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Volker Muller - One of the best experts on this subject based on the ideXlab platform.
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the pyruvate ferredoxin oxidoreductase of the thermophilic Acetogen thermoanaerobacter kivui
FEBS Open Bio, 2021Co-Authors: Alexander Katsyv, Mirko Basen, Marie Charlotte Schoelmerich, Volker MullerAbstract:Pyruvate:ferredoxin oxidoreductase (PFOR) is a key enzyme in bacterial anaerobic metabolism. Since a low-potential ferredoxin (Fd2- ) is used as electron carrier, PFOR allows for hydrogen evolution during heterotrophic growth as well as pyruvate synthesis during lithoautotrophic growth. The thermophilic Acetogenic model bacterium Thermoanaerobacter kivui can use both modes of lifestyle, but the nature of the PFOR in this organism was previously unestablished. Here, we have isolated PFOR to apparent homogeneity from cells grown on glucose. Peptide mass fingerprinting revealed that it is encoded by pfor1. PFOR uses pyruvate as an electron donor and methylene blue (1.8 U·mg-1 ) and ferredoxin (Fd; 27.2 U·mg-1 ) as electron acceptors, and the reaction is dependent on thiamine pyrophosphate, pyruvate, coenzyme A, and Fd. The pH and temperature optima were 7.5 and 66 °C, respectively. We detected 13.6 mol of iron·mol of protein-1 , consistent with the presence of three predicted [4Fe-4S] clusters. The ability to provide reduced Fd makes PFOR an interesting auxiliary enzyme for enzyme assays. To simplify and speed up the purification procedure, we established a protocol for homologous protein production in T. kivui. Therefore, pfor1 was cloned and expressed in T. kivui and the encoded protein containing a genetically engineered His-tag was purified in only two steps to apparent homogeneity. The homologously produced PFOR1 had the same properties as the enzyme from T. kivui. The enzyme can be used as auxiliary enzyme in enzymatic assays that require reduced Fd as electron donor, such as electron-bifurcating enzymes, to keep a constant level of reduced Fd.
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homologous production one step purification and proof of na transport by the rnf complex from acetobacterium woodii a model for Acetogenic conversion of c1 substrates to biofuels
Biotechnology for Biofuels, 2020Co-Authors: Anja Wiechmann, Dragan Trifunovic, Sophie Klein, Volker MullerAbstract:Capture and storage of the energy carrier hydrogen as well as of the greenhouse gas carbon dioxide are two major problems that mankind faces currently. Chemical catalysts have been developed, but only recently a group of anaerobic bacteria that convert hydrogen and carbon dioxide to acetate, formate, or biofuels such as ethanol has come into focus, the Acetogenic bacteria. These biocatalysts produce the liquid organic hydrogen carrier formic acid from H2 + CO2 or even carbon monoxide with highest rates ever reported. The autotrophic, hydrogen-oxidizing, and CO2-reducing Acetogens have in common a specialized metabolism to catalyze CO2 reduction, the Wood–Ljungdahl pathway (WLP). The WLP does not yield net ATP, but is hooked up to a membrane-bound respiratory chain that enables ATP synthesis coupled to CO2 fixation. The nature of the respiratory enzyme has been an enigma since the discovery of these bacteria and has been unraveled in this study. We have produced a His-tagged variant of the ferredoxin:NAD oxidoreductase (Rnf complex) from the model Acetogen Acetobacterium woodii, solubilized the enzyme from the cytoplasmic membrane, and purified it by Ni2+–NTA affinity chromatography. The enzyme was incorporated into artificial liposomes and catalyzed Na+ transport coupled to ferredoxin-dependent NAD reduction. Our results using the purified enzyme do not only verify that the Rnf complex from A. woodii is Na+-dependent, they also demonstrate for the first time that this membrane-embedded molecular engine creates a Na+ gradient across the membrane of A. woodii which can be used for ATP synthesis. We present a protocol for homologous production and purification for an Rnf complex. The enzyme catalyzed electron-transfer driven Na+ export and, thus, our studies provided the long-awaited biochemical proof that the Rnf complex is a respiratory enzyme.
