rTCA Cycle Research Articles

Identification of carbon fixation microorganisms and pathways in an aquifer contaminated with long-chain petroleum hydrocarbons.

Petroleum hydrocarbons (PHCs) can be biodegraded into CO2, and PHC-contaminated aquifers are always deemed as carbon sources. Fortunately, some carbon fixation microorganisms have been found in PHC-contaminated sites. However, most of the studies are related to volatile short-chain PHC, and few studies focus on long-chain PHC-contaminated sites. To reveal the carbon fixation microorganisms in these sites, in the study, a long-chain PHC polluted site in North China was selected. Through hydrochemical and metagenomics analysis, the structure and capacity of carbon fixing microorganisms in the site were revealed. Results showed that there were many kinds of carbon fixed microorganisms that were identified such as Flavobacterium, Pseudomonas. HP/4HB, rTCA, and DC/4HB cycles were dominated carbon fixation pathways. The long-chain PHC were weakly correlated with carbon fixation microorganisms, but it may stimulate the growth of some carbon fixation microorganisms, such as microorganisms involved in rTCA cycle. PRACTITIONER POINTS: The microorganisms with carbon fixation gene exist in the aquifer contaminated by long-chain petroleum hydrocarbon. Microorganisms that have the ability to degrade petroleum also have the ability to carbon fixation. Long-chain petroleum hydrocarbon may promote the growth of carbon fixation microorganisms.

Bacterial functions are main driving factors on paddy soil organic carbon in both surface soil and subsoil

Subsoil contains a significant amount of soil organic carbon (SOC) in rice paddies, playing a crucial role in global carbon (C) cycling. However, the mechanisms driving SOC dynamics in topsoil and subsoil remain elusive, as the vertical distribution of soil microbes and their associated functions varies. In our study, we explored the linkages between SOC dynamics and microbial functions across different soil depths (0–10 cm for topsoil, and 10–20 and 20–30 cm for subsoil) within a framework of contrasting tillage managements, including conventional tillage (CT), reduced tillage (RT), and no-tillage (NT). In the topsoil, bacterial biosynthesis and degradation functions negatively contributed (P < 0.05) to SOC accumulation, whereas in the subsoil layers, these functions exhibited a positive effect. Notably, both partial least squares path modeling (PLSPM) and stepwise regression showed that bacterial biosynthesis and degradation functions exert a stronger influence on SOC accumulation than fungal functions. Metagenomic sequencing revealed that the cellulose degradation (K01179) and reductive tricarboxylic acid cycle (rTCA cycle) (K00174 and K00175) were main factors driving SOC variation in topsoil. Starch degradation (K00705 and K01187) and rTCA cycle (K00175 and K00031) contributed to 93 % of the SOC variation in subsoil. Therefore, the rTCA cycle contributed to SOC variation in paddy soil across 0–30 cm soil profile. Moreover, tillage management could influence the abundances of enzymes associated with starch degradation and rTCA cycle. The contrasting driving factors influencing SOC in topsoil and subsoil could be associated with soil microbial functions, which would response differently to tillage managements.

Elevation Shapes Soil Microbial Diversity and Carbon Cycling in Platycladus orientalis Plantations

Diversified soil microbiomes are the key drivers of carbon fixation and plant residue decomposition in forest ecosystems. Revealing the elevation patterns of soil microbial carbon cycling in forests is essential for utilization of forest ecological resources. However, the soil microbial diversity and carbon cycle processes in Platycladus orientalis plantations across different elevations are still unclear. Here, we established a gradient with three elevations (118 m, 300 m, and 505 m) on the Beijing Ming Dynasty Tombs Forest Farm, which is located in Changping District, Beijing. The metagenomics method was applied to study the soil microbiome, with a special focus on the carbon cycle process at each elevation. We found the diversity and composition of the soil microbiomes significantly varied across the elevation gradients. The structure of bacteria and archaea was mainly driven by soil total potassium, pH and NH4+, but the eukaryota had no significant relationship with the environmental factors. The relative abundance of genes involved in microbial carbon fixation and decomposition of organic carbon were also significantly impacted by elevation, with the former showing increasing, u-shaped, or hump trends with increasing elevation, but the latter only showing hump trends. The rTCA cycle and 3-hydroxypropionate pathway were the dominant carbon fixation pathways in the Platycladus orientalis plantations. The elevation gradient shaped the microbial decomposition of plant-derived organic carbon by changing soil properties and, furthermore, led to soil organic carbon stock losses. These findings increase our understanding of soil microbial diversity and the carbon cycle across different elevations and provide a theoretical basis for the utilization of forest ecological resources to promote carbon sequestration.

