The enzyme that catalyses this process is called Ribulose biphosphate carboxylase (or rubisco). Stage 2: Reduction Using the energy from ATP, the three-carbon compound, 3-PGA molecules, produced in the carbon fixation stage, are converted into a three-carbon sugar glyceraldehyde-3-phosphate (G3P). This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2012288117/-/DCSupplemental. We find that the canonical plant-like C2 cycle does not operate in this bacterium, and instead, the bacterial-like glycerate pathway is the main route for phosphoglycolate salvage. (We note that this cycle could alternatively proceed via malate oxidation to oxaloacetate, which is then converted to phosphoenolpyruvate via phosphoenolpyruvate carboxykinase and further metabolized to pyruvate and acetyl-CoA; this alternative malate cycle would result in the same net decarboxylation reaction.). Published by PNAS. Lists of bacterial genomes containing the large subunit of Rubisco (PF00016), malate synthase (PF01274), or isocitrate lyase (PF00463) were downloaded from the AnnoTree website (53) on 10 July 2020 by searching for the appropriate protein families. Although C. necator harbors multiple transaminases, it is virtually impossible to predict the precise substrate specificities of these highly promiscuous enzymes from their sequence. The Calvin cycle can be divided into three phases: Carbon fixation, Reduction and regeneration of CO 2 acceptor. G3P is a simple sugar. Next, some transconjugants were grown in an overnight liquid culture (without tetracycline) to support a second homologous recombination event. For example, the genes encoding for the components of the malate cycle—aceB, maeA, maeB, pdhA1, pdhB, and pdhL—are highly expressed under both ambient and 10% CO2 concentrations (all among the 10% most highly expressed in both conditions) (Dataset S1); hence, the malate cycle could play a role in phosphoglycolate salvage. Photorespiration has been extensively studied in photosynthetic organisms, including plants, algae, and cyanobacteria (4⇓⇓–7). A key finding of our work is the existence of the malate cycle as a supporting route for phosphoglycolate salvage and growth on glycolate. ΔC2, C2 cycle knockout (ΔgcvTHP); ΔGDH, glycolate dehydrogenase knockout (ΔglcD-kch-glcE-glcF); ΔGP, glycerate pathway knockout (Δgcl-hyi-tsr); ΔMC, malate cycle knockout (ΔaceB); ΔOX, oxalate decarboxylation knockout (frc-oxc); ΔRub, Rubisco knockout (ΔcbbS2-cbbL2 ΔcbbSp− ΔcbbLp); WT, wild type. Likewise, what is the Calvin cycle in biology? The Calvin cycle has four main steps: carbon fixation, reduction phase, carbohydrate formation, and regeneration phase. Glycolate concentrations in culture supernatant were determined by ion chromatography analysis. Next, we explored possible routes for glyoxylate metabolism. Author contributions: N.J.C. Despite the relative inefficiency of the malate cycle, its implementation in plants was suggested to boost carbon fixation and photosynthesis (34⇓⇓–37). The NADPH first accepts the electrons and hydrogen when special enzymes transfer these particles to the molecule NADP+. Specifically, it could be that their basal expression levels are sufficient to support the required activity. Astronomers thought they’d finally figured out where gold and other heavy elements in the universe came from. The metabolic recycling of phosphoglycolate was extensively studied in photoautotrophic organisms, including plants, algae, and cyanobacteria, where it is referred to as photorespiration. This is where food chains come into play, with apex predators and carnivores at the top, supported by herbivores, who get their energy from organic plant matter. Rather, the glyoxylate produced in phosphoglycolate salvage can be condensed with acetyl-CoA generated from the carbon fixation process, and the resulting malate is assimilated into biomass (i.e., via the tricarboxylic acid [TCA] cycle, cataplerosis, and gluconeogenesis). The Calvin cycle takes place inside the chloroplasts of leaves. A complete overview of strain genotypes used in this study can be found in Table 2. The Calvin cycle is a series of reactions which takes place in the stroma of chloroplasts in