocultures are favoured only under stress conditions. This leads to the question what the potential ecological role of this pathway might be, because these experiments were performed in anaerobic digesters or samples taken from it, where such conditions more likely occur. Recent experiments show that this reaction may take place also in other habitats such as rice fields, profundal sediment or biofilms of an oil facility19-21. These experiments also indicate that while acetoclastic methanogens are dominant, increasing temperatures lead to a shift of the microbial community to the SAO. This suggests that while the aceticlastic pathway is the dominant one in certain habitats, the SAO microorganisms are dormant, but start to degrade acetate under stress conditions. So far several of the SAO microorganism are acetogenic bacteria (Strain AOR22, Clostridium ultunense13, Thermoacetogenium phaeum23), which are quite versatile with respect to the substrate usage24, and this may be another hint that those SAO grow on other substrates in these habitats and take over the acetate degradation if necessary. In contrast to the above mentioned SAO, Thermotoga lettingae grow syntrophically on acetate but is not considered as an acetogen25. Acetogenic prokaryotes Acetogens or homoacetogenic bacteria have been defined as anaerobes which use the acetyl- CoA pathway for CO2 reduction to form acetyl CoA either for assimilation purposes or to conserve energy26, which means that acetate as a product is not important to define a bacterium as an acetogen. For example Synthrophomonas wolfei growing in a syntrophic culture on butyrate converts 27 butyrate to acetate and H2 but acetate is formed by ß-oxidation via the crotonyl-CoA pathway . 28 Homoacetogens were first found in the 1930s in a sewage plant where it was shown that H2 dependent reduction of CO2 to acetate took place. In the 1980s research interest increased after it became apparent that acetogens are widely distributed and they use a new pathway for CO2 reduction and the key enzyme, acetyl-CoA synthase, was also found in sulfate reducing bacteria and aceticlastic methanogens24,29. The huge disadvantage in competing with other microorganism, e.g. sulfate reducers or methanogens, is the relatively low energy supply of that reaction, which can be seen by comparing the half-cell reaction of CO2/acetate (-290 mV) with CO2/methane (-240 mV) or sulfate/sulfide (-220 mV). However homoacetogens are able to use several different substrates as electron donor, such as CO, H2, carbohydrates, alcohols, fatty acids or

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