Phenacetin promoted the rapid start-up and stable maintenance of partial nitrification: Responses of nitrifiers and antibiotic resistance genes

Phenacetin (PNCT) belongs to one of the earliest synthetic antipyretics. However, impact of PNCT on nitrifying microorganisms in wastewater treatment plants and its potential microbial mechanism was still unclear. In this study, PN could be initiated within six days by PNCT anaerobic soaking treatment (8 mg/L). In order to improve the stable performance of PN, 21 times of PNCT aerobic soaking treatment every three days was conducted and PN was stabilized for 191 days. After PN was damaged, ten times of PNCT aerobic soaking treatment every three days was conducted and PN was recovered after once soaking, maintained over 88 days. Ammonia oxidizing bacteria might change the dominant oligotype to gradually adjust to PNCT, and the increase of abundance and activity of Nitrosomonas promoted the initiation of PN. For nitrite-oxidizing bacteria (NOB), the increase of Candidatus Nitrotoga and Nitrospira destroyed PN, but PN could be recovered after once aerobic soaking illustrating NOB was not resistant to PNCT. KEGG and COG analysis suggested PNCT might disrupt rTCA cycle of Nitrospira, resulting in the decrease of relative abundance of Nitrospira. Moreover, PNCT did not lead to the sharp increase of absolute abundances of antibiotic resistance genes (ARGs), and the risk of ARGs transmission was negligible.

The Impact of Aboveground Epichloë Endophytic Fungi on the Rhizosphere Microbial Functions of the Host Melica transsilvanica.

In nature, the symbiotic relationship between plants and microorganisms is crucial for ecosystem balance and plant growth. This study investigates the impact of Epichloë endophytic fungi, which are exclusively present aboveground, on the rhizosphere microbial functions of the host Melica transsilvanica. Using metagenomic methods, we analyzed the differences in microbial functional groups and functional genes in the rhizosphere soil between symbiotic (EI) and non-symbiotic (EF) plants. The results reveal that the presence of Epichloë altered the community structure of carbon and nitrogen cycling-related microbial populations in the host's rhizosphere, significantly increasing the abundance of the genes (porA, porG, IDH1) involved in the rTCA cycle of the carbon fixation pathway, as well as the abundance of nxrAB genes related to nitrification in the nitrogen-cycling pathway. Furthermore, the presence of Epichloë reduces the enrichment of virulence factors in the host rhizosphere microbiome, while significantly increasing the accumulation of resistance genes against heavy metals such as Zn, Sb, and Pb. This study provides new insights into the interactions among endophytic fungi, host plants, and rhizosphere microorganisms, and offers potential applications for utilizing endophytic fungi resources to improve plant growth and soil health.

Quantifying genome-specific carbon fixation in a 750-meter deep subsurface hydrothermal microbial community.

Dissolved inorganic carbon has been hypothesized to stimulate microbial chemoautotrophic activity as a biological sink in the carbon cycle of deep subsurface environments. Here, we tested this hypothesis using quantitative DNA stable isotope probing of metagenome-assembled genomes (MAGs) at multiple 13C-labeled bicarbonate concentrations in hydrothermal fluids from a 750-m deep subsurface aquifer in the Biga Peninsula (Turkey). The diversity of microbial populations assimilating 13C-labeled bicarbonate was significantly different at higher bicarbonate concentrations, and could be linked to four separate carbon-fixation pathways encoded within 13C-labeled MAGs. Microbial populations encoding the Calvin-Benson-Bassham cycle had the highest contribution to carbon fixation across all bicarbonate concentrations tested, spanning 1-10mM. However, out of all the active carbon-fixation pathways detected, MAGs affiliated with the phylum Aquificae encoding the reverse tricarboxylic acid (rTCA) pathway were the only microbial populations that exhibited an increased 13C-bicarbonate assimilation under increasing bicarbonate concentrations. Our study provides the first experimental data supporting predictions that increased bicarbonate concentrations may promote chemoautotrophy via the rTCA cycle and its biological sink for deep subsurface inorganic carbon.

