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It has recently been shown that in anaerobic microorganisms the tricarboxylic acid (TCA) cycle, including the seemingly irreversible citrate synthase reaction, can be reversed and used for autotrophic fixation of carbon 1 , 2 . This reversed oxidative TCA cycle requires ferredoxin-dependent 2-oxoglutarate synthase instead of the NAD-dependent dehydrogenase as well as extremely high levels of citrate synthase (more than 7% of the proteins in the cell). In this pathway, citrate synthase replaces ATP-citrate lyase of the reductive TCA cycle, which leads to the spending of one ATP-equivalent less per one turn of the cycle. Here we show, using the thermophilic sulfur-reducing deltaproteobacterium Hippea maritima , that this route is driven by high partial pressures of CO 2 . These high partial pressures are especially important for the removal of the product acetyl coenzyme A (acetyl-CoA) through reductive carboxylation to pyruvate, which is catalysed by pyruvate synthase. The reversed oxidative TCA cycle may have been functioning in autotrophic CO 2 fixation in a primordial atmosphere that is assumed to have been rich in CO 2 . In the deltaproteobacterium Hippea maritima , the tricarboxylic acid (TCA) cycle can be reversed by high partial pressures of CO 2 for the autotrophic fixation of carbon.

Classically, it is thought that citrate synthase only works in one direction: to catalyze the production of citrate from acetyl coenzyme A and oxaloacetate in the tricarboxylic acid (TCA) cycle. The TCA cycle can run in reverse to cleave citrate and fix carbon dioxide autotrophically, but this was thought to occur only with alternative enzymes, such as citrate lyase. Now Nunoura et al. and Mall et al. have discovered thermophilic bacteria with highly efficient and reversible citrate synthase that requires reduced ferredoxin (see the Perspective by Ragsdale). This function is undetectable by metagenomics, but classical biochemistry filled in the gaps seen between the genome sequences and the phenotypes of the organisms. The direction of catalysis depends on the availability of organic versus inorganic carbon and reflects a flexible bet-hedging strategy for survival in fluctuating environments. In evolutionary terms, this capacity might predate the classical TCA cycle and is likely to occur in a wide range of anaerobic microorganisms. Science , this issue p. 559 , p. 563 ; see also p. 517 Classical biochemistry reveals the occurrence of an unexpected capacity to reverse the tricarboxylic acid cycle in anaerobic microbes. Biological inorganic carbon fixation proceeds through a number of fundamentally different autotrophic pathways that are defined by specific key enzymatic reactions. Detection of the enzymatic genes in (meta)genomes is widely used to estimate the contribution of individual organisms or communities to primary production. Here we show that the sulfur-reducing anaerobic deltaproteobacterium Desulfurella acetivorans is capable of both acetate oxidation and autotrophic carbon fixation, with the tricarboxylic acid cycle operating either in the oxidative or reductive direction, respectively. Under autotrophic conditions, the enzyme citrate synthase cleaves citrate adenosine triphosphate independently into acetyl coenzyme A and oxaloacetate, a reaction that has been regarded as impossible under physiological conditions. Because this overlooked, energetically efficient carbon fixation pathway lacks key enzymes, it may function unnoticed in many organisms, making bioinformatical predictions difficult, if not impossible.

Archaea of the phylum Thaumarchaeota are among the most abundant prokaryotes on Earth and are widely distributed in marine, terrestrial, and geothermal environments. All studied Thaumarchaeota couple the oxidation of ammonia at extremely low concentrations with carbon fixation. As the predominant nitrifiers in the ocean and in various soils, ammonia-oxidizing archaea contribute significantly to the global nitrogen and carbon cycles. Here we provide biochemical evidence that thaumarchaeal ammonia oxidizers assimilate inorganic carbon via a modified version of the autotrophic hydroxypropionate/hydroxybutyrate cycle of Crenarchaeota that is far more energy efficient than any other aerobic autotrophic pathway. The identified genes of this cycle were found in the genomes of all sequenced representatives of the phylum Thaumarchaeota, indicating the environmental significance of this efficient CO ₂-fixation pathway. Comparative phylogenetic analysis of proteins of this pathway suggests that the hydroxypropionate/hydroxybutyrate cycle emerged independently in Crenarchaeota and Thaumarchaeota, thus supporting the hypothesis of an early evolutionary separation of both archaeal phyla. We conclude that high efficiency of anabolism exemplified by this autotrophic cycle perfectly suits the lifestyle of ammonia-oxidizing archaea, which thrive at a constantly low energy supply, thus offering a biochemical explanation for their ecological success in nutrient-limited environments.