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revealing formate production from carbon monoxide in wild type and mutants of rnf and ech containing Acetogens acetobacterium woodii and thermoanaerobacter kivui
Microbial Biotechnology, 2020Co-Authors: Fabian M Schwarz, Anja Wiechmann, Sarah Ciurus, Surbhi Jain, Christoph Baum, Mirko Basen, Volker MullerAbstract:Acetogenic bacteria have gained much attraction in recent years as they can produce different biofuels and biochemicals from H2 plus CO2 or even CO alone, therefore opening a promising alternative route for the production of biofuels from renewable sources compared to existing sugar-based routes. However, CO metabolism still raises questions concerning the biochemistry and bioenergetics in many Acetogens. In this study, we focused on the two Acetogenic bacteria Acetobacterium woodii and Thermoanaerobacter kivui which, so far, are the only identified Acetogens harbouring a H2 -dependent CO2 reductase and furthermore belong to different classes of 'Rnf'- and 'Ech-Acetogens'. Both strains catalysed the conversion of CO into the bulk chemical acetate and formate. Formate production was stimulated by uncoupling the energy metabolism from the Wood-Ljungdahl pathway, and specific rates of 1.44 and 1.34 mmol g-1 h-1 for A. woodii ∆rnf and T. kivui wild type were reached. The demonstrated CO-based formate production rates are, to the best of our knowledge, among the highest rates ever reported. Using mutants of ∆hdcr, ∆cooS, ∆hydBA, ∆rnf and ∆ech2 with deficiencies in key enzyme activities of the central metabolism enabled us to postulate two different CO utilization pathways in these two model organisms.
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energetics and application of heterotrophy in Acetogenic bacteria
Applied and Environmental Microbiology, 2016Co-Authors: Kai Schuchmann, Volker MullerAbstract:Acetogenic bacteria are a diverse group of strictly anaerobic bacteria that utilize the Wood-Ljungdahl pathway for CO2 fixation and energy conservation. These microorganisms play an important part in the global carbon cycle and are a key component of the anaerobic food web. Their most prominent metabolic feature is autotrophic growth with molecular hydrogen and carbon dioxide as the substrates. However, most members also show an outstanding metabolic flexibility for utilizing a vast variety of different substrates. In contrast to autotrophic growth, which is hardly competitive, metabolic flexibility is seen as a key ability of Acetogens to compete in ecosystems and might explain the almost-ubiquitous distribution of Acetogenic bacteria in anoxic environments. This review covers the latest findings with respect to the heterotrophic metabolism of Acetogenic bacteria, including utilization of carbohydrates, lactate, and different alcohols, especially in the model Acetogen Acetobacterium woodii Modularity of metabolism, a key concept of pathway design in synthetic biology, together with electron bifurcation, to overcome energetic barriers, appears to be the basis for the amazing substrate spectrum. At the same time, Acetogens depend on only a relatively small number of enzymes to expand the substrate spectrum. We will discuss the energetic advantages of coupling CO2 reduction to fermentations that exploit otherwise-inaccessible substrates and the ecological advantages, as well as the biotechnological applications of the heterotrophic metabolism of Acetogens.
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CO Metabolism in the Thermophilic Acetogen Thermoanaerobacter kivui.
Applied and environmental microbiology, 2016Co-Authors: Marie Charlotte Weghoff, Volker MullerAbstract:ABSTRACT The thermophilic Acetogenic bacterium Thermoanaerobacter kivui, previously described not to use carbon monoxide as a carbon and energy source, was adapted to grow on CO. This was achieved by using a preculture grown on H 2 plus CO 2 and by increasing the CO concentration in small, 10% increments. T. kivui was finally able to grow within a 100% CO atmosphere. Growth on CO was found in complex and mineral media, and vitamins were not required. Carbon monoxide consumption was accompanied by acetate and hydrogen production. Cells also grew on synthesis gas (syngas) with the simultaneous use of CO and H 2 coupled to acetate production. CO oxidation in resting cells was coupled to hydrogen and acetate production and accompanied by the synthesis of ATP. A protonophore abolished ATP synthesis but stimulated H 2 production, which is consistent with a chemiosmotic mechanism of ATP synthesis. Hydrogenase activity was highest in crude extracts of CO-grown cells, and carbon monoxide dehydrogenase (CODH) activity was highest in H 2 -plus-CO 2 - or CO-grown cells. The genome of T. kivui harbors two CODH gene clusters, and both CODH proteins were present in crude extracts, but one CODH was more prevalent in crude extracts from CO-grown cells.
Harold L Drake - One of the best experts on this subject based on the ideXlab platform.