Changes in soil microbial carbon fixation pathways along the oasification process in arid desert region: A confirmation based on metagenome analysis

Microbial carbon fixation has been proved to play a key role in soil carbon accumulation in arid desert oasis, but the fixation mechanism remains unclear. To solve it, we used metagenomic sequencing technology to measure the changes of soil microbial community structure, carbon fixation–related genes and carbon fixation pathway, as well as their relationships with soil properties between natural desert shrubbery (CK) and four oasis farmlands with different cultivation years (i.e., 3, 18 and 30 years) in the arid desert region of northwest China. The results showed that the contents of four soil carbon fraction (soil organic carbon/SOC, soil recalcitrant organic carbon/ROC, soil water solution carbon /WSC, and microbial biomass carbon/MBC) all significantly increased with the cultivation years (30 years ≥ 18 years > 6 years ≥ 3 years ≥ CK) (P < 0.05). Microbial composition was significant difference among CK, 3-, 6-, 18- and 30-years cultivated farmlands. Bacteria were the soil dominant taxa, and its relative abundance gradually decreased with the cultivation years. Proteobacteria, Actinobacteria, Bacteroidetes and Acidobacteria were the dominant phylum of soil microorganisms in desert shrubbery and oasis farmlands. Their relative abundance changed obviously with cultivation years. Six microbial carbon fixation pathways. i.e., the reductive tricarboxylic acid cycle (rTCA), dicarboxylate/4–hydroxybutyrate cycle (DC/4–HB cycle), 3–hydroxypropionate bicycle (3–HP bi–cycle), 3–hydroxypropionate/4–hydroxybutyrate cycle (3–HP/4–HB cycle), Calvin cycle and the Wood–Ljungdahl pathway (WL pathway), were found in all soils, but only the rTCA, DC/4–HB, 3–HP/4–HB cycles and WL pathway significantly changed with the cultivation years. The rTCA cycle was the most common fixation pathway, followed by DC/4–HB cycle and 3–HP/4–HB cycle, while WL pathway was least detected. Soil properties, especially pH, recalcitrant organic carbon, bulk density, total salinity and soil water contents, affected microbial community composition and carbon fixation–related genes in the oasification process. Our results suggested that oasification affected microbial carbon fixation by changing the soil environment due to human agricultural activities. Limited by energy supply in the nutrient–poor soils, rTCA and DC/4–HB cycles were mainly used by microorganisms to fix carbon in oasis farmlands.

Deciphering the microbial functional potential of aeration drip irrigation to enhance soil organic carbon content

Aerated drip irrigation (ADI) is an important practice for promoting soil fertility and crop productivity in greenhouse vegetable production, yet little research has comprehensively investigated the effects on functional traits of carbon (C) cycling microorganisms. In this study, we sought to assess the potential efficacy of the ADI in enhancing soil organic carbon (SOC) content mediated via changes in microbial communities and the expression of genes associated with the carbon cycle. To this end, we adopted a metagenomics approach to compare the effects of ADI treatments with three dissolved oxygen concentrations (10, 15, and 20 mg L-1) during a three-season tomato cultivation experiment in northern China. The results revealed that the 10 mg L-1 treatment led to a significant increase in the abundance of KorA/B genes (associated with the rTCA cycle) in the C fixation pathway, whereas the 15 mg L-1 treatment increased the abundances of cbbL/R and coxL/S genes (associated with the Calvin cycle and carbon monoxide oxidation, respectively). In addition, based on a co-occurrence network analysis, we observed a positive correlation between cbbL and coxS. Interestingly, r-selected microorganisms, such as Proteobacteria and Actinomycetes, characterized by rapid cell multiplication and high biomass production, were identified as the primary contributors to C fixation and were the main predictors of SOC pools. In contrast, the 20 mg L-1 treatment was found to adversely influence C fixation, although enhanced C degradation could be attributed to the breakdown of organic matter by extracellular enzymes secreted by K-selected microorganisms. Collectively, our findings indicate that ADI with dissolved oxygen concentrations ≤15 mg L-1 can promote SOC content by altering the life history strategies of r-selected microorganisms and genes associated with C fixation. These findings will provide valuable insights into agroecosystem irrigation management, specifically in improving soil fertility and promoting sustainable production.