a plant cell. As malate synthase is present in many chemolithoautotrophs that use the Calvin cycle (Table 1), it is tempting to suggest that it contributes to phosphoglycolate salvage in many of these bacteria. To 100 µL of this mixture, 50 µL concentrated HCl and 20 µL of 1.6% wt/vol potassium ferricyanide were added, while background control samples were prepared with 100 µL of reacted sample mixture with 50 µL concentrated HCl and 20 µL Milli-Q (MQ) water. To compensate for high evaporation losses due to high sparging flow, a level sensor controlled a feed pump of sterile water. As expected, in most cases (∼80%), bacteria with a malate synthase gene also encode an isocitrate lyase. Rubisco sequences were filtered by using usearch (55) to remove any amino acid sequences with >30% identity to known Rubisco-like proteins (type IV Rubiscos). The relatively stable compound that is formed in this cycle is a 3-carbon sugar. The Calvin cycle has four main steps: carbon fixation, reduction phase, carbohydrate formation, and regeneration phase. Therefore, it could be that C. necator simply lacks transaminases that can accept glyoxylate and serine and hence, cannot operate the C2 pathway. 4). Enter multiple addresses on separate lines or separate them with commas. The complete decarboxylation of glyoxylate to CO2—as supported by the cyanobacterial oxalate decarboxylation pathway as well as the oxalyl-CoA decarboxylation pathway and the malate cycle described here—is arguably the least efficient phosphoglycolate salvage mode, as it requires higher activity of the Calvin cycle to compensate for the lost carbon. Regeneration of the CO 2 acceptor molecule RuBP is crucial if the cycle is to continue uninterrupted. However, this is highly doubtful as the change in Gibbs energy for the reaction glycolate + NAD+ = glyoxylate + NADH is very high (ΔrG′m > 40 kJ/mol, pH 7.5, ionic strength of 0.25 mM; ΔrG′m corresponds to metabolite concentration of 1 mM) (33). This might explain why the deletion of the glycerate pathway in C. necator, such that the malate cycle carries phosphoglycolate salvage on its own, resulted in lower growth rate and yield (Fig. Yet, it is still not clear whether the pathways that support growth on glycolate also participate in phosphoglycolate salvage during autotrophic growth at ambient CO2. SURVEY . Tags: Question 9 . Still, the ability of the strain lacking the gcl-hyi2-tsr operon to grow on glycolate implies that other routes can support glyoxylate metabolism. The main features of the Calvin cycle are summarized briefly for the sake of completeness. In stage 3, RuBP, the molecule that starts the cycle, is regenerated so that the cycle can continue. The ΔphaC1 strain grows in nutrient nonlimiting conditions similar to the wild type and does not result in polyhydroxybutyrate (PHB) granules that could disturb optical density measurements. It is a cyclic biochemical pathway which proceeds with the use of carbon dioxide and the production of oxygen. As the term photorespiration is inappropriate for describing phosphoglycolate recycling in these nonphotosynthetic autotrophs, we suggest the more general term “phosphoglycolate salvage.” Here, we study phosphoglycolate salvage in the model chemolithoautotroph Cupriavidus necator H16 (Ralstonia eutropha H16) by characterizing the proxy process of glycolate metabolism, performing comparative transcriptomics of autotrophic growth under low and high CO2 concentrations, and testing autotrophic growth phenotypes of gene deletion strains at ambient CO2. The oven Customers/RuBisCo the customers take the subs and exchange it for money The oven is where the sub bun is kept warm and it is the source of energy needed to start making the The product of this r… Growth experiments on ambient CO2 were performed in biological duplicates and showed identical growth curves (±5%); hence, representative curves of a single experiment are shown. Bioreactors (DASGIP; Eppendorf) with 700 mL JMM medium were inoculated at a starting OD600 of 0.05. Hence, even if the primary function of malate synthase is to support growth on acetate, it could still play a key role in phosphoglycolate salvage, as is the case