Deconstructing Methanosarcina acetivorans into an acetogenic archaeon

The reductive acetyl-coenzyme A (acetyl-CoA) pathway, whereby carbon dioxide is sequentially reduced to acetyl-CoA via coenzyme-bound C1 intermediates, is the only autotrophic pathway that can at the same time be the means for energy conservation. A conceptually similar metabolism and a key process in the global carbon cycle is methanogenesis, the biogenic formation of methane. All known methanogenic archaea depend on methanogenesis to sustain growth and use the reductive acetyl-CoA pathway for autotrophic carbon fixation. Here, we converted a methanogen into an acetogen and show that Methanosarcina acetivorans can dispense with methanogenesis for energy conservation completely. By targeted disruption of the methanogenic pathway, followed by adaptive evolution, a strain was created that sustained growth via carbon monoxide–dependent acetogenesis. A minute flux (less than 0.2% of the carbon monoxide consumed) through the methane-liberating reaction remained essential, indicating that currently living methanogens utilize metabolites of this reaction also for anabolic purposes. These results suggest that the metabolic flexibility of methanogenic archaea might be much greater than currently known. Also, our ability to deconstruct a methanogen into an acetogen by merely removing cellular functions provides experimental support for the notion that methanogenesis could have evolved from the reductive acetyl-coenzyme A pathway.

The metabolic network of the last bacterial common ancestor

Bacteria are the most abundant cells on Earth. They are generally regarded as ancient, but due to striking diversity in their metabolic capacities and widespread lateral gene transfer, the physiology of the first bacteria is unknown. From 1089 reference genomes of bacterial anaerobes, we identified 146 protein families that trace to the last bacterial common ancestor, LBCA, and form the conserved predicted core of its metabolic network, which requires only nine genes to encompass all universal metabolites. Our results indicate that LBCA performed gluconeogenesis towards cell wall synthesis, and had numerous RNA modifications and multifunctional enzymes that permitted life with low gene content. In accordance with recent findings for LUCA and LACA, analyses of thousands of individual gene trees indicate that LBCA was rod-shaped and the first lineage to diverge from the ancestral bacterial stem was most similar to modern Clostridia, followed by other autotrophs that harbor the acetyl-CoA pathway.

Key bacterial taxa and metabolic pathways affecting gut short-chain fatty acid profiles in early life

Infant gut microbiota development affects the host physiology throughout life, and short-chain fatty acids (SCFAs) are promising key metabolites mediating microbiota-host relationships. Here, we investigated dense longitudinally collected faecal samples from 12 subjects during the first 2 years (n = 1048) to identify early life gut SCFA patterns and their relationships with the microbiota. Our results revealed three distinct phases of progression in the SCFA profiles: early phase characterised by low acetate and high succinate, middle-phase characterised by high lactate and formate and late-phase characterised by high propionate and butyrate. Assessment of the SCFA–microbiota relationships revealed that faecal butyrate is associated with increased Clostridiales and breastfeeding cessation, and that diverse and personalised assemblage of Clostridiales species possessing the acetyl-CoA pathway play major roles in gut butyrate production. We also found an association between gut formate and some infant-type bifidobacterial species, and that human milk oligosaccharides (HMO)-derived fucose is the substrate for formate production during breastfeeding. We identified genes upregulated in fucose and fucosylated HMO utilisation in infant-type bifidobacteria. Notably, bifidobacteria showed interspecific and intraspecific variation in the gene repertoires, and cross-feeding of fucose contributed to gut formate production. This study provides an insight into early life SCFA–microbiota relationships, which is an important step for developing strategies for modulating lifelong health.

Formate-Dependent Acetogenic Utilization of Glucose by the Fecal Acetogen Clostridium bovifaecis