Effect of soil flooding and drying on the metabolic pathways of CO2 fixing microorganisms along an elevation gradient in the Three Gorges Reservoir drawdown area

AbstractThe construction of dams and subsequent water level fluctuations significantly alters the environmental conditions of reservoir ecosystems, affecting the metabolic pathways of CO2 fixing microorganisms (CFMs) and carbon storage in drawdown areas. We investigated the response of soil bacterial communities and carbon fixation pathways to periodic flooding and drying at different elevations by collecting surface soil samples (0–10 cm) in the drawdown area of the Three Gorges Reservoir. The results show that periodic flooding and drying increased the complexity of bacterial co‐occurrence networks. We identified the reductive citrate cycle (rTCA cycle), dicarboxylate‐hydroxybutyrate cycle (DC/4‐HB cycle), 3‐Hydroxypropionate cycle (3‐HP cycle) and reductive pentose phosphate cycle (Calvin cycle) as the main carbon fixation pathways. Notably, as the inundation duration increased, the abundance of the Calvin cycle and its key gene (cbbL) gradually decreased. We also found that soil moisture, pH and the different organic carbon components affected the composition of bacterial communities and the abundance of carbon fixation pathways. These findings elucidate evolutionary trends bacterial communities and the impacts of water level fluctuations on microbial‐mediated carbon cycling processes because of dam construction.

Aqueous microdroplets promote C-C bond formation and sequences in the reverse tricarboxylic acid cycle.

The reverse tricarboxylic acid cycle (rTCA) is a central anabolic network that uses carbon dioxide (CO2) and may have provided complex carbon substrates for life before the advent of RNA or enzymes. However, non-enzymatic promotion of the rTCA cycle, in particular carbon fixation, remains challenging, even with primordial metal catalysis. Here, we report that the fixation of CO2 by reductive carboxylation of succinate and α-ketoglutarate was achieved in aqueous microdroplets under ambient conditions without the use of catalysts. Under identical conditions, the aqueous microdroplets also facilitated the sequences in the rTCA cycle, including reduction, hydration, dehydration and retro-aldol cleavage and linked with the glyoxylate cycle. These reactions of the rTCA cycle were compatible with the aqueous microdroplets, as demonstrated with two-reaction and four-reaction sequences. A higher selectivity giving higher product yields was also observed. Our results suggest that the microdroplets provide an energetically favourable microenvironment and facilitate a non-enzymatic version of the rTCA cycle in prebiotic carbon anabolism.

Late acquisition of the rTCA carbon fixation pathway by Chlorobi.

The reverse tricarboxylic acid (rTCA) cycle is touted as a primordial mode of carbon fixation due to its autocatalytic propensity and oxygen intolerance. Despite this inferred antiquity, however, the earliest rock record affords scant supporting evidence. In fact, based on the chimeric inheritance of rTCA cycle steps within the Chlorobiaceae, even the use of the chemical fossil record of this group is now subject to question. While the 1.64-billion-year-old Barney Creek Formation contains chemical fossils of the earliest known putative Chlorobiaceae-derived carotenoids, interferences from the accompanying hydrocarbon matrix have hitherto precluded the carbon isotope measurements necessary to establish the physiology of the organisms that produced them. Overcoming this obstacle, here we report a suite of compound-specific carbon isotope measurements identifying a cyanobacterially dominated ecosystem featuring heterotrophic bacteria. We demonstrate chlorobactane is 13C-depleted when compared to contemporary equivalents, showing only slight 13C-enrichment over co-existing cyanobacterial carotenoids. The absence of this diagnostic isotopic fingerprint, in turn, confirms phylogenomic hypotheses that call for the late assembly of the rTCA cycle and, thus, the delayed acquisition of autotrophy within the Chlorobiaceae. We suggest that progressive oxygenation of the Earth System caused an increase in the marine sulfate inventory thereby providing the selective pressure to fuel the Neoproterozoic shift towards energy-efficient photoautotrophy within the Chlorobiaceae.

