Explore the vast range of titles available.

Despite decades of study, electron flow and energy conservation in methanogenic Archaea are still not thoroughly understood. For methanogens without cytochromes, flavin-based electron bifurcation has been proposed as an essential energy-conserving mechanism that couples exergonic and endergonic reactions of methanogenesis. However, an alternative hypothesis posits that the energy-converting hydrogenase Eha provides a chemiosmosis-driven electron input to the endergonic reaction. In vivo evidence for both hypotheses is incomplete. By genetically eliminating all nonessential pathways of H₂ metabolism in the model methanogen Methanococcus maripaludis and using formate as an additional electron donor, we isolate electron flow for methanogenesis from flux through Eha. We find that Eha does not function stoichiometrically for methanogenesis, implying that electron bifurcation must operate in vivo. We show that Eha is nevertheless essential, and a substoichiometric requirement for H₂ suggests that its role is anaplerotic. Indeed, H₂ via Eha stimulates methanogenesis from formate when intermediates are not otherwise replenished. These results fit the model for electron bifurcation, which renders the methanogenic pathway cyclic, and as such requires the replenishment of intermediates. Defining a role for Eha and verifying electron bifurcation provide a complete model of methanogenesis where all necessary electron inputs are accounted for.

The rate of production of methane in many environments depends upon mutualistic interactions between sulfate‐reducing bacteria and methanogens. To enhance our understanding of these relationships, we took advantage of the fully sequenced genomes of Desulfovibrio vulgaris and Methanococcus maripaludis to produce and analyze the first multispecies stoichiometric metabolic model. Model results were compared to data on growth of the co‐culture on lactate in the absence of sulfate. The model accurately predicted several ecologically relevant characteristics, including the flux of metabolites and the ratio of D. vulgaris to M. maripaludis cells during growth. In addition, the model and our data suggested that it was possible to eliminate formate as an interspecies electron shuttle, but hydrogen transfer was essential for syntrophic growth. Our work demonstrated that reconstructed metabolic networks and stoichiometric models can serve not only to predict metabolic fluxes and growth phenotypes of single organisms, but also to capture growth parameters and community composition of simple bacterial communities. Synopsis Biological communities present tremendous modeling challenges because of the complex network of diverse interactions between species. A class of communities that has not been extensively modeled—but is of particular interest from ecological, geological, and engineering perspectives—is represented by microbial communities that thrive in oxygen‐free (anoxic) environments. These communities are vital components in numerous environments ranging from freshwater sediments and guts of insects and animals to wastewater treatment plants. They play a significant role in global cycling of carbon. Unlike communities of macro‐organisms, the flow of carbon through anaerobic communities depends to a large extent on the transfer of metabolites between species. Thus, it is essential that modelers of these communities first consider the metabolic networks determining the interactions among species. We report here on a metabolic model of a simple anaerobic community. This simple community consists of a bacterium and an archaeon that cooperate in a special mutualistic interaction called ‘syntrophy’ to degrade lactate into acetate and methane as the sole means of gaining energy for growth. The cooperation is based on the transfer of electrons from the bacterium to the archaeon in the form of hydrogen or formate. The archaeon uses the electrons to reduce carbon dioxide into methane. If the electrons are not transferred, then the archaeon will not have an energy source for growth. In turn, the bacteria will be unable to gain energy by oxidizing lactate unless their metabolites, primarily hydrogen and acetate, are kept at sufficiently low concentrations to make the reaction thermodynamically favorable. Such methanogenic syntrophies often form the final step of anaerobic trophic cascades and are therefore necessary for maintaining a continuous flux of metabolites through the community (Figure 1 ). Although a variety of anaerobic syntrophic associations have been examined, they have been characterized primarily in terms of bulk system properties and not as an integrated metabolic network (Schink and Stams, 2002 ). To develop a foundation for a more mechanistic understanding of syntrophic growth, we established a syntrophic interaction between two species whose genomes have been sequenced— Desulfovibrio vulgaris and Methanococcus maripaludis. D. vulgaris is a bacterium that can oxidize and obtain energy from a wide variety of carbon sources by sulfate respiration. M. maripaludis is an archaeon that obtains energy for growth by producing methane from carbon dioxide and hydrogen. The complete genome sequences of these two organisms were used as a basis to construct flux‐balance models of the central metabolisms of each growing independently and in syntrophic association. The models consist of a series of linear equations, each expressing a relationship between metabolites and the rate of flux through the reaction (Edwards et al , 2002 ). Because of the number of unknowns in the model, fluxes were calculated using linear optimization of a specific reaction, usually biomass production (Price et al , 2004 ). The single‐organism flux‐balance models were first refined by comparing the predicted biomass yields to experimental observations. These analyses provided insight into the physiology of growth of each species. Specifically, analysis of the D. vulgaris submodel suggested that two protons must be translocated to produce one ATP during respiration of sulfate. The M. maripaludis model provided insight into the likely number of protons that must be translocated during methane production and generated predictions about the influence of acetate consumption on biomass yield. The two models were then combined to form one model describing growth and metabolite accumulation when the organisms were growing syntrophically. A flux diagram showing the metabolic networks contained in the combined two‐species model, and an example of a simulation result is shown in Figure 2 . Experimental data for lactate and hydrogen uptake rates during growth of the syntrophic culture were used as inputs to the model to test whether it could accurately capture the main features of syntrophic growth. The model was able to predict fluxes of acetate, methane, and carbon dioxide. In addition, the model consistently predicted roughly two‐fold more D. vulgaris biomass than M. maripaludis biomass even when it was set to optimize M. maripaludis biomass. This result demonstrates the stoichiometric coupling of growth of this association and is consistent with our experimental data which showed a ratio of D. vulgaris to M. maripaludis cells of approximately 2–2.5 throughout growth in batch culture. The model and experimental studies were also used to address a long‐standing and central question concerning the mediator of electron transfer between the sulfate reducer and the methanogen. In addition to hydrogen, formate is thought to be a significant mediator under some growth conditions or in alternative species pairings. Additional simulations using the syntrophic model demonstrated that although either formate or hydrogen could serve as electron carriers, a minimum exchange of hydrogen was essential for syntrophic growth. Formate cannot serve as the exclusive mediator. A dominant role of hydrogen was confirmed by establishment of a syntrophic association between D. vulgaris and M. maripaludis strain MM 709 (Wood et al , 2003 ), which was mutated such that it could not obtain energy from formate. To our knowledge, this is the first flux‐balance model of an association involving two species. We have shown that even a relatively simple multispecies flux‐balance model is able to predict key features of community dynamics and provide a means to test the physiological parameters responsible for the observed growth characteristics. Flux‐balance modeling of even more complex microbial communities may therefore facilitate investigations of the links between gene content and community characteristics, helping us to understand and predict microbial processes in natural and engineered systems. Metabolic models of the central metabolism of Desulfovibrio vulgaris, Methanococcus maripaludis, and a combined model of their mutualistic interaction was developed and tested. The relatively simple multi‐species flux‐balance model was able to predict key features of microbial community dynamics. Analysis of the multi‐species model indicated that hydrogen is an essential and dominant mediator of the interaction between species but formate may complement hydrogen transfer.