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competing formate and carbon dioxide utilizing prokaryotes in an anoxic methane emitting fen soil
Applied and Environmental Microbiology, 2011Co-Authors: Sindy Hunger, Oliver Schmidt, Maik Hilgarth, Marcus A Horn, Steffen Kolb, Ralf Conrad, Harold L DrakeAbstract:Methanogenesis in wetlands is dependent on intermediary substrates derived from the degradation of biopolymers. Formate is one such substrate and is stimulatory to methanogenesis and Acetogenesis in anoxic microcosms of soil from the fen Schloppnerbrunnen. Formate dissimilation also yields CO2 as a potential secondary substrate. The objective of this study was to resolve potential differences between anaerobic formate- and CO2-utilizing prokaryotes of this fen by stable isotope probing. Anoxic soil microcosms were pulsed daily with low concentrations of [13C]formate or 13CO2 (i.e., [13C]bicarbonate). Taxa were evaluated by assessment of 16S rRNA genes, mcrA (encoding the alpha-subunit of methyl-coenzyme M reductase), and fhs (encoding formyltetrahydrofolate synthetase). Methanogens, Acetogens, and formate-hydrogen lyase-containing taxa appeared to compete for formate. Genes affiliated with Methanocellaceae, Methanobacteriaceae, Acetobacteraceae, and Rhodospirillaceae were 13C enriched (i.e., labeled) in [13C]formate treatments, whereas genes affiliated with Methanosarcinaceae, Conexibacteraceae, and Solirubrobacteraceae were labeled in 13CO2 treatments. [13C]acetate was enriched in [13C]formate treatments, but labeling of known Acetogenic taxa was not detected. However, several phylotypes were affiliated with Acetogen-containing taxa (e.g., Sporomusa). Methanosaetaceae-affiliated methanogens appeared to participate in the consumption of acetate. Twelve and 58 family-level archaeal and bacterial 16S rRNA phylotypes, respectively, were detected, approximately half of which had no isolated representatives. Crenarchaeota constituted half of the detected archaeal 16S rRNA phylotypes. The results highlight the unresolved microbial diversity of the fen Schloppnerbrunnen, suggest that differing taxa competed for the same substrate, and indicate that Methanocellaceae, Methanobacteriaceae, Methanosarcinaceae, and Methanosaetaceae were linked to the production of methane, but they do not clearly resolve the taxa responsible for the apparent conversion of formate to acetate.
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carbon metabolism of the moderately acid tolerant Acetogen clostridium drakei isolated from peat
Fems Microbiology Letters, 2008Co-Authors: Anita S Gosner, Ralph S Tanner, Flynn W Picardal, Harold L DrakeAbstract:A moderately acid-tolerant, malodorous bacterium, strain FP, was isolated from peat that had a pore water pH of c. 4.2. The 16S rRNA gene sequence of FP was closely related to that of Acetogens Clostridium drakei, Clostridium scatologenes, and Clostridium carboxidivorans. The DNA-DNA reassociation values obtained with DNA from FP and that of these three Acetogens approximated 80%, 64%, and 59%, respectively, indicating that FP was a new strain of C. drakei. FP had broad pH and temperature ranges (3.6-7.4 and 5-40 degrees C, respectively), and metabolized a wide range of substrates, including cellobiose, glucose, xylose, vanillate, ferulate, lactate, propanol, formate, H(2)-CO(2), and CO-CO(2). Acetate was the primary reduced end product, and substrate/product stoichiometries were indicative of Acetogenesis at circumneutral pH. Butyrate and H(2) became significant products from glucose at low pH. FP tolerated and could consume moderate amounts of O(2). These results (1) demonstrate that peat can harbor Acetogens with a broad substrate range and tolerance to transient exposure to O(2), and (2) confirm that C. drakei, the type strain of which was originally isolated from an acidic coal mine pond, occurs in moderately acidic habitats.
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trophic interaction of the aerotolerant anaerobe clostridium intestinale and the Acetogen sporomusa rhizae sp nov isolated from roots of the black needlerush juncus roemerianus
Microbiology, 2006Co-Authors: Anita S Gosner, Charles R. Lovell, Daria Schulz, Sonja Trenz, George Acker, Kirsten Kusel, Harold L DrakeAbstract:Acetogens were enumerated from root homogenates of the black needlerush Juncus roemerianus obtained from a nearly pristine salt marsh. An isolated colony, ST1, yielded Acetogenic activity and was initially thought to be a pure culture; however, ST1 was subsequently found to be composed of an aerotolerant fermentative anaerobe (RC) and an Acetogen (RST) (T indicates type strain). The two spore-forming mesophiles were separated by selective cultivation under conditions favouring the growth of either RC or RST. The 16S rRNA gene sequence of RC was 99 % similar to that of Clostridium intestinale, indicating that RC was a new isolate of this clostridial species. The rRNA gene sequence most similar to that of RST was only 96 % similar to that of RST and was from a species of the Acetogenic genus Sporomusa, indicating that RST was a new sporomusal species; the name Sporomusa rhizae sp. nov. is proposed. RC grew at the expense of saccharides. H2-forming butyrate fermentation was the primary catabolism utilized by RC under anoxic conditions, while homolactate fermentation was the primary catabolism under oxic conditions. RC consumed O2 and tolerated 20 % O2 in the headspace of shaken broth cultures. In contrast, RST was Acetogenic, utilized H2, lactate and formate, did not utilize saccharides, and could not tolerate high concentrations of O2. RST grew by trophic interaction with RC on saccharides via the uptake of H2, and, to a lesser extent, lactate and formate produced by RC. Co-cultures of the two organisms yielded high amounts of acetate. These results indicate that (i) previously uncharacterized species of Sporomusa are associated with Juncus roots and (ii) trophic links to O2-consuming aerotolerant anaerobes might contribute to the in situ activities and survival strategies of Acetogens in salt marsh rhizospheres, a habitat subject to gradients of plant-derived O2.