Metagenomics reveals the effect of long-term fertilization on carbon cycle in the maize rhizosphere.

Long-term fertilization can result in the changes in carbon (C) cycle in the maize rhizosphere soil. However, there have been few reports on the impacts of microbial regulatory mechanisms on the C cycle in soil. In the study, we analyzed the response of functional genes that regulate the C fixation, decomposition and methane (CH4) metabolism in maize rhizosphere soil to different fertilization treatments using metagenomics analysis. As the dominant C fixation pathway in maize rhizosphere soil, the abundance of the functional genes regulating the reductive citrate cycle (rTCA cycle) including korA, korB, and IHD1 was higher under the chemical nitrogen (N) fertilizer treatments [nitrogen fertilizer (N), compound chemical fertilization (NPK), the combination of compound chemical fertilizer with maize straw (NPKS)] than maize straw return treatments [maize straw return (S), the combination of phosphorus and potassium fertilizer with maize straw (PKS)]. The NPK treatment decreased the abundance of functional genes involved in 3-hydroxypropionate bicycle (3-HP cycle; porA, porB, and porD), which was one of the major C fixation pathways in soil aside from dicarboxylate-hydroxybutyrate (DC/4-HB cycle) and Calvin cycle. The abundance of functional genes related to C degradation was higher in S, PKS and NPKS treatments than N and NPK treatments, and chemical N fertilizer application had a significant effect on C degradation. The dominant Methanaogenesis pathway in maize rhizosphere soil, used acetate as a substrate, and was significantly promoted under chemical N fertilizer application. The functional genes that were related to CH4 oxidation (i.e., pmoA and pmoB) were reduced under N and NPK treatments. Moreover, soil chemical properties had a significant impact on the functional genes related to C fixation and degradation, with SOC (r2 = 0.79) and NO3--N (r2 = 0.63) being the main regulators. These results implied that N fertilization rather than maize straw return had a greater influence on the C cycle in maize rhizosphere soil.

Carbon Fixation in the Chemolithoautotrophic Bacterium Aquifex aeolicus Involves Two Low-Potential Ferredoxins as Partners of the PFOR and OGOR Enzymes

Aquifex aeolicus is a microaerophilic hydrogen- and sulfur -oxidizing bacterium that assimilates CO2 via the reverse tricarboxylic acid cycle (rTCA). Key enzymes of this pathway are pyruvate:ferredoxin oxidoreductase (PFOR) and 2-oxoglutarate:ferredoxin oxidoreductase (OGOR), which are responsible, respectively, for the reductive carboxylation of acetyl-CoA to pyruvate and of succinyl-CoA to 2-oxoglutarate, two energetically unfavorable reactions that require a strong reduction potential. We have confirmed, by biochemistry and proteomics, that A. aeolicus possesses a pentameric version of these enzyme complexes ((αβγδε)2) and that they are highly abundant in the cell. In addition, we have purified and characterized, from the soluble fraction of A. aeolicus, two low redox potential and oxygen-stable [4Fe-4S] ferredoxins (Fd6 and Fd7, E0 = -440 and -460 mV, respectively) and shown that they can physically interact and exchange electrons with both PFOR and OGOR, suggesting that they could be the physiological electron donors of the system in vivo. Shotgun proteomics indicated that all the enzymes assumed to be involved in the rTCA cycle are produced in the A. aeolicus cells. A number of additional enzymes, previously suggested to be part of a putative partial Wood-Ljungdahl pathway used for the synthesis of serine and glycine from CO2 were identified by mass spectrometry, but their abundance in the cell seems to be much lower than that of the rTCA cycle. Their possible involvement in carbon assimilation is discussed.

Metagenomic insights into the functions of microbial communities in sulfur-rich sediment of a shallow-water hydrothermal vent off Kueishan Island.