The assimilation of carbon dioxide (CO₂) into organic material is quantitatively the most important biosynthetic process. We discovered that an autotrophic member of the archaeal order Sulfolobales, Metallosphaera sedula, fixed CO₂ with acetyl-coenzyme A (acetyl-CoA)/propionyl-CoA carboxylase as the key carboxylating enzyme. In this system, one acetyl-CoA and two bicarbonate molecules were reductively converted via 3-hydroxypropionate to succinyl-CoA. This intermediate was reduced to 4-hydroxybutyrate and converted into two acetyl-CoA molecules via 4-hydroxybutyryl-CoA dehydratase. The key genes of this pathway were found not only in Metallosphaera but also in Sulfolobus, Archaeoglobus, and Cenarchaeum species. Moreover, the Global Ocean Sampling database contains half as many 4-hydroxybutyryl-CoA dehydratase sequences as compared with those found for another key photosynthetic CO₂-fixing enzyme, ribulose-1,5-bisphosphate carboxylase-oxygenase. This indicates the importance of this enzyme in global carbon cycling.

The prokaryotic cell is traditionally seen as a “bag of enzymes,” yet its organization is much more complex than in this simplified view. By now, various microcompartments encapsulating metabolic enzymes or pathways are known for Bacteria. These microcompartments are usually small, encapsulating and concentrating only a few enzymes, thus protecting the cell from toxic intermediates or preventing unwanted side reactions. The hyperthermophilic, strictly anaerobic Crenarchaeon Ignicoccus hospitalis is an extraordinary organism possessing two membranes, an inner and an energized outer membrane. The outer membrane (termed here outer cytoplasmic membrane) harbors enzymes involved in proton gradient generation and ATP synthesis. These two membranes are separated by an intermembrane compartment, whose function is unknown. Major information processes like DNA replication, RNA synthesis, and protein biosynthesis are located inside the “cytoplasm” or central cytoplasmic compartment. Here, we show by immunogold labeling of ultrathin sections that enzymes involved in autotrophic CO₂ assimilation are located in the intermembrane compartment that we name (now) a peripheric cytoplasmic compartment. This separation may protect DNA and RNA from reactive aldehydes arising in the I. hospitalis carbon metabolism. This compartmentalization of metabolic pathways and information processes is unprecedented in the prokaryotic world, representing a unique example of spatiofunctional compartmentalization in the second domain of life.

Mesaconase catalyzes the hydration of mesaconate (methylfumarate) to (S)-citramalate. The enzyme participates in the methylaspartate pathway of glutamate fermentation as well as in the metabolism of various C5-dicarboxylic acids such as mesaconate or L-threo-β-methylmalate. We have recently shown that Burkholderia xenovorans uses a promiscuous class I fumarase to catalyze this reaction in the course of mesaconate utilization. Here we show that classical Escherichia coli class I fumarases A and B (FumA and FumB) are capable of hydrating mesaconate with 4% (FumA) and 19% (FumB) of the catalytic efficiency kcat/Km, compared to the physiological substrate fumarate. Furthermore, the genomes of 14.8% of sequenced Enterobacteriaceae (26.5% of E. coli, 90.6% of E. coli O157:H7 strains) possess an additional class I fumarase homologue which we designated as fumarase D (FumD). All these organisms are (opportunistic) pathogens. fumD is clustered with the key genes for two enzymes of the methylaspartate pathway of glutamate fermentation, glutamate mutase and methylaspartate ammonia lyase, converting glutamate to mesaconate. Heterologously produced FumD was a promiscuous mesaconase/fumarase with a 2- to 3-fold preference for mesaconate over fumarate. Therefore, these bacteria have the genetic potential to convert glutamate to (S)-citramalate, but the further fate of citramalate is still unclear. Our bioinformatic analysis identified several other putative mesaconase genes and revealed that mesaconases probably evolved several times from various class I fumarases independently. Most, if not all iron-dependent fumarases, are capable to catalyze mesaconate hydration.