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physiology of the thermophilic Acetogen moorella thermoacetica
Research in Microbiology, 2004Co-Authors: Harold L Drake, Steven L. DanielAbstract:Moorella thermoacetica (originally isolated as Clostridium thermoaceticum) has served as the primary Acetogenic bacterium for the resolution of the acetyl coenzyme A (acetyl-CoA) or Wood-Lijungdahl pathway, a metabolic pathway that (i) autotrophically assimilates CO2 and (ii) is centrally important to the turnover of carbon in many habitats. The purpose of this article is to highlight the diverse physiological features of this model Acetogen and to examine some of the consequences of its metabolic capabilities.
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thermicanus aegyptius gen nov sp nov isolated from oxic soil a fermentative microaerophile that grows commensally with the thermophilic Acetogen moorella thermoacetica
Applied and Environmental Microbiology, 1999Co-Authors: Anita S Gosner, Richard Devereux, Nadja Ohnemuller, Georg Acker, Erko Stackebrandt, Harold L DrakeAbstract:A thermophilic, fermentative microaerophile (ET-5b) and a thermophilic Acetogen (ET-5a) were coisolated from oxic soil obtained from Egypt. The 16S rRNA gene sequence of ET-5a was 99.8% similar to that of the classic Acetogen Moorella thermoacetica. Further analyses confirmed that ET-5a was a new strain of M. thermoacetica. For ET-5b, the nearest 16S rRNA gene sequence similarity value to known genera was approximately 88%. ET-5b was found to be a motile rod with a genomic G+C content of 50.3 mol%. Cells were weakly gram positive and lacked spores. Growth was optimal at 55 to 60°C and pH 6.5 to 7.0. ET-5b grew under both oxic and anoxic conditions, but growth was erratic under atmospheric concentrations of O2. Utilizable substrates included oligosaccharides and monosaccharides. Acetate, formate, and succinate supported growth only under oxic conditions. Saccharides yielded succinate, lactate, ethanol, acetate, formate, and H2 under anoxic conditions; fermentation products were also formed under oxic conditions. A new genus is proposed, the type strain being Thermicanus aegyptius ET-5b gen. nov., sp. nov. (DSMZ 12793). M. thermoacetica ET-5a (DSMZ 12797) grew commensally with T. aegyptius ET-5b on oligosaccharides via the interspecies transfer of H2 formate, and lactate. In support of this interaction, uptake hydrogenase and formate dehydrogenase specific activities were fundamentally greater in M. thermoacetica ET-5a than in T. aegyptius ET-5b. These results demonstrate that (i) soils subject to high temperatures harbor uncharacterized thermophilic microaerophiles, (ii) the classic Acetogen M. thermoacetica resides in such soils, and (iii) trophic links between such soil bacteria might contribute to their in situ activities.
Emma J. Gagen - One of the best experts on this subject based on the ideXlab platform.
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hydrogenotrophic culture enrichment reveals rumen lachnospiraceae and ruminococcaceae Acetogens and hydrogen responsive bacteroidetes from pasture fed cattle
Fems Microbiology Letters, 2015Co-Authors: Emma J. Gagen, Jagadish Padmanabha, C S McsweeneyAbstract:Molecular information suggests that there is a broad diversity of Acetogens in the rumen, distinct from any currently isolated Acetogens. We combined molecular analysis with enrichment culture techniques to investigate this diversity further. Methane-inhibited, hydrogenotrophic enrichment cultures produced acetate as the dominant end product. Acetyl-CoA synthase gene analysis revealed putative Acetogens in the cultures affiliated with the Lachnospiraceae and Ruminococcaceae as has been found in other rumen studies. No formyltetrahydrofolate synthetase genes affiliating with Acetogens or with 'homoAcetogen similarity' scores >90% were identified. To further investigate the hydrogenotrophic populations in these cultures and link functional gene information with 16S rRNA gene identity, cultures were subcultured quickly, twice, through medium without exogenous hydrogen, followed by incubation without exogenous hydrogen. Comparison of cultures lacking hydrogen and their parent cultures revealed novel Lachnospiraceae and Ruminococcaceae that diminished in the absence of hydrogen, supporting the hypothesis that they were likely the predominant Acetogens in the enrichments. Interestingly, a range of Bacteroidetes rrs sequences that demonstrated <86% identity to any named isolate also diminished in cultures lacking hydrogen. Acetogens or sulphate reducers from the Bacteroidetes have not been reported previously; therefore this observation requires further investigation.
