Carbon Fixation Metabolism Research Articles

Metabolomics uncovers adaptation discrepancy among anammox granular sludge with different granule size: Metabolic pathway regulation by consortia cooperation

The relationship between granular size and anaerobic ammonium oxidation (anammox) performance in the anammox granular sludge (AnGS) system has been extensively observed. However, the metabolic pathways regulated by communication and cross-feedings among anammox consortia remain unclear. The reactor operation and metabolomics analyses were combined to explore the influence of microbiota cooperation on metabolic pathways and granule properties under low temperature (18 °C) and nitrite inhibition. Anammox activity was sustained under challenging circumstances by active quorum sensing among anammox consortia in AnGS with diameters larger than 1.4 mm, which promoted nucleotide metabolism. Cross-feedings among anammox consortia increased the levels of molybdopterin cofactor and folate meanwhile decreasing the cost of carbon fixation metabolism, which supported anabolism and maintained the content of heme c and extracellular polymeric substance. These metabolic insights into the AnGS system provide a new view for anammox process overcoming the low temperature and nitrite stress.

Engineering Cupriavidus necator H16 for enhanced lithoautotrophic poly(3-hydroxybutyrate) production from CO2

BackgroundA representative hydrogen-oxidizing bacterium Cupriavidus necator H16 has attracted much attention as hosts to recycle carbon dioxide (CO2) into a biodegradable polymer, poly(R)-3-hydroxybutyrate (PHB). Although C. necator H16 has been used as a model PHB producer, the PHB production rate from CO2 is still too low for commercialization.ResultsHere, we engineer the carbon fixation metabolism to improve CO2 utilization and increase PHB production. We explore the possibilities to enhance the lithoautotrophic cell growth and PHB production by introducing additional copies of transcriptional regulators involved in Calvin Benson Bassham (CBB) cycle. Both cbbR and regA-overexpressing strains showed the positive phenotypes for 11% increased biomass accumulation and 28% increased PHB production. The transcriptional changes of key genes involved in CO2—fixing metabolism and PHB production were investigated.ConclusionsThe global transcriptional regulator RegA plays an important role in the regulation of carbon fixation and shows the possibility to improve autotrophic cell growth and PHB accumulation by increasing its expression level. This work represents another step forward in better understanding and improving the lithoautotrophic PHB production by C. necator H16.

Prokaryotic diversity and biogeochemical characteristics of field living and laboratory cultured stromatolites from the hypersaline Laguna Interna, Salar de Atacama (Chile).

Stromatolites are organo-sedimentary structures found principally in seas and saline lakes that contain sheets of sediments and minerals formed by layers of microbial communities, which trap sediments and induce the precipitation of minerals.A living stromatolite from the alkaline Laguna Interna in the Salar de Atacama was collected and one of the fragments was deposited in an experimental aquarium for 18 months. We used Illumina sequencing of PCR-amplified V4 regions of 16S rRNA genes from total extracted DNA to identify the microbial populations. The chemical structure was studied using X-Ray Diffraction (XRD) and bench chemical methods. We found that members belonging to the Proteobacteria, Planctomycetes, Chloroflexi and Bacteroidetes phyla dominated the bacterial communities of the living and aquarium cultured samples. The potential metabolic functionality of the prokaryotic community reveals that sulfur, nitrogen, methane and carbon fixation metabolism functions are present in the samples. This study is the first to provide new insights into the prokaryotic community composition from this unusual aquatic desert site. Further studies will be helpful to obtain a better understanding of the biotic and abiotic mechanisms residing in stromatolites from Laguna Interna, as well as to have better knowledge about the formation of these biosignatures.

Phosphoglycolate salvage in a chemolithoautotroph using the Calvin cycle

Carbon fixation via the Calvin cycle is constrained by the side activity of Rubisco with dioxygen, generating 2-phosphoglycolate. The metabolic recycling of phosphoglycolate was extensively studied in photoautotrophic organisms, including plants, algae, and cyanobacteria, where it is referred to as photorespiration. While receiving little attention so far, aerobic chemolithoautotrophic bacteria that operate the Calvin cycle independent of light must also recycle phosphoglycolate. 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 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. Upon disruption of the glycerate pathway, we find that an oxidative pathway, which we term the malate cycle, supports phosphoglycolate salvage. In this cycle, glyoxylate is condensed with acetyl coenzyme A (acetyl-CoA) to give malate, which undergoes two oxidative decarboxylation steps to regenerate acetyl-CoA. When both pathways are disrupted, autotrophic growth is abolished at ambient CO2 We present bioinformatic data suggesting that the malate cycle may support phosphoglycolate salvage in diverse chemolithoautotrophic bacteria. This study thus demonstrates a so far unknown phosphoglycolate salvage pathway, highlighting important diversity in microbial carbon fixation metabolism.