Hydrothermal vent (HTV) systems are important habitats for understanding the biological processes of extremophiles on Earth and their relative contributions to material and energy cycles in the ocean. Current understanding on hydrothermal systems have been primarily focused on deep-sea HTVs, and little is known about the functions and metabolisms of microorganisms in shallow-water HTVs (SW-HTVs), which are distinguished from deep-sea HTVs by a depth limit of 200 m. In this study, we analyzed metagenomes of sulfur-rich sediment samples collected from a SW-HTV of Kueishan Island, located in a marginal sea of the western Pacific Ocean. Comparing with a previously published report of pelagic samples from the nearby sampling site, microbial communities in the SW-HTV sediments enriching with genes of both aerobic and anaerobic respiration inferred variable environments in the tested sediments. Abundant genes of energy metabolism encoding sulfur oxidation, H2 oxidation, and carbon fixation were detected from the sediment samples. Sixty-eight metagenome-assembled-genomes (MAGs) were reconstructed to further understand the metabolism and potential interactions between different microbial taxa in the SW-HTVs sediment. MAGs with the highest abundant were chemolithotrophic sulfur-oxidization bacteria, including Sulfurovum represented Campylobacteria involved sox multienzyme, sulfide oxidation genes and rTCA cycle, and Gammaproteobacteria involved dsr gene and CBB cycle. In addition, Desulfobacterota with the potential to participate in sulfur-disproportionating processes also had higher abundance than the sample's overall mean value. The interaction of these bacterial groups allows the microbial communities to efficiently metabolize a large variety of sulfur compounds. In addition, the potential to use simple organic carbon, such as acetate, was found in chemolithotrophic Campylobacterial MAGs. Collectively, our results revealed the complexity of environmental conditions of the vent sediment and highlight the interactive relationships of the dominant microbial populations in driving sulfur cycles in the SW-HTV sediments off Kueishan Island.

Chimeric inheritance and crown-group acquisitions of carbon fixation genes within Chlorobiales: Origins of autotrophy in Chlorobiales and implication for geological biomarkers.

The geological record of microbial metabolisms and ecologies primarily consists of stable isotope fractionations and the diagenetic products of biogenic lipids. Carotenoid lipid biomarkers are particularly useful proxies for reconstructing this record, providing information on microbial phototroph primary productivity, redox couples, and oxygenation. The biomarkers okenane, chlorobactane, and isorenieratene are generally considered to be evidence of anoxygenic phototrophs, and provide a record that extends to 1.64 Ga. The utility of the carotenoid biomarker record may be enhanced by examining the carbon isotopic ratios in these products, which are diagnostic for specific pathways of biological carbon fixation found today within different microbial groups. However, this joint inference assumes that microbes have conserved these pathways across the duration of the preserved biomarker record. Testing this hypothesis, we performed phylogenetic analyses of the enzymes constituting the reductive tricarboxylic acid (rTCA) cycle in Chlorobiales, the group of anoxygenic phototrophic bacteria usually implicated in the deposition of chlorobactane and isorenieretane. We find phylogenetically incongruent patterns of inheritance across all enzymes, indicative of horizontal gene transfers to both stem and crown Chlorobiales from multiple potential donor lineages. This indicates that a complete rTCA cycle was independently acquired at least twice within Chlorobiales and was not present in the last common ancestor. When combined with recent molecular clock analyses, these results predict that the Mesoproterzoic lipid biomarker record diagnostic for Chlorobiales should not preserve isotopic fractionations indicative of a full rTCA cycle. Furthermore, we conclude that coupling isotopic and biomarker records is insufficient for reliably reconstructing microbial paleoecologies in the absence of a complementary and consistent phylogenomic narrative.