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Investigation of a new Acetogen isolated from an enrichment of the tammar wallaby forestomach.
BMC microbiology, 2014Co-Authors: Emma J. Gagen, Jagadish Padmanabha, Rafat Al Jassim, Mark Morrison, Jiakun Wang, Jing Liu, Isabela Pena Carvalho De Carvalho, Jianxin Liu, Richard I. Webb, Stuart E. DenmanAbstract:Background Forestomach fermentation in Australian marsupials such as wallabies and kangaroos, though analogous to rumen fermentation, results in lower methane emissions. Insights into hydrogenotrophy in these systems could help in devising strategies to reduce ruminal methanogenesis. Reductive Acetogenesis may be a significant hydrogen sink in these systems and previous molecular analyses have revealed a novel diversity of putative Acetogens in the tammar wallaby forestomach.
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investigation of a new Acetogen isolated from an enrichment of the tammar wallaby forestomach
BMC Microbiology, 2014Co-Authors: Emma J. Gagen, Jagadish Padmanabha, Jiakun Wang, Jing Liu, Isabela Pena Carvalho De Carvalho, Jianxin Liu, Richard I. WebbAbstract:Background: Forestomach fermentation in Australian marsupials such as wallabies and kangaroos, though analogous to rumen fermentation, results in lower methane emissions. Insights into hydrogenotrophy in these systems could help in devising strategies to reduce ruminal methanogenesis. Reductive Acetogenesis may be a significant hydrogen sink in these systems and previous molecular analyses have revealed a novel diversity of putative Acetogens in the tammar wallaby forestomach. Results: Methanogen-inhibited enrichment cultures prepared from tammar wallaby forestomach contents consumed hydrogen and produced primarily acetate. Functional gene (formyltetrahydrofolate synthetase and acetyl-CoA synthase) analyses revealed a restricted diversity of Clostridiales species as the putative Acetogens in the cultures. A new Acetogen (growth on H2/CO2 with acetate as primary end product) designated isolate TWA4, was obtained from the cultures. Isolate TWA4 classified within the Lachnospiraceae and demonstrated >97% rrs identity to previously isolated kangaroo Acetogens. Isolate TWA4 was a potent hydrogenotroph and demonstrated excellent mixotrophic growth (concomitant consumption of hydrogen during heterotrophic growth) with glycerol. Mixotrophic growth of isolate TWA4 on glycerol resulted in increased cell densities and acetate production compared to autotrophic growth. Co-cultures with an autotrophic methanogen Methanobrevibacter smithii revealed that isolate TWA4 performed reductive Acetogenesis under high hydrogen concentration (>5 mM), but not at low concentrations. Under heterotrophic growth conditions, isolate TWA4 did not significantly stimulate methanogenesis in a co-culture with M. smithii contrary to the expectation for organisms growing fermentatively. Conclusions: The unique properties of tammar wallaby Acetogens might be contributing factors to reduced methanogen numbers and methane emissions from tammar wallaby forestomach fermentation, compared to ruminal fermentation. The macropod forestomach may be a useful source of Acetogens for future strategies to reduce methane emissions from ruminants, particularly if these strategies also include some level of methane suppression and/or Acetogen stimulation, for example by harnessing mixotrophic growth capabilities
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Methanogen colonisation does not significantly alter Acetogen diversity in lambs isolated 17 h after birth and raised aseptically
Microbial Ecology, 2012Co-Authors: Emma J. Gagen, Stuart E. Denman, Christopher S. Mcsweeney, Pascale Mosoni, Rafat Jassim, Evelyne ForanoAbstract:Reductive Acetogenesis is not competitive with methanogenesis in adult ruminants, whereas Acetogenic bacteria are the dominant hydrogenotrophs in the early rumen microbiota. The ecology of hydrogenotrophs in the developing rumen was investigated using young lambs, raised in sterile isolators, and conventional adult sheep. Two lambs were born naturally, left with their dams for 17 h and then placed into a sterile isolator and reared aseptically. They were inoculated with cellulolytic bacteria and later with Methanobrevibacter sp. 87.7 to investigate the effect of methanogen establishment on the rumen Acetogen population since they lacked cultivable representatives of methanogens. Putative Acetogens were investigated by acetyl-CoA synthase and formyltetrahydrofolate synthetase gene analysis and methanogens by methyl coenzyme reductase A gene analysis. Unexpectedly, a low abundant but diverse population of methanogens (predominantly Methanobrevibacter spp.) was identified in isolated lambs pre-inoculation with Mbb. sp 87.7, which was similar to the community structure in conventional sheep. In contrast, potential Acetogen diversity in isolated lambs and conventional sheep was different. Potential Acetogens affiliated between the Lachnospiraceae and Clostridiaceae in conventional sheep and with the Blautia genus and the Lachnospiraceae in isolated lambs. The establishment of Mbb. sp. 87.7 (1,000-fold increase in methanogens) did not substantially affect Acetogen diversity.