Host cells respond to bacterial infection by producing itaconate, an inhibitor of bacterial metabolism, among other strategies. Biochemical characterization now defines genes known to be important for bacterial virulence as a new pathway that degrades itaconate into metabolic building blocks. Itaconate (methylenesuccinate) was recently identified as a mammalian metabolite whose production is substantially induced during macrophage activation. This compound is a potent inhibitor of isocitrate lyase, a key enzyme of the glyoxylate cycle, which is a pathway required for the survival of many pathogens inside the eukaryotic host. Here we show that numerous bacteria, notably many pathogens such as Yersinia pestis and Pseudomonas aeruginosa , have three genes for itaconate degradation. They encode itaconate coenzyme A (CoA) transferase, itaconyl-CoA hydratase and ( S )-citramalyl-CoA lyase, formerly referred to as CitE-like protein. These genes are known to be crucial for survival of some pathogens in macrophages. The corresponding enzymes convert itaconate into the cellular building blocks pyruvate and acetyl-CoA, thus enabling the bacteria to metabolize itaconate and survive in macrophages. The itaconate degradation and detoxification pathways of Yersinia and Pseudomonas are the result of convergent evolution. This work revealed a common persistence factor operating in many pathogenic bacteria.

Key Points The Archaea form the third domain of life alongside the other two domains, the Bacteria and Eukarya. Most cultivated autotrophic archaea live under conditions resembling the conditions of early life (no oxygen, high temperature and purely inorganic substrates), and their autotrophic pathways can serve as models for an ancestral metabolism. None of the autotrophic archaea seems to use the Calvin cycle for CO 2 fixation. Instead, they use three different CO 2 fixation mechanisms to generate acetyl-coenzyme A (acetyl-CoA), from which the biosynthesis of building blocks can start. The reductive acetyl-CoA pathway function in Euryarchaeota, notably in methanogens, and has the lowest energetic costs among the autotrophic CO 2 fixation pathways. However, the demanding requirements for metals, cofactors, anaerobiosis and substrates with low reducing potential restrict this pathway to a limited set of anoxic niches. Two recently discovered cycles function in Crenarchaeota, the dicarboxylate–hydroxybutyrate cycle and the hydroxypropionate–hydroxybutyrate cycle. They have in common the synthesis of succinyl-CoA from acetyl-CoA and two inorganic carbons, although this is accomplished in different ways and using different carboxylases. However, the regeneration of acetyl-CoA, the primary CO 2 acceptor, from succinyl-CoA is similar in both pathways. The oxygen-sensitive dicarboxylate–hydroxybutyrate cycle is restricted to the anaerobic Thermoproteales and Desulfurococcales, whereas the oxygen-insensitive hydroxypropionate–hydroxybutyrate cycle is restricted to the mostly aerobic Sulfolobales and possibly marine Crenarchaeota. The two lifestyles presuppose different electron donors with different redox potentials and different oxygen sensitivity of cofactors and enzymes. The distribution of an autotrophic pathway in bacteria and archaea depends on both the genetic predisposition (phylogeny) of the organisms and the constraints of their occupied niches (ecology). The main external factors are the presence of oxygen in the environment, but also the availability of trace metals and C 1 compounds. The energy demand of the autotrophic pathways is decisive under energy limitation and caused mainly by the costs for synthesizing autotrophy-related auxiliary enzymes. Further determinants are the main metabolic fluxes in an organism, the usage of CO 2 or HCO 3 − by the carboxylases