ABSTRACT Acetogenic bacteria are a diverse group of anaerobes that use the reductive acetyl coenzyme A (acetyl-CoA) (Wood-Ljungdahl) pathway for CO2 fixation and energy conservation. The conversion of 2 mol CO2 into acetyl-CoA by using the Wood-Ljungdahl pathway as the terminal electron accepting process is the most prominent metabolic feature for these microorganisms. However, here, we describe that the fecal acetogen Clostridium bovifaecis strain BXX displayed poor metabolic capabilities of autotrophic acetogenesis, and acetogenic utilization of glucose occurred only with the supplementation of formate. Genome analysis of Clostridium bovifaecis revealed that it contains almost the complete genes of the Wood-Ljungdahl pathway but lacks the gene encoding formate dehydrogenase, which catalyzes the reduction of CO2 to formate as the first step of the methyl branch of the Wood-Ljungdahl pathway. The lack of a gene encoding formate dehydrogenase was verified by PCR, reverse transcription-PCR analysis, enzyme activity assay, and its formate-dependent acetogenic utilization of glucose on DNA, RNA, protein, and phenotype level, respectively. The lack of a formate dehydrogenase gene may be associated with the adaption to a formate-rich intestinal environment, considering the isolating source of strain BXX. The formate-dependent acetogenic growth of Clostridium bovifaecis provides insight into a unique metabolic feature of fecal acetogens. IMPORTANCE The acetyl-CoA pathway is an ancient pathway of CO2 fixation, which converts 2 mol of CO2 into acetyl-CoA. Autotrophic growth with H2 and CO2 via the acetyl-CoA pathway as the terminal electron accepting process is the most unique feature of acetogenic bacteria. However, the fecal acetogen Clostridium bovifaecis strain BXX displayed poor metabolic capabilities of autotrophic acetogenesis, and acetogenic utilization of glucose occurred only with the supplementation of formate. The formate-dependent acetogenic growth of Clostridium bovifaecis was associated with its lack of a gene encoding formate dehydrogenase, which may result from adaption to a formate-rich intestinal environment. This study gave insight into a unique metabolic feature of fecal acetogens. Because of the requirement of formate for the acetogenic growth of certain acetogens, the ecological impact of acetogens could be more complex and important in the formate-rich environment due to their trophic interactions with other microbes.

A hydrogen dependent geochemical analogue of primordial carbon and energy metabolism

Hydrogen gas, H2, is generated in alkaline hydrothermal vents from reactions of iron containing minerals with water during a geological process called serpentinization. It has been a source of electrons and energy since there was liquid water on the early Earth, and it fuelled early anaerobic ecosystems in the Earth’s crust1–3. H2is the electron donor for the most ancient route of biological CO2fixation, the acetyl-CoA (or Wood-Ljungdahl) pathway, which unlike any other autotrophic pathway simultaneously supplies three key requirements for life: reduced carbon in the form of acetyl groups, electrons in the form of reduced ferredoxin, and ion gradients for energy conservation in the form of ATP4,5. The pathway is linear, not cyclic, it releases energy rather than requiring energy input, its enzymes are replete with primordial metal cofactors6,7, it traces to the last universal common ancestor8and abiotic, geochemical organic syntheses resembling segments of the pathway occur in hydrothermal vents today9,10. Laboratory simulations of the acetyl-CoA pathway’s reactions include the nonenzymatic synthesis of thioesters from CO and methylsulfide11, the synthesis of acetate12and pyruvate13from CO2using native iron or external electrochemical potentials14as the electron source. However, a full abiotic analogue of the acetyl-CoA pathway from H2and CO2as it occurs in life has not been reported to date. Here we show that three hydrothermal minerals — awaruite (Ni3Fe), magnetite (Fe3O4) and greigite (Fe3S4) — catalyse the fixation of CO2with H2at 100 °C under alkaline aqueous conditions. The product spectrum includes formate (100 mM), acetate (100 μM), pyruvate (10 μM), methanol (100 μM), and methane. With these simple catalysts, the overall exergonic reaction of the acetyl-CoA pathway is facile, shedding light on both the geochemical origin of microbial metabolism and on the nature of abiotic formate and methane synthesis in modern hydrothermal vents.

Energy conservation by a hydrogenase-dependent chemiosmotic mechanism in an ancient metabolic pathway