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Functional gene analysis suggests different Acetogen populations in the Bovine Rumen and Tammar Wallaby Forestomach
Applied and environmental microbiology, 2010Co-Authors: Emma J. Gagen, Stuart E. Denman, Jagadish Padmanabha, Someshwar Zadbuke, Rafat Al Jassim, Mark Morrison, Christopher S. McsweeneyAbstract:Reductive Acetogenesis via the acetyl coenzyme A (acetyl-CoA) pathway is an alternative hydrogen sink to methanogenesis in the rumen. Functional gene-based analysis is the ideal approach for investigating organisms capable of this metabolism (Acetogens). However, existing tools targeting the formyltetrahydrofolate synthetase gene (fhs) are compromised by lack of specificity due to the involvement of formyltetrahydrofolate synthetase (FTHFS) in other pathways. Acetyl-CoA synthase (ACS) is unique to the acetyl-CoA pathway and, in the present study, acetyl-CoA synthase genes (acsB) were recovered from a range of Acetogens to facilitate the design of acsB-specific PCR primers. fhs and acsB libraries were used to examine Acetogen diversity in the bovine rumen and forestomach of the tammar wallaby (Macropus eugenii), a native Australian marsupial demonstrating foregut fermentation analogous to rumen fermentation but resulting in lower methane emissions. Novel, deduced amino acid sequences of acsB and fhs affiliated with the Lachnospiraceae in both ecosystems and the Ruminococcaeae/Blautia group in the rumen. FTHFS sequences that probably originated from nonAcetogens were identified by low "homoAcetogen similarity" scores based on analysis of FTHFS residues, and comprised a large proportion of FTHFS sequences from the tammar wallaby forestomach. A diversity of FTHFS and ACS sequences in both ecosystems clustered between the Lachnospiraceae and Clostridiaceae Acetogens but without close sequences from cultured isolates. These sequences probably originated from novel Acetogens. The community structures of the acsB and fhs libraries from the rumen and the tammar wallaby forestomach were different (LIBSHUFF, P < 0.001), and these differences may have significance for overall hydrogenotrophy in both ecosystems.
V Müller - One of the best experts on this subject based on the ideXlab platform.
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Ethylene glycol metabolism in the Acetogen Acetobacterium woodii
Journal of bacteriology, 2016Co-Authors: Dragan Trifunović, Kai Schuchmann, V MüllerAbstract:ABSTRACT The Acetogenic bacterium Acetobacterium woodii is able to grow by the oxidation of diols, such as 1,2-propanediol, 2,3-butanediol, or ethylene glycol. Recent analyses demonstrated fundamentally different ways for oxidation of 1,2-propanediol and 2,3-butanediol. Here, we analyzed the metabolism of ethylene glycol. Our data demonstrate that ethylene glycol is dehydrated to acetaldehyde, which is then disproportionated to ethanol and acetyl coenzyme A (acetyl-CoA). The latter is further converted to acetate, and this pathway is coupled to ATP formation by substrate-level phosphorylation. Apparently, the product ethanol is in part further oxidized and the reducing equivalents are recycled by reduction of CO2 to acetate in the Wood-Ljungdahl pathway. Biochemical data as well as the results of protein synthesis analysis are consistent with the hypothesis that the propane diol dehydratase (PduCDE) and CoA-dependent propionaldehyde dehydrogenase (PduP) proteins, encoded by the pdu gene cluster, also catalyze ethylene glycol dehydration to acetaldehyde and its CoA-dependent oxidation to acetyl-CoA. Moreover, genes encoding bacterial microcompartments as part of the pdu gene cluster are also expressed during growth on ethylene glycol, arguing for a dual function of the Pdu microcompartment system. IMPORTANCE Acetogenic bacteria are characterized by their ability to use CO2 as a terminal electron acceptor by a specific pathway, the Wood-Ljungdahl pathway, enabling in most Acetogens chemolithoautotrophic growth with H2 and CO2. However, Acetogens are very versatile and can use a wide variety of different substrates for growth. Here we report on the elucidation of the pathway for utilization of ethylene glycol by the model Acetogen Acetobacterium woodii. This diol is degraded by dehydration to acetaldehyde followed by a disproportionation to acetate and ethanol. We present evidence that this pathway is catalyzed by the same enzyme system recently described for the utilization of 1,2-propanediol. The enzymes for ethylene glycol utilization seem to be encapsulated in protein compartments, known as bacterial microcompartments.