of the pathway and the possibility of co-assimilating traces of organic compounds present in the environment. According to the 'metabolism first' theory, life started in a hydrothermal vent setting in the Hadean ocean with catalytic metal sulphide surfaces or compartments. The structural (and catalytic) similarity between the minerals themselves and the catalytic metal or Fe–S-containing centres of the enzymes or cofactors in the acetyl-CoA pathway suggests that minerals catalysed a primitive acetyl-CoA pathway. The unique features of this pathway indeed indicate that it might be close to an ancestral autotrophic carbon fixation mechanism. Recently a highly conserved, heat-stabile and bifunctional fructose 1,6-bisphosphate aldolase–phosphatase was identified in archaea and deep-branching lineages of bacteria. This enzyme is regarded as the pace-making ancestral gluconeogenic enzyme. The finding supports the idea that in evolution gluconeogenesis preceded glycolysis. The distribution pattern of this enzyme, its phylogenetic tree and the unidirectional catalysis lend further support to the theory of a chemolithoautotrophic origin of life. To date, three different autotrophic carbon fixation mechanisms have been found in archaea. Here, Georg Fuchs and colleagues describe these mechanisms and their phylogenetic distribution. As most cultivated autotrophic archaea live in conditions that resemble the conditions of early life, these pathways can serve as models for an ancestral autotrophic carbon fixation pathway. The acquisition of cellular carbon from inorganic carbon is a prerequisite for life and marked the transition from the inorganic to the organic world. Recent theories of the origins of life assume that chemoevolution took place in a hot volcanic flow setting through a transition metal-catalysed, autocatalytic carbon fixation cycle. Many archaea live in volcanic habitats under such constraints, in high temperatures with only inorganic substances and often under anoxic conditions. In this Review, we describe the diverse carbon fixation mechanisms that are found in archaea. These reactions differ fundamentally from those of the well-known Calvin cycle, and their distribution mirrors the phylogenetic positions of the archaeal lineages and the needs of the ecological niches that they occupy.

Ignicoccus hospitalis is an anaerobic, autotrophic, hyperthermophilic Archaeum that serves as a host for the symbiotic/parasitic Archaeum Nanoarchaeum equitans. It uses a yet unsolved autotrophic CO₂ fixation pathway that starts from acetyl-CoA (CoA), which is reductively carboxylated to pyruvate. Pyruvate is converted to phosphoenol-pyruvate (PEP), from which glucogenesis as well as oxaloacetate formation branch off. Here, we present the complete metabolic cycle by which the primary CO₂ acceptor molecule acetyl-CoA is regenerated. Oxaloacetate is reduced to succinyl-CoA by an incomplete reductive citric acid cycle lacking 2-oxoglutarate dehydrogenase or synthase. Succinyl-CoA is reduced to 4-hydroxybutyrate, which is then activated to the CoA thioester. By using the radical enzyme 4-hydroxybutyryl-CoA dehydratase, 4-hydroxybutyryl-CoA is dehydrated to crotonyl-CoA. Finally, β-oxidation of crotonyl-CoA leads to two molecules of acetyl-CoA. Thus, the cyclic pathway forms an extra molecule of acetyl-CoA, with pyruvate synthase and PEP carboxylase as the carboxylating enzymes. The proposal is based on in vitro transformation of 4-hydroxybutyrate, detection of all enzyme activities, and in vivo-labeling experiments using [1-¹⁴C]4-hydroxybutyrate, [1,4-¹³C₂], [U-¹³C₄]succinate, or [1-¹³C]pyruvate as tracers. The pathway is termed the dicarboxylate/4-hydroxybutyrate cycle. It combines anaerobic metabolic modules to a straightforward and efficient CO₂ fixation mechanism.