Combining isotopically non-stationary metabolic flux analysis with proteomics to unravel the regulation of the Calvin-Benson-Bassham cycle in Synechocystis sp. PCC 6803

Photosynthetic microorganisms are increasingly being investigated as a sustainable alternative to existing bio-industrial processes, converting CO2 into desirable end products without the use of carbohydrate feedstock. The Calvin-Benson-Bassham (CBB) cycle is the main pathway of carbon fixation metabolism in photosynthetic organisms. In this study, we analyzed the metabolic fluxes in two strains of the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) that overexpressed fructose-1,6/sedoheptulose-1,7-bisphosphatase (FBP/SBPase) and transketolase (TK), respectively. These two potential carbon flux control enzymes in the CBB cycle had previously been shown to improve biomass accumulation when overexpressed under air and low light (15 μmol m−2 s−1) conditions (Liang and Lindblad, 2016). We measured the growth rates of Synechocystis under atmospheric and high (3% v/v) CO2 conditions at 80 μmol m−2 s−1. Surprisingly, the cells overexpressing transketolase (tktA) demonstrated no significant increase in growth rates when CO2 was increased, suggesting an altered carbon flux distribution and a potential metabolic bottleneck in carbon fixation. Moreover, the tktA strain had an increased susceptibility to oxidative stress under high light as revealed by its chlorotic phenotype under high light conditions. In contrast, the fructose-1,6/sedoheptulose-1,7-bisphosphatase (70glpX) and wild-type cells demonstrated increases in growth rates as expected.To investigate the disparate phenotypical responses of these different Synechocystis strains, isotopically non-stationary metabolic flux analysis (INST-MFA) was used to estimate the carbon flux distribution of tktA, 70glpX, and a kanamycin-resistant control (Km), under atmospheric conditions. In addition, untargeted label-free proteomics, which can detect changes in relative enzymatic abundance, was employed to study the possible effects caused by overexpressing each enzyme. Fluxomic and proteomic results indicated a decrease in oxidative pentose phosphate pathway activity when either FBP/SBPase or TK were overexpressed, resulting in increased carbon fixation efficiency. These results are an example of the integration of multiple omic-level experimental techniques and can be used to guide future metabolic engineering efforts to improve performances and efficiencies.

Description of carbon fixation pathway based on <italic>Skeletonema marinoi</italic> transcriptome

Diatoms can fix approximately 1016 g CO2 into organic carbon every year, equivalent to roughly 20% of global primary production. They are the basis for important food webs, having considerable impacts on carbon cycle and biological carbon pump that draws down CO2 from the atmosphere to the ocean interior. Nevertheless, the K 1/2 of their ribulose- 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is 40~60 μ mol L - 1, higher than the apparent photosynthetic K 1/2 (CO2) for ambient CO2. Growth and kinetic studies have shown that diatoms can avoid CO2 limitation through CO2 concentrating mechanisms. Recently the carbon fixation metabolism pathway in diatoms is further elucidated by the presence of a complete set of genes essential for this metabolic route in Thalassiosira pseudonana and Phaeodactylum tricornutum because of their entire availablegenome information. In this study, we used RNA-Seq technology to confirm the existence of necessary genes for plant-like C4 activity in Skeletonema marinoi , which is a complex biological trait that enables S. marinoi to either has high water-use efficiency or accumulate biomass at a fast rate. The carbon fixation metabolism pathway of S. marinoi involves 20 enzymes and 34 coding genes, including the complete key enzymes especially for C4 pathway, which are phosphoenolpyruvate carboxylase (PEPC), phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate orthophosphate dikinase (PPDK). Comparing with the homologous genes of carbon fixation metabolism pathway in T. pseudonana and P. tricornutum , these genes showed little divergence. Besides, the gene differential expression of S. marinoi in carbon fixation metabolism pathway was identified at growth stages using digital gene expression profiling. The results indicated the number of differential expression genes was 7 and 3 in C3 and C4 pathway, respectively. As for the differential expression of coding genes in growth stages, there were 8 and 10 coding genes in the stationary and decline phase, respectively versus to those in the exponential phase. The gene expression of fructose-1,6-bisphosphatase (FBP), fructose-bisphosphate aldolase (ALDO) and PPDK was changed significantly after the exponential phase. The FBP and ALDO are not only responsible for the carbon fixation metabolism pathway, but also as the key enzymes in the glycolytic metabolism pathway, which links closely with the flow direction of carbon and the synthesis of carbohydrates. The PPDK, a cardinal enzyme of the C4 pathway, catalyzing the regeneration of phosphoenol pyruvate(PEP), evolved as an adaptation to high light intensity, high temperature, low concentration of CO2 and dryness. Therefore, the formation of PEP through PPDK is considered to be very important in the C4 pathway. This study will contribute to analyze the gene regulation of key enzymes involved in the carbon metabolism of S. marinoi , explain the reason for huge amounts of primary production by diatom, and enhance our knowledge on the carbon fixation mechanism, which provides a new insight into understanding carbon biogeochemical cycle.