In methanogenic Archaea, the final step of methanogenesis generates methane and a heterodisulfide of coenzyme M and coenzyme B (CoM-S-S-CoB). Reduction of this heterodisulfide by heterodisulfide reductase to regenerate HS-CoM and HS-CoB is an exergonic process. Thauer et al. [Thauer, et al. 2008 Nat Rev Microbiol 6:579—591] recently suggested that in hydrogenotrophic methanogens the energy of heterodisulfide reduction powers the most endergonic reaction in the pathway, catalyzed by the formylmethanofuran dehydrogenase, via flavin-based electron bifurcation. Here we present evidence that these two steps in methanogenesis are physically linked. We identify a protein complex from the hydrogenotrophic methanogen, Methanococcus maripaludis, that contains heterodisulfide reductase, formylmethanofuran dehydrogenase, F₄₂₀-nonreducing hydrogenase, and formate dehydrogenase. In addition to establishing a physical basis for the electron-bifurcation model of energy conservation, the composition of the complex also suggests that either H₂ or formate (two alternative electron donors for methanogenesis) can donate electrons to the heterodisulfide-H₂ via F₄₂₀-nonreducing hydrogenase or formate via formate dehydrogenase. Electron flow from formate to the heterodisulfide rather than the use of H₂ as an intermediate represents a previously unknown path of electron flow in methanogenesis. We further tested whether this path occurs by constructing a mutant lacking F₄₂₀-nonreducing hydrogenase. The mutant displayed growth equal to wild-type with formate but markedly slower growth with hydrogen. The results support the model of electron bifurcation and suggest that formate, like H₂, is closely integrated into the methanogenic pathway.