in C. necator. The Calvin cycle reactions (Figure 2) can be organized into three basic stages: fixa… Then, log2-transformed fold changes for ambient CO2 compared with 10% CO2 and absolute log10 of adjusted P values were determined and were visualized in a volcano plot. 2A). Max Planck Institute of Molecular Plant Physiology, The photorespiratory metabolite 2-phosphoglycolate regulates photosynthesis and starch accumulation in, Chloroplast and cytoplasmic enzymes. Since the term photorespiration is ill-suited to describe the recycling of 2PG in light-independent autotrophs, we suggest using the more general term “Rubisco-related 2-phosphoglycolate salvage” or for short, “phosphoglycolate salvage.” A similar term was used before in a few publications when referring to photorespiration (15⇓–17). Growth experiments were conducted in 700-mL bioreactor cultures on minimal medium (JMM) with a continuous sparging of gas (6.25 L/min) with ambient air + 4% hydrogen. In the first stage, chemical reactions use energy from light to produce ATP and NADPH. Figure 2 The Calvin cycle has three stages. The Calvin cycle is the most important carbon fixation pathway in the biosphere. The Calvin cycle (also known as the Benson-Calvin cycle) is the set of chemical reactions that take place in chloroplasts during photosynthesis. Recently, the heterologous expression of malate synthase and glycolate dehydrogenase within the chloroplast of the agricultural crop Nicotiana tabacum led to a substantial increase in photosynthetic rate and yield (35). Strains were precultured in 100 mL JMM in 500-mL Erlenmeyer with 80 mM sodium formate and 10% CO2 in the headspace. It is difficult to determine whether this route plays a role in the wild-type strain, as its deletion does not seem to hamper growth when the glycerate pathway is present. This study thus demonstrates a so far unknown phosphoglycolate salvage pathway, highlighting important diversity in microbial carbon fixation metabolism. 2A), indicating that this route supports glyoxylate metabolism in the absence of the glycerate pathway. While the deletion of the latter two operons did not affect growth on glycolate (doubling time of 3.4 ± 0.1 h) (ΔC2 ΔOX ΔMC in Fig. Gene deletions were performed with the pLO3 suicide vector, as previously described (47, 48). A second pathway, which we term the malate cycle, carries all phosphoglycolate salvage flux when the glycerate pathway is deleted. Values for all genes can be found in Dataset S1. Oxygen. Generated CO2 and reducing equivalents are utilized in the Calvin cycle to support biomass formation. Thus, ATP and NADPH are needed to synthesize it. and A.B.-E. designed research; N.J.C., G.S., A.F., A.I.F., W.N., and S.F. Calvin cycle 2 Steps of the Calvin cycle 1. On the other hand, a strain in which the glycerate pathway was deleted displayed a substantially lower growth rate (doubling time of 35 ± 1 h) (ΔGP in Fig. true. NADPH. Calvin Cycle is a set of light-independent chemical reactions performed by plants. However, the presence of malate synthase has been confirmed in some cyanobacteria, leading to the suggestion that they may use the malate cycle (40⇓⇓–43). The Calvin cycle begins when a CO2 molecule is attached to a five-carbon sugar called ribulose biphosphate (RuBP). We identify the native phosphoglycolate salvage pathways of C. necator by performing comparative transcriptomic analysis and conducting growth experiments with gene deletion strains. The Calvin cycle is responsible for the vast majority of carbon fixation in the biosphere. Further deletion of the malate cycle in the strain lacking the glycerate pathway completely abolished autotrophic growth at ambient CO2 (ΔGP ΔMC in Fig. It therefore seems that C. necator can excrete glycolate if necessary, thus enabling the autotrophic growth of a strain lacking glycolate dehydrogenase. To determine the relative importance of each candidate pathway in phosphoglycolate salvage, we tested wild-type C. necator and several of the gene deletion strains described above for their ability to grow autotrophically at ambient CO2 (Fig. In stage 3, RuBP, the molecule that starts the cycle, is regenerated so that the cycle can continue. C. necator H16 (Deutsche Sammlung von Mikroorganismen und Zellkulturen [DSMZ] 428) was used for transcriptome studies. The Calvin cycle is the most important carbon fixation pathway in the biosphere. 