Deciphering the mechanisms involved in Portulaca oleracea (C4) response to drought: metabolic changes including crassulacean acid‐like metabolism induction and reversal upon re‐watering

Portulaca oleracea is a C(4) plant; however, under drought it can change its carbon fixation metabolism into a crassulacean acid metabolism (CAM)-like one. While the C(3) -CAM shift is well known, the C(4) -CAM transition has only been described in Portulaca. Here, a CAM-like metabolism was induced in P. oleracea by drought and then reversed by re-watering. Physiological and biochemical approaches were undertaken to evaluate the drought and recovery responses. In CAM-like plants, chlorophyll fluorescence parameters were transitory affected and non-radiative energy dissipation mechanisms were induced. Induction of flavonoids, betalains and antioxidant machinery may be involved in photosynthetic machinery protection. Metabolic analysis highlights a clear metabolic shift, when a CAM-like metabolism is induced and then reversed. Increases in nitrogenous compounds like free amino acids and urea, and of pinitol could contribute to withstand drought. Reciprocal variations in arginase and urease in drought-stressed and in re-watered plants suggest urea synthesis is strictly regulated. Recovery of C(4) metabolism was accounted by CO(2) assimilation pattern and malate levels. Increases in glycerol and in polyamines would be of importance of re-watered plants. Collectively, in P. oleracea multiple strategies, from induction of several metabolites to the transitory development of a CAM-like metabolism, participate to enhance its adaptation to drought.

Effects of light exposure on the retention of kleptoplastic photosynthetic activity in the sacoglossan mollusc Elysia viridis

The effects of light exposure on the photosynthetic activity of kleptoplasts were studied in the sacoglossan mollusc Elysia viridis. The photosynthetic activity of ingested chloroplasts was assessed in vivo by non-destructively measuring photophysiological parameters using pulse amplitude modulation (PAM) fluorometry. Animals kept under starvation were exposed to two contrasting light conditions, 30 μmol photons m−2 s−1 (low light, LL), and 140 μmol photons m−2 s−1 (high light, HL), and changes in photosynthetic activity were monitored by measuring the maximum quantum yield of photosystem II (PSII), Fv/Fm, the minimum fluorescence, Fo, related to chlorophyll a content, and by measuring rapid light-response curves (RLC) of relative electron transport rate (rETR). RLCs were characterised by the initial slope of the curve, αRLC, related to efficiency of light capture, and the maximum rETR level, rETRm,RLC, determined by the carbon-fixation metabolism. Starvation induced the decrease of all photophysiological parameters. However, the retention of photosynthetic activity (number of days for Fv/Fm > 0), as well as the rate and the patterns of its decrease over time, varied markedly with light exposure. Under HL conditions, a rapid, exponential decrease was observed for Fv/Fm, αRLC and rETRm,RLC, Fo not showing any consistent trend of variation, and retention times ranged between 6 and 15 days. These results suggested that the retention of chloroplast functionality is limited by photoinactivation of PSII reaction center protein D1. In contrast, under LL conditions, a slower decrease in all parameters was found, with retention times varying from 15 to 57 days. Fv/Fm, αRLC and rETRm,RLC exhibited a bi-phasic pattern composed by a long phase of slow decrease in values followed by a rapid decline, whilst Fo decayed exponentially. These results were interpreted as resulting from lower rates of D1 photoinactivation under low light and from the gradual decrease in carbon provided by photosynthesis due to reduction of functional photosynthetic units.

Barium Toxicity Effects in Soybean Plants

Barium (Ba)-induced phytotoxicity at 100, 1000, or 5000 microM Ba in soybean plants (Glycine max) was investigated under hydroponic culture conditions. Soybean growth and leaf photosynthetic activity were significantly inhibited by all three levels of Ba treatments. In the case of photosynthetic activity, 5000 microM Ba treatment shutdown stomatal opening and perturbed carbon fixation metabolism and translocation. However, 100 and 1000 microM Ba treatments shut down stomatal opening and inhibited carbon fixation, but without perturbation of leaf carbon fixation-related metabolism. Potassium (K) absorption by soybean roots was also reduced in all three Ba treatments. This decreased K absorption reduced K localization at guard cells. Barium accumulation in guard cells also inhibited K transport from epidermal cells to guard cells. This lack of K in guard cells resulted in stomatal closure. As a result of inhibition of K transport into guard cells and stomatal shutdown, photosynthetic activity and plant productivity were inhibited. Our experiment indicates that Ba has phytotoxic effects on soybean plants by inhibiting photosynthesis.