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CO Metabolism in the Acetogen Acetobacterium woodii
Applied and environmental microbiology, 2015Co-Authors: Johannes Bertsch, V MüllerAbstract:The Wood-Ljungdahl pathway allows Acetogenic bacteria to grow on a number of one-carbon substrates, such as carbon dioxide, formate, methyl groups, or even carbon monoxide. Since carbon monoxide alone or in combination with hydrogen and carbon dioxide (synthesis gas) is an increasingly important feedstock for third-generation biotechnology, we studied CO metabolism in the model Acetogen Acetobacterium woodii. When cells grew on H2-CO2, addition of 5 to 15% CO led to higher final optical densities, indicating the utilization of CO as a cosubstrate. However, the growth rate was decreased by the presence of small amounts of CO, which correlated with an inhibition of H2 consumption. Experiments with resting cells revealed that the degree of inhibition of H2 consumption was a function of the CO concentration. Since the hydrogen-dependent CO2 reductase (HDCR) of A. woodii is known to be very sensitive to CO, we speculated that cells may be more tolerant toward CO when growing on formate, the product of the HDCR reaction. Indeed, addition of up to 25% CO did not influence growth rates on formate, while the final optical densities and the production of acetate increased. Higher concentrations (75 and 100%) led to a slight inhibition of growth and to decreasing rates of formate and CO consumption. Experiments with resting cells revealed that the HDCR is a site of CO inhibition. In contrast, A. woodii was not able to grow on CO as a sole carbon and energy source, and growth on fructose-CO or methanol-CO was not observed.
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2,3-Butanediol Metabolism in the Acetogen Acetobacterium woodii.
Applied and environmental microbiology, 2015Co-Authors: Verena Hess, Dragan Trifunović, Olga Oyrik, V MüllerAbstract:ABSTRACT The Acetogenic bacterium Acetobacterium woodii is able to reduce CO2 to acetate via the Wood-Ljungdahl pathway. Only recently we demonstrated that degradation of 1,2-propanediol by A. woodii was not dependent on Acetogenesis, but that it is disproportionated to propanol and propionate. Here, we analyzed the metabolism of A. woodii on another diol, 2,3-butanediol. Experiments with growing and resting cells, metabolite analysis and enzymatic measurements revealed that 2,3-butanediol is oxidized in an NAD+-dependent manner to acetate via the intermediates acetoin, acetaldehyde, and acetyl coenzyme A. Ethanol was not detected as an end product, either in growing cultures or in cell suspensions. Apparently, all reducing equivalents originating from the oxidation of 2,3-butanediol were funneled into the Wood-Ljungdahl pathway to reduce CO2 to another acetate. Thus, the metabolism of 2,3-butanediol requires the Wood-Ljungdahl pathway.
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NonAcetogenic Growth of the Acetogen Acetobacterium woodii on 1,2-Propanediol
Journal of bacteriology, 2014Co-Authors: Kai Schuchmann, Silke Schmidt, Antonio Martinez Lopez, Christina Kaberline, Martin Kuhns, Wolfram Lorenzen, Helge B. Bode, Friederike Joos, V MüllerAbstract:Acetogenic bacteria can grow by the oxidation of various substrates coupled to the reduction of CO2 in the Wood-Ljungdahl pathway. Here, we show that growth of the Acetogen Acetobacterium woodii on 1,2-propanediol (1,2-PD) as the sole carbon and energy source is independent of Acetogenesis. Enzymatic measurements and metabolite analysis revealed that 1,2-PD is dehydrated to propionaldehyde, which is further oxidized to propionyl coenzyme A (propionyl-CoA) with concomitant reduction of NAD. NADH is reoxidized by reducing propionaldehyde to propanol. The potential gene cluster coding for the responsible enzymes includes genes coding for shell proteins of bacterial microcompartments. Electron microscopy revealed the presence of microcompartments as well as storage granules in cells grown on 1,2-PD. Gene clusters coding for the 1,2-PD pathway can be found in other Acetogens as well, but the distribution shows no relation to the phylogeny of the organisms.
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discovery of a ferredoxin nad oxidoreductase rnf in acetobacterium woodii
Annals of the New York Academy of Sciences, 2008Co-Authors: V Müller, Silke Schmidt, Eva Biegel, Frank Imkamp, Sabrina DillingAbstract:Acetogens use the Wood-Ljungdahl pathway for reduction of carbon dioxide to acetate. This pathway not only allows reoxidation of reducing equivalents during heterotrophic growth but also supports chemolithoautotrophic growth on H(2) + CO(2). The latter argues for this pathway being a source for net energy conservation, but the mechanism involved remains unknown. In addition to CO(2), Acetogens can use alternative electron acceptors, such as nitrate or caffeate. Caffeate respiration in the model Acetogen Acetobacterium woodii is coupled to energy conservation via a chemiosmotic mechanism, with Na(+) as coupling ion. The pathway and its bioenergetics were solved in some detail very recently. This review focuses on the regulation of caffeate respiration, describes the enyzmes involved, summarizes the evidence for a potential Na(+)-translocating ferredoxin:NAD(+)-oxidoreductase (Rnf complex) as a new coupling site, and hypothesizes on the role of this Rnf complex in the Wood-Ljungdahl pathway.