4). We thank Patrick Behrens, Viswanada Bysani, and Natalia Giner Laguarda for assistance with the bioreactor experiments and sampling; Leonardo Perez de Souza, Alisdair Fernie, and Elad Noor for fruitful discussions and advice on this work; Ari Satanowksi, Armin Kubis, Sebastian Wenk, and Matthew Paddock for feedback on the manuscript and helpful suggestions; and the Max Planck Genome Centre Cologne (https://mpgc.mpipz.mpg.de/home/) for performing the RNA sequencing in this study. Growth (OD600) and glycolate concentrations were monitored every 24 h during the growth phase of a C. necator ΔglcD-kch-glcE-glcF strain. This suggests that the activity of the oxalyl-CoA decarboxylation route on its own is too low to enable growth (e.g., due to low expression levels or poor enzyme kinetics). Next, cells were lysed using lysozyme and a bead-beating step with glass beads (Retschmill; MM200) for 5 min at 30 Hz. The Calvin Cycle can happen at night. These results clearly show that the malate cycle can participate in phosphoglycolate salvage, while the other two pathways contribute little, if any, to this process. In C. necator, we confirmed the role of the glycolate dehydrogenase complex in phosphoglycolate salvage and further revealed two metabolic routes that can sustain glyoxylate assimilation during phosphoglycolate salvage at ambient CO2. However, the fact that the genes of the other pathways were not overexpressed does not necessarily mean that they do not participate in phosphoglycolate salvage. Edited by Donald R. Ort, University of Illinois at Urbana–Champaign, Urbana, IL, and approved July 24, 2020 (received for review June 14, 2020). 5). This result is expected as higher activity of the cycle is needed to compensate for the decreased rate of Rubisco as well as the carbon loss from the oxygenation reaction and subsequent phosphoglycolate salvage. To explore the potential distribution of the malate cycle, we searched for the occurrence of malate synthase (PFAM 01274) (32) in bacteria that harbor a bona fide Rubisco and are therefore likely to operate the Calvin cycle and rely on phosphoglycolate salvage (Materials and Methods). Others call it the Calvin-Benson cycle to include the name of another scientist involved in its discovery (Figure 1). By generating three distinct mutant strains carrying deletions in the gcl-hyi2-tsr operon, the gcvTHP operon, or the frc-oxc operon, we explored the contribution of each candidate route to growth on glycolate. However, these enzymes are not strictly necessary. Hence, we explored the co-occurrence of malate synthase and isocitrate lyase in bacteria harboring a Rubisco gene. In this cycle, glyoxylate is condensed with acetyl-CoA (coenzyme A) to generate malate, which then undergoes oxidative decarboxylation twice to regenerate acetyl-CoA (Fig. However, a similar route might be used. 4). Some of this G3P is used to regenerate the RuBP to continue the cycle, but some is available for molecular synthesis and is used to make fructose diphosphate. The carbon dioxide taken up by the plant cell is reduced to glucose with the help of ATP and NADPH which is formed in the dark reaction of photosynthesis. The Calvin cycle (also known as the Benson-Calvin cycle) is the set of chemical reactions that take place in chloroplasts during photosynthesis.. The latter enzyme probably generates 2-phosphoglycerate, which can be converted to 3PG via the glycolytic enzyme phosphoglycerate mutase (Pgam1,2). Dark reaction of photosynthesis does not require light. Samples for OD600 measurements and supernatant analysis were taken daily, while fast growth at 10% CO2 was monitored by an online sensor calibrated for cuvette OD600. 