Adaptation of thermophilic enzymes from an ancestral reductive TCA cycle for carbon fixation in plants

The reductive TCA cycle (rTCA) is one of 5 carbon‐fixing cycles in prokaryotes other than the Calvin‐Benson cycle ubiquitous in plants. Adaptation of these cycles to biofuel crops is one strategy for reducing the greenhouse gas CO2 and simultaneously increasing crop yields. A minimal 5‐enzyme synthetic rTCA cycle was designed at NCSU utilizing 2 unique enzymes to carboxylate and reduce 2‐oxoglutarate to isocitrate. The enzymes are derived from an evolutionarily primitive configuration of the rTCA cycle that persists in the order Aquificales in hot springs and deep ocean thermal vents. Two strategies are being pursued to engineer mesophilic forms of these enzymes: 1) reducing the temperature of activity of the Aquificales enzymes, and 2) modifying the substrate specificity of homologous mesophilic enzymes. The first enzyme, 2‐oxoglutarate carboxylase (OGC), is a member of the biotin‐dependent carboxylase family homologous to pyruvate carboxylase. Biotin‐dependent carboxylases are multi‐subunit complexes. So far, we have identified the temperature‐dependent steps of the biotin carboxylase domain of OGC and predicted mutations to reduce the temperature of activity based on the crystal structure. Phylogenetic analysis suggests that OGC and pyruvate carboxylase diverged prior to Aquificales evolution. An ancestral promiscuous enzyme has been proposed to carboxylate both pyruvate and 2‐oxoglutarate. Ancestral enzymes are being reconstructed from phylogenetic analyses to switch the substrate specificity of mesophilic pyruvate carboxylase to carboxylate 2‐oxoglutarate. The second enzyme, oxalosuccinate reductase (OSR), is homologous with isocitrate dehydrogenase. In fact, isocitrate dehydrogenase has replaced OGC/OSR in most organisms utilizing the rTCA cycle. Isocitrate dehydrogenase favors the oxidative decarboxylase of isocitrate in the TCA cycle, while OGC/OSR favors the reductive cycle. OSR is an ancestral, non‐decarboxylating isocitrate dehydrogenase. We propose to engineer a mesophilic OSR by mutating isocitrate dehydrogenase to inhibit decarboxylation, based on phylogenic and structural studies of OSR. Ultimately engineering a mesophilic OGC/OSR will be utilized to increase carbon capture in seed oil crops like Camelina sativa that are carbon limited. Oil from Camelina seeds can be converted to jet fuels.

Metagenomics reveals divergent functional profiles of soil carbon and nitrogen cycling under long-term addition of chemical and organic fertilizers in the black soil region

The long-term effects of different fertilization regimes on the microbial functional potential of soils involving nutrient cycling remain largely unknown. Here, metagenomic sequencing was applied to investigate the influences of long-term chemical and organic fertilization on soil microbial C and N cycling across southern, middle and northern sites of black soil region in Northeast China. The results showed that biogeographic distance induced the most influential on the microbial functional profiles of soil C and N cycling, and significant effects of manure fertilization were detected across three experimental sites. Organic fertilization enriched the relative abundances of Proteobacteria and Planctomycetes that carry C and N cycling genes, while inhibited the growth of oligotrophic groups such as Verrucomicrobia. Chemical fertilization increased the gene abundances involved in methane oxidation, but had little effect on soil C degradation and fixation. Contrarily, manure fertilization, particularly the combination of chemical and organic fertilizers (CFM), significantly decreased the abundance of cooC (reductive acetyl-CoA pathway) and coxS (CO oxidation) while enhanced the abundance of icd (rTCA cycle), which are involved in C fixation. Additionally, chemical fertilization enriched the gene abundance that involved in soil N degradation, nitrification and anammox, whereas manure fertilization was beneficial for the functional potentials of assimilatory and dissimilatory nitrate reductions across the black soils. However, CFM significantly promoted the soil denitrification potential, possibly due to excess N input, which might result in soil N loss via the emission of nitrogenous gas in this region. Furthermore, the substantial enhancement in soil P contents induced by manure addition predominantly affected the C and N cycling profiles, abundance of functional genes and microbial taxa. Moreover, diverse correlations between C and N cycling genes suggested the synergetic or antagonistic interactions of C and N metabolic potentials in the black soils. Overall, this study provided in-depth insights into distinct microbial functional potentials under long-term chemical and organic fertilization that may have predictable consequences for soil nutrient cycling in agroecosystems of black soil region.