Hydrogenotrophic methanogenic Archaea require reduced ferredoxin as an anaplerotic source of electrons for methanogenesis. H(2) oxidation by the hydrogenase Eha provides these electrons, consistent with an H(2) requirement for growth. Here we report the identification of alternative pathways of ferredoxin reduction in Methanococcus maripaludis that operate independently of Eha to stimulate methanogenesis. A suppressor mutation that increased expression of the glycolytic enzyme glyceraldehyde-3-phosphate:ferredoxin oxidoreductase resulted in a strain capable of H(2)-independent ferredoxin reduction and growth with formate as the sole electron donor. In this background, it was possible to eliminate all seven hydrogenases of M. maripaludis. Alternatively, carbon monoxide oxidation by carbon monoxide dehydrogenase could also generate reduced ferredoxin that feeds into methanogenesis. In either case, the reduced ferredoxin generated was inefficient at stimulating methanogenesis, resulting in a slow growth phenotype. As methanogenesis is limited by the availability of reduced ferredoxin under these conditions, other electron donors, such as reduced coenzyme F(420), should be abundant. Indeed, when F(420)-reducing hydrogenase was reintroduced into the hydrogenase-free mutant, the equilibrium of H(2) production via an F(420)-dependent formate:H(2) lyase activity shifted markedly toward H(2) compared to the wild type. Hydrogenotrophic methanogens are thought to require H(2) as a substrate for growth and methanogenesis. Here we show alternative pathways in methanogenic metabolism that alleviate this H(2) requirement and demonstrate, for the first time, a hydrogenotrophic methanogen that is capable of growth in the complete absence of H(2). The demonstration of alternative pathways in methanogenic metabolism suggests that this important group of organisms is metabolically more versatile than previously thought.

Background Methanogenic Archaea play key metabolic roles in anaerobic ecosystems, where they use H 2 and other substrates to produce methane. Methanococcus maripaludis is a model for studies of the global response to nutrient limitations. Results We used high-coverage quantitative proteomics to determine the response of M. maripaludis to growth-limiting levels of H 2 , nitrogen, and phosphate. Six to ten percent of the proteome changed significantly with each nutrient limitation. H 2 limitation increased the abundance of a wide variety of proteins involved in methanogenesis. However, one protein involved in methanogenesis decreased: a low-affinity [Fe] hydrogenase, which may dominate over a higher-affinity mechanism when H 2 is abundant. Nitrogen limitation increased known nitrogen assimilation proteins. In addition, the increased abundance of molybdate transport proteins suggested they function for nitrogen fixation. An apparent regulon governed by the euryarchaeal nitrogen regulator NrpR is discussed. Phosphate limitation increased the abundance of three different sets of proteins, suggesting that all three function in phosphate transport. Conclusion The global proteomic response of M. maripaludis to each nutrient limitation suggests a wider response than previously appreciated. The results give new insight into the function of several proteins, as well as providing information that should contribute to the formulation of a regulatory network model.

NrpR is a transcriptional repressor of nitrogen assimilation genes that was recently discovered and characterized in the methanogenic archaeon Methanococcus maripaludis. NrpR homologues are widely distributed in Euryarchaeota and present in a few bacterial species. They exist in three different domain configurations: a single ORF encoding one NrpR domain following an N-terminal helix-turn-helix (HTH); a single ORF encoding two NrpR domains fused in tandem following an N-terminal HTH; and two separate ORFs, one with a single domain following an N-terminal HTH and one with a single domain without a HTH. Phylogenetic analysis indicated that the NrpR family forms five distinct groups: the single domain HTH type, the two domains of the double domain HTH type and the two separately encoded domains. To determine the function of diverse NrpR homologues, representative genes in were expressed an Methanococcus maripaludis nrpR deletion mutant. Homologues from species that possess a single gene restored regulated repression, regardless of domain structure. In the case of Methanosarcina acetivorans that contains two genes, both were required. The results show that distantly related NrpR homologues that are present in widely dispersed phyla regulate the expression of nitrogen assimilation genes in a similar fashion.

Hydrogenotrophic methanogenic Archaea require reduced ferredoxin as an anaplerotic source of electrons for methanogenesis. H 2 oxidation by the hydrogenase Eha provides these electrons, consistent with an H 2 requirement for growth. Here we report the identification of alternative pathways of ferredoxin reduction in Methanococcus maripaludis that operate independently of Eha to stimulate methanogenesis. A suppressor mutation that increased expression of the glycolytic enzyme glyceraldehyde-3-phosphate:ferredoxin oxidoreductase resulted in a strain capable of H 2 -independent ferredoxin reduction and growth with formate as the sole electron donor. In this background, it was possible to eliminate all seven hydrogenases of M. maripaludis . Alternatively, carbon monoxide oxidation by carbon monoxide dehydrogenase could also generate reduced ferredoxin that feeds into methanogenesis. In either case, the reduced ferredoxin generated was inefficient at stimulating methanogenesis, resulting in a slow growth phenotype. As methanogenesis is limited by the availability of reduced ferredoxin under these conditions, other electron donors, such as reduced coenzyme F 420 , should be abundant. Indeed, when F 420 -reducing hydrogenase was reintroduced into the hydrogenase-free mutant, the equilibrium of H 2 production via an F 420 -dependent formate:H 2 lyase activity shifted markedly toward H 2 compared to the wild type. IMPORTANCE Hydrogenotrophic methanogens are thought to require H 2 as a substrate for growth and methanogenesis. Here we show alternative pathways in methanogenic metabolism that alleviate this H 2 requirement and demonstrate, for the first time, a hydrogenotrophic methanogen that is capable of growth in the complete absence of H 2 . The demonstration of alternative pathways in methanogenic metabolism suggests that this important group of organisms is metabolically more versatile than previously thought. Hydrogenotrophic methanogens are thought to require H 2 as a substrate for growth and methanogenesis. Here we show alternative pathways in methanogenic metabolism that alleviate this H 2 requirement and demonstrate, for the first time, a hydrogenotrophic methanogen that is capable of growth in the complete absence of H 2 . The demonstration of alternative pathways in methanogenic metabolism suggests that this important group of organisms is metabolically more versatile than previously thought.