Photosynthesis in Plants of Four Tropical Species Growing under Elevated CO<sub>2</sub>

We studied the responses of leaf gas exchange and growth to an increase in atmospheric CO2 concentration in four tropical deciduous species differing in carbon fixation metabolism: Alternanthera crucis, C3-C4; Ipomoea carnea, C3; Jatropha gossypifolia, C3; and Talinum triangulare, inducible-CAM. In the first stage, plants were grown in one open-top chamber at a CO2 concentration of 560±40 μmol mol-1 (EC), one ambient CO2 concentration chamber (AC), and one unenclosed plot (U). In the second stage, plants were grown in five EC chambers (CO2 concentration = 680±30 μmol mol-1), five AC chambers, and five unenclosed plots. During the first weeks under EC in the first stage, plants of all the species had a very marked increase in their maximal net photosynthetic rates (Pmax) of 3.5 times on average; this stimulatory effect was maintained for 11-15 weeks, rates dampening afterward to values still higher than controls for 37 weeks. After a suspension of CO2 enrichment for 6 weeks, an increase in Pmax of EC plants over the controls was found in plants of all the species until week 82 of the experiment. Stomatal conductance (g) showed no response to EC. Carboxylation efficiency decreased in all the species under EC

Effects of micro‐environmental factors on photosynthetic CO2 uptake and carbon fixation metabolism in a spring ephemeral, Erythronium japonicum, growing in native and open habitats

Microenvironmental factors and physiological parameters (such as water potential, activity of ribulose 1,5‐bisphosphate carboxylase (RuBPcase), levels of ribulose 1,5‐bisphosphate (RuBP), 3‐phosphoglyceric acid (PGA) and sucrose in leaves) affecting photosynthetic processes of the typical vernal species Erythronium japonicum Decne. were examined on the floor of a deciduous broad‐leaved Quercus mongolica forest (Q.m. stand) and on bare land left undisturbed for 7 years after forest clearing (bare stand). Daytime solar radiation and the air and leaf temperatures at the bare stand were significantly higher than those at the Q.m. stand. The relative air humidity was very low and did not differ much between the stands, whereas the leaf–air vapor pressure difference (VPD) at the bare stand was twice as high as that at the Q.m. stand. The water potential in leaves at the bare stand was lower than two times that at the Q.m. stand. Therefore, the aboveground parts of the plants at the bare stand were subjected to much more severe heat stress than those at the Q.m. stand. When these environmental factors observed at the bare stand were reproduced in an assimilation chamber, the rate of photosynthetic CO2 uptake, stomatal conductance and water potential in leaves were significantly low in comparison with those when the factors at the Q.m. stand were simulated. The internal CO2 partial pressure in leaves at the bare stand was considerably lower than that at the Q.m. stand. Consequently, the decrease in the photosynthetic rate of the plants at the bare stand was caused mainly by a decrease in stomatal conductance through a lowering of water potential due to subjection of the aboveground parts to much more severe heat stress than that at the Q.m. stand. The possibility that an inhibition of the photosynthetic carbon fixation metabolism induced by the decrease in water potential contributes to the reduction in photosynthetic CO2 uptake in the plants at the bare stand is also discussed in light of physiological characteristics such as the activity of RuBPcase and levels of PGA, RuBP and sucrose in the leaves.

A carbonic anhydrase gene is induced in the nodule primordium and its cell-specific expression is controlled by the presence of Rhizobium during development.

Under nitrogen starvation, Rhizobium meliloti is able to induce nitrogen-fixing nodules on alfalfa roots. Certain alfalfa cultivars spontaneously develop pseudonodules in the absence of bacteria. A transcript, Msca1, expressed in spontaneous and R. meliloti-induced nodules, that codes for a carbonic anhydrase (CA), an enzyme catalyzing the hydration of CO2 has been identified. This is the first CA gene cloned from a non-photosynthetic tissue in plants. Msca1 was activated initially in all cells of the bacterium-induced nodule primordium and was also induced by cytokinin treatment of alfalfa roots. The presence of CA enzymatic activity in different nodule types was demonstrated. Thus, Msca1 is a new early nodulin gene with a function possibly related to the increased amyloplast deposition of the dividing cortical cells. Msca1 transcripts were subsequently found mainly in a peripheral envelope of cells in developing and mature nodules. This novel pattern of gene expression is controlled by the presence of the bacterium inside the nodule. Sucrose synthase and phosphoenol pyruvate carboxylase (PEPC), other genes of the carbon fixation metabolism, were expressed in the same peripheral cells and even more strongly in the nitrogen-fixing region. Analysis of expression patterns of these genes indicated that early CA function may not be related to carbon fixation through PEPC. CA might be acting in pH regulation and/or CO2/HCO3-transport during nodule initiation. Thus, carbonic anhydrase may play different roles at several stages of nodule development and function.