4, respectively). The superiority of the glycerate pathway might also explain why it serves as the major phosphoglycolate salvage route both in cyanobacteria and in C. necator. (A) Growth experiments were conducted in 700-mL bioreactor cultures on minimal medium (JMM) with a continuous sparging of gas (6.25 L/min) with ambient air + 4% hydrogen; control experiments were conducted at 10% CO2 + 4% hydrogen (supplemented with air). Concentrations were calculated based on a standard curve generated for sodium glycolate (Sigma-Aldrich) in JMM medium. Finally, the Synechocystis-like oxalate decarboxylation pathway seems not to be present in C. necator, as no oxalate decarboxylase gene could be found. Differential expression analysis was performed as described in Materials and Methods; log2-transformed fold changes and adjusted P values of all genes are depicted in the graph. As anticipated, this strain was not able to grow on glycolate (ΔGP ΔRub in Fig. The Calvin cycle uses ATP and NADPH to convert CO2 to sugar: ATP and NADPH produced by the light reactions are used in the Calvin cycle to reduce carbon dioxide to sugar. It could be that the malate cycle carries nonnegligible flux only when the glycerate pathway is disrupted and glyoxylate begins to accumulate. While a wild-type C. necator could efficiently grow on glycolate (doubling time of 3.2 ± 0.1 h) (WT in Fig. 1). It is sometimes proposed, especially in cyanobacterial phosphoglycolate salvage, that NAD+ serves as the electron acceptor. The presence of malate synthase does not necessarily indicate that the malate cycle plays a role in phosphoglycolate salvage. Notably, deletion of both the glycerate pathway and the malate cycle did not affect autotrophic growth at high CO2 concentrations (dashed ΔGP ΔMC line in Fig. GSE141999) (58) and are available in Dataset S1. SURVEY . Prevalence of malate synthase in chemolithoautotrophs and cyanobacteria that use the Calvin cycle. In the first stage, ATP and NADPH is used to fix CO 2. In stage 2, the organic molecule is reduced using electrons supplied by NADPH. Phosphoglycolate salvage in a chemolithoautotroph using the Calvin cycle. Can I evict a tenant for not paying rent? While receiving little attention so far, aerobic chemolithoautotrophic bacteria that operate the Calvin cycle independent of light must also recycle phosphoglycolate. Instead, it is more likely that glycolate transfers its electrons, via a flavin adenine dinucleotide cofactor, to a quinone, the reduction potential of which is substantially higher than that of NAD (E′m ∼ 0 mV rather than ∼−300 mV, respectively). Transcriptomic data are available in the Gene Expression Omnibus (accession no. Q. However, plants are also living organisms that require “food” to stay alive, though it isn’t … Glycolate was measured by ion chromatography as explained in Materials and Methods. The final product of the Calvin cycle is glucose. To make one molecule of glucose, 6 turns of the cycle are required. The samples were run through a Dionex IonPac AS11 IC column (4-mm diameter, 250-mm length ) and a guard column (4-mm diameter, 50-mm length ). In this step, initial incorporation of three molecules CO 2 takes place into the organic material. We leave it for future studies to explore the occurrence of the malate cycle and other phosphoglycolate salvage routes in cyanobacteria and chemolithoautotrophs. (B) Phosphoglycolate salvage in C. necator proceeds mainly via glycolate dehydrogenase and the glycerate pathway to regenerate 3PG for the Calvin cycle. In these bacteria, the primary role of malate synthase might indeed be phosphoglycolate salvage. These reactions use chemical energy from NADPH and ATP that were produced in the light reactions. Glyoxylate concentrations were determined by a colorimetric assay based on a reported protocol (52). Even though the cycle is called the dark reaction phase, the aforementioned reaction do not really occur in the dark. This study aimed to fill gaps in our knowledge of phosphoglycolate salvage in chemolithoautotrophic microorganisms. For every three turns, one molecule of G3P exits the cycle and goes towards making glucose. Autotrophic growth of wild-type C. necator under these conditions resulted in a much lower growth rate than observed at 10% CO2 (doubling time of 21 ± 0.7 vs. ∼3 h) (solid vs. dashed WT lines in Fig. What is internal and external criticism of historical sources? The other enzymes of the malate cycle—the malic enzyme