Despite decades of study, electron flow and energy conservation in methanogenic Archaea are still not thoroughly understood. For methanogens without cytochromes, flavin-based electron bifurcation has been proposed as an essential energy-conserving mechanism that couples exergonic and endergonic reactions of methanogenesis. However, an alternative hypothesis posits that the energy-converting hydrogenase Eha provides a chemiosmosis-driven electron input to the endergonic reaction. In vivo evidence for both hypotheses is incomplete. By genetically eliminating all nonessential pathways of H2 metabolism in the model methanogen Methanococcus maripaludis and using formate as an additional electron donor, we isolate electron flow for methanogenesis from flux through Eha. We find that Eha does not function stoichiometrically for methanogenesis, implying that electron bifurcation must operate in vivo. We show that Eha is nevertheless essential, and a substoichiometric requirement for H2 suggests that its role is anaplerotic. Indeed, H2 via Eha stimulates methanogenesis from formate when intermediates are not otherwise replenished. These results fit the model for electron bifurcation, which renders the methanogenic pathway cyclic, and as such requires the replenishment of intermediates. Defining a role for Eha and verifying electron bifurcation provide a complete model of methanogenesis where all necessary electron inputs are accounted for.

The gastrointestinal tract is a multi-organ system crucial for efficient nutrient uptake and barrier immunity. Advances in genomics and a surge in gastrointestinal diseases 1 , 2 has fuelled efforts to catalogue cells constituting gastrointestinal tissues in health and disease 3 . Here we present systematic integration of 25 single-cell RNA sequencing datasets spanning the entire healthy gastrointestinal tract in development and in adulthood. We uniformly processed 385 samples from 189 healthy controls using a newly developed automated quality control approach (scAutoQC), leading to a healthy reference atlas with approximately 1.1 million cells and 136 fine-grained cell states. We anchor 12 gastrointestinal disease datasets spanning gastrointestinal cancers, coeliac disease, ulcerative colitis and Crohn’s disease to this reference. Utilizing this 1.6 million cell resource (gutcellatlas.org), we discover epithelial cell metaplasia originating from stem cells in intestinal inflammatory diseases with transcriptional similarity to cells found in pyloric and Brunner’s glands. Although previously linked to mucosal healing 4 , we now implicate pyloric gland metaplastic cells in inflammation through recruitment of immune cells including T cells and neutrophils. Overall, we describe inflammation-induced changes in stem cells that alter mucosal tissue architecture and promote further inflammation, a concept applicable to other tissues and diseases. The study provides a comprehensive transcriptomic atlas of the human gastrointestinal tract across the lifespan, highlighting inflammation-induced changes in epithelial stem cells that alter mucosal architecture and promote further inflammation.

Repair of defects in the common bile duct is hampered by a lack of healthy donor tissue. Developing human extrahepatic cholangiocyte organoids and testing them in mouse models may provide a way to overcome this limitation. The treatment of common bile duct (CBD) disorders, such as biliary atresia or ischemic strictures, is restricted by the lack of biliary tissue from healthy donors suitable for surgical reconstruction. Here we report a new method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree in the form of extrahepatic cholangiocyte organoids (ECOs) for regenerative medicine applications. The resulting ECOs closely resemble primary cholangiocytes in terms of their transcriptomic profile and functional properties. We explore the regenerative potential of these organoids in vivo and demonstrate that ECOs self-organize into bile duct–like tubes expressing biliary markers following transplantation under the kidney capsule of immunocompromised mice. In addition, when seeded on biodegradable scaffolds, ECOs form tissue-like structures retaining biliary characteristics. The resulting bioengineered tissue can reconstruct the gallbladder wall and repair the biliary epithelium following transplantation into a mouse model of injury. Furthermore, bioengineered artificial ducts can replace the native CBD, with no evidence of cholestasis or occlusion of the lumen. In conclusion, ECOs can successfully reconstruct the biliary tree, providing proof of principle for organ regeneration using human primary cholangiocytes expanded in vitro .