Lars K. Nielsen - One of the best experts on this subject based on the ideXlab platform.
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redox controls metabolic robustness in the gas fermenting Acetogen clostridium autoethanogenum
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Vishnuvardhan Mahamkali, Renato De Souza Pinto Lemgruber, Ryan Tappel, Sean Dennis Simpson, Michael Kopke, Kaspar Valgepea, Lars K. Nielsen, Manuel R PlanAbstract:Living biological systems display a fascinating ability to self-organize their metabolism. This ability ultimately determines the metabolic robustness that is fundamental to controlling cellular behavior. However, fluctuations in metabolism can affect cellular homeostasis through transient oscillations. For example, yeast cultures exhibit rhythmic oscillatory behavior in high cell-density continuous cultures. Oscillatory behavior provides a unique opportunity for quantitating the robustness of metabolism, as cells respond to changes by inherently compromising metabolic efficiency. Here, we quantify the limits of metabolic robustness in self-oscillating autotrophic continuous cultures of the gas-fermenting Acetogen Clostridium autoethanogenum Online gas analysis and high-resolution temporal metabolomics showed oscillations in gas uptake rates and extracellular byproducts synchronized with biomass levels. The data show initial growth on CO, followed by growth on CO and H2 Growth on CO and H2 results in an accelerated growth phase, after which a downcycle is observed in synchrony with a loss in H2 uptake. Intriguingly, oscillations are not linked to translational control, as no differences were observed in protein expression during oscillations. Intracellular metabolomics analysis revealed decreasing levels of redox ratios in synchrony with the cycles. We then developed a thermodynamic metabolic flux analysis model to investigate whether regulation in Acetogens is controlled at the thermodynamic level. We used endo- and exo-metabolomics data to show that the thermodynamic driving force of critical reactions collapsed as H2 uptake is lost. The oscillations are coordinated with redox. The data indicate that metabolic oscillations in Acetogen gas fermentation are controlled at the thermodynamic level.
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quantitative analysis of tetrahydrofolate metabolites from clostridium autoethanogenum
Metabolomics, 2018Co-Authors: Renato De Souza Pinto Lemgruber, Mark P Hodson, Ryan Tappel, Sean Dennis Simpson, Michael Kopke, Kaspar Valgepea, Lars K. Nielsen, Esteban MarcellinAbstract:Introduction Quantification of tetrahydrofolates (THFs), important metabolites in the Wood–Ljungdahl pathway (WLP) of Acetogens, is challenging given their sensitivity to oxygen.
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arginine deiminase pathway provides atp and boosts growth of the gas fermenting Acetogen clostridium autoethanogenum
Metabolic Engineering, 2017Co-Authors: Kaspar Valgepea, Renato De Souza Pinto Lemgruber, Mark P Hodson, Michael Kopke, Lars K. Nielsen, Manuel R Plan, Kim Q Loi, James B Y H Behrendorff, Esteban MarcellinAbstract:Acetogens are attractive organisms for the production of chemicals and fuels from inexpensive and non-food feedstocks such as syngas (CO, CO2 and H2). Expanding their product spectrum beyond native compounds is dictated by energetics, particularly ATP availability. Acetogens have evolved sophisticated strategies to conserve energy from reduction potential differences between major redox couples, however, this coupling is sensitive to small changes in thermodynamic equilibria. To accelerate the development of strains for energy-intensive products from gases, we used a genome-scale metabolic model (GEM) to explore alternative ATP-generating pathways in the gas-fermenting Acetogen Clostridium autoethanogenum. Shadow price analysis revealed a preference of C. autoethanogenum for nine amino acids. This prediction was experimentally confirmed under heterotrophic conditions. Subsequent in silico simulations identified arginine (ARG) as a key enhancer for growth. Predictions were experimentally validated, and faster growth was measured in media containing ARG (tD~4h) compared to growth on yeast extract (tD~9h). The growth-boosting effect of ARG was confirmed during autotrophic growth. Metabolic modelling and experiments showed that acetate production is nearly abolished and fast growth is realised by a three-fold increase in ATP production through the arginine deiminase (ADI) pathway. The involvement of the ADI pathway was confirmed by metabolomics and RNA-sequencing which revealed a ~500-fold up-regulation of the ADI pathway with an unexpected down-regulation of the Wood-Ljungdahl pathway. The data presented here offer a potential route for supplying cells with ATP, while demonstrating the usefulness of metabolic modelling for the discovery of native pathways for stimulating growth or enhancing energy availability.