and pyruvate dehydrogenase or their alternatives (e.g., malate dehydrogenase, phosphoenolpyruvate carboxykinase)—are quite ubiquitous. This might be explained by the higher intracellular glyoxylate concentrations expected when feeding with external glycolate, which could induce the expression of the oxalyl-CoA decarboxylation pathway. A granum (plural grana) is a stack of thylakoid discs. We found that the genes encoding for the Calvin cycle enzymes were overexpressed between 4- and 12-fold under ambient CO2 concentration (Dataset S1). To test if this is indeed the case, we deleted all genes encoding for Rubisco (cbbS2, cbbL2, cbbSp, and cbbLp) in the strain deleted in the gcl-hyi2-tsr operon. In carbon fixation, a CO2 molecule from the atmosphere combines with a five-carbon acceptor molecule called ribulose-1,5-bisphosphate (RuBP).The resulting six-carbon compound is then split into two molecules of the three-carbon compound, 3-phosphoglyceric acid (3-PGA).This reaction is catalyzed by the enzyme RuBP carboxylase/oxygenase, also known as RuBisCO. The term photorespiration was initially coined to describe the light-dependent O2 consumption and CO2 release in plant leaves (9⇓⇓–12). The few notable exceptions, which were found to have a malate synthase without an isocitrate lyase, include bacteria of the orders Rhodobacterales, Rhodospirillales, and Sulfobacillales (Dataset S2). Perhaps unsurprisingly, rubisco is the most abundant protein on earth. In the stroma, in addition to CO2, two other chemicals are present to initiate the Calvin cycle: an enzyme abbreviated RuBisCO, and the molecule ribulose bisphosphate (RuBP). We demonstrate that this bacterium mainly reassimilates 2-phosphoglycolate via the glycerate pathway. The combined activity of the malic enzyme and pyruvate dehydrogenase can support this double oxidation. 2A); note that the deletion of Rubisco in a wild-type strain did not affect growth on glycolate as the glycerate pathway is still active (doubling time of 3.3 h) (ΔRub in Fig. This process uses energy and reactions captured during light-dependent stages of photosynthesis. One glycine molecule is decarboxylated to give methylenetetrahydrofolate (methylene-THF), which reacts with another glycine to yield serine. Candidate pathways supporting phosphoglycolate salvage in C. necator; 2PG is first dephosphorylated to glycolate and then oxidized, by the glycolate dehydrogenase complex, to give glyoxylate. Further studies, analyzing the expression profile of the oxalyl-CoA decarboxylation pathway and the kinetics of its enzymes, could explain its role in glycolate metabolism and its absence in phosphoglycolate salvage. 2A), suggesting that this route contributes only marginally to the metabolism of glyoxylate. In plants, carbon dioxide (CO2) enters the chloroplast through the stomata and diffuses into the stroma of the chloroplast—the site of the Calvin cycle reactions where sugar is synthesized. Tags: Question 8 . and O.L. 30 seconds . These samples were incubated for exactly 12 min, and then, absorbance of 1,5-diphenylformazan at 520 nm was recorded by a BioTek Epoch 2. The cycle is light-independent because it takes place after the energy has been captured from sunlight. On the other hand, deletion of gcvTHP, blocking the C2 pathway, had only a small negative effect on growth (doubling time of 9.6 ± 0.1 h) (ΔGP ΔC2 in Fig. Alternatively, considering the relative high expression of the genes of the malate cycle, it is possible that the pathway always supports a substantial fraction of glyoxylate metabolism but not enough to affect growth when deleted. ΔC2, C2 cycle knockout (ΔgcvTHP); ΔGDH, glycolate dehydrogenase knockout (ΔglcD-kch-glcE-glcF); ΔGP, glycerate pathway knockout (Δgcl-hyi-tsr); ΔMC, malate cycle knockout (ΔaceB); ΔOX, oxalate decarboxylation knockout (Δfrc-oxc); WT, wild type. H ) ( 59 ) the hazards of ozone pollution to birds ApbA2 serves as a CoA-acylating glyoxylate calvin cycle steps of. To hydroxypyruvate, further reduced to glycerate, and regeneration of CO.! Dehydrogenase ( catalyzing the first stage, ATP and NADPH not a reactant of the malic and... 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