The ancient reductive acetyl-CoA pathway is employed by acetogenic bacteria to form acetate from inorganic energy sources. Since the central pathway does not gain net ATP by substrate-level phosphorylation, chemolithoautotrophic growth relies on the additional formation of ATP via a chemiosmotic mechanism. Genome analyses indicated that some acetogens only have an energy-converting, ion-translocating hydrogenase (Ech) as a potential respiratory enzyme. Although the Ech-encoding genes are widely distributed in nature, the proposed function of Ech as an ion-translocating chemiosmotic coupling site has neither been demonstrated in bacteria nor has it been demonstrated that it can be the only energetic coupling sites in microorganisms that depend on a chemiosmotic mechanism for energy conservation. Here, we show that the Ech complex of the thermophilic acetogenic bacteriumThermoanaerobacter kivuiis indeed a respiratory enzyme. Experiments with resting cells prepared fromT. kivuicultures grown on carbon monoxide (CO) revealed CO oxidation coupled to H2formation and the generation of a transmembrane electrochemical ion gradient (Δµ∼ion). Inverted membrane vesicles (IMVs) prepared from CO-grown cells also produced H2and ATP from CO (via a loosely attached CO dehydrogenase) or a chemical reductant. Finally, we show that Ech activity led to the translocation of both H+and Na+across the membrane of the IMVs. The H+gradient was then used by the ATP synthase for energy conservation. These data demonstrate that the energy-converting hydrogenase in concert with an ATP synthase may be the simplest form of respiration; it combines carbon dioxide fixation with the synthesis of ATP in an ancient pathway.

Sulfur reduction coupled with anaerobic ammonium oxidation drove prebiotic proto-anabolic networks

SUMMARYThe geochemical energy that drove the transition from geochemistry to biochemistry on early Earth remains unknown. Here, we show that the reduction of sulfurous species, such as thiosulfate, sulfite, elemental sulfur, and sulfate, coupled with anaerobic ammonium oxidation (Sammox), could have provided the primordial redox equivalents and proton potential for prebiotic proto-anabolic networks consisting of the reductive acetyl-CoA pathway combined with the incomplete reductive tricarboxylic acid (rTCA) cycle under mild hydrothermal conditions. Sammox-driven prebiotic proto-anabolic networks (SPPN) include CO2 reduction, esterification, reductive amination, pyrrole synthesis, and peptides synthesis, in one geochemical setting. Iron-sulfur (FeS) minerals, as the proto-catalysts, enhanced the efficiency of SPPN. Thiols/thioesters were used as the energy currency in non-enzymatic phosphate-independent metabolism and accelerated SPPN. Peptides that consisted of 15 proteinogenic amino acids were the end products of SPPN with bicarbonate as the only source of carbon. Most peptides shared high similarity with the truly minimal protein content (TMPC) of the last universal common ancestor (LUCA). The peptides and/or proteinogenic amino acids might have endowed SPPN with autocatalysis and homochirality. Thus, Sammox drove the coupling transformation of carbon, hydrogen, oxygen, nitrogen, sulfur, and/or iron simultaneously in the far-from-equilibrium environment, thereby initiating the emergence of biochemistry. The existing Sammox microorganisms might belong to the phylum of Planctomycetes, and might be transitional forms between the three domains of life.

Serpentinization: Connecting Geochemistry, Ancient Metabolism and Industrial Hydrogenation

Rock–water–carbon interactions germane to serpentinization in hydrothermal vents have occurred for over 4 billion years, ever since there was liquid water on Earth. Serpentinization converts iron(II) containing minerals and water to magnetite (Fe3O4) plus H2. The hydrogen can generate native metals such as awaruite (Ni3Fe), a common serpentinization product. Awaruite catalyzes the synthesis of methane from H2 and CO2 under hydrothermal conditions. Native iron and nickel catalyze the synthesis of formate, methanol, acetate, and pyruvate—intermediates of the acetyl-CoA pathway, the most ancient pathway of CO2 fixation. Carbon monoxide dehydrogenase (CODH) is central to the pathway and employs Ni0 in its catalytic mechanism. CODH has been conserved during 4 billion years of evolution as a relic of the natural CO2-reducing catalyst at the onset of biochemistry. The carbide-containing active site of nitrogenase—the only enzyme on Earth that reduces N2—is probably also a relic, a biological reconstruction of the naturally occurring inorganic catalyst that generated primordial organic nitrogen. Serpentinization generates Fe3O4 and H2, the catalyst and reductant for industrial CO2 hydrogenation and for N2 reduction via the Haber–Bosch process. In both industrial processes, an Fe3O4 catalyst is matured via H2-dependent reduction to generate Fe5C2 and Fe2N respectively. Whether serpentinization entails similar catalyst maturation is not known. We suggest that at the onset of life, essential reactions leading to reduced carbon and reduced nitrogen occurred with catalysts that were synthesized during the serpentinization process, connecting the chemistry of life and Earth to industrial chemistry in unexpected ways.