Insights into carbon-fixation pathways through metagonomics in the sediments of deep-sea cold seeps

Carbon fixation by chemoautotrophic microorganisms in the dark ocean has a major impact on global carbon cycling and ecological relationships in the ocean's interior. At present, six pathways of autotrophic carbon fixation have been found: the Calvin cycle, the reductive Acetyl-CoA or Wood-Ljungdahl pathway (rAcCoA), the reductive tricarboxylic acid cycle (rTCA), the 3-hydroxypropionate bicycle (3HP), the 3-hydroxypropionate/4-hydroxybutyrate cycle (3HP/4HB), and the dicarboxylate/4-hydroxybutyrate cycle (DC/4HB). Although our knowledge about carbon fixation pathways in the ocean has increased significantly, carbon fixation pathways in the cold seeps are still unknown. In this study, we collected sediment samples from two cold seeps and one trough in the south China sea (SCS), and investigated with metagenomic and metagenome assembled genomes (MAGs). We found that six autotrophic carbon fixation pathways present in the cold seeps and trough with rTCA cycle was the most common pathway, whose genes were particularly high in the cold seeps and increased with sediment depths; the rAcCoA cycle mainly occurred in the cold seep regions, and the abundance of module genes increased with sediment depths. We also elucidated members of chemoautotrophic microorganisms involved in these six carbon-fixation pathways. The rAcCoA, rTCA and DC/4-HB cycles required significantly less energy probably play an important role in the deep-sea environments, especially in the cold seeps. This study provided metabolic insights into the carbon fixation pathways in the cold seeps, and laid the foundation for future detailed study on processes and rates of carbon fixation in the deep-sea ecosystems.

Growth Enhancement Facilitated by Gaseous CO2 through Heterologous Expression of Reductive Tricarboxylic Acid Cycle Genes in Escherichia coli

The enzymatic mechanisms of carbon fixation by autotrophs, such as the reductive tricarboxylic acid cycle (rTCA), have inspired biotechnological approaches to producing bio-based chemicals directly through CO2. To explore the possibility of constructing an rTCA cycle in Escherichia coli and to investigate their potential for CO2 assimilation, a total of ten genes encoding the key rTCA cycle enzymes, including α-ketoglutarate:ferredoxin oxidoreductase, ATP-dependent citrate lyase, and fumarate reductase/succinate dehydrogenase, were cloned into E. coli. The transgenic E. coli strain exhibited enhanced growth and the ability to assimilate external inorganic carbon with a gaseous CO2 supply. Further experiments conducted in sugar-free medium containing hydrogen as the electron donor and dimethyl sulfoxide (DMSO) as the electron acceptor proved that the strain is able to undergo anaerobic respiration, using CO2 as the major carbon source. The transgenic stain demonstrated CO2-enhanced growth, whereas the genes involved in chemotaxis, flagellar assembly, and acid-resistance were upregulated under the anaerobic respiration. Furthermore, metabolomic analysis demonstrated that the total concentrations of ATP, ADP, and AMP in the transgenic strain were higher than those in the vector control strain and these results coincided with the enhanced growth. Our approach offers a novel strategy to engineer E. coli for assimilating external gaseous CO2.

The Composition and Primary Metabolic Potential of Microbial Communities Inhabiting the Surface Water in the Equatorial Eastern Indian Ocean.

Simple SummaryMarine microbes are regarded as the most diverse organisms in the biosphere and drive biogeochemical cycles through their metabolism. It is essential to understand the structure and metabolic function of microbial communities. The Indian Ocean is the third largest ocean in the world, and it possesses unique hydrographical properties. So far, assessments of microbial diversity and metabolism need to be improved in the Indian Ocean. Therefore, we carried out a series of investigations in the equatorial eastern Indian Ocean in order to clarify the local microbial communities and detect the genetic potential for microbial functions. The obtained results suggested Cyanobacteria was the dominant microbial group, and predicted the Calvin cycle and the assimilatory nitrate and nitrite reduction played important role in the pathway of carbon fixation and nitrogen metabolism respectively. This study provides insights into microbial community structures as well as the metabolic potential that may be active in the local environment, and lays the groundwork for understanding the roles of microbes in energy and resource cycling in this habitat.Currently, there is scant information about the biodiversity and functional diversity of microbes in the eastern Indian Ocean (EIO). Here, we used a combination of high-throughput sequencing of 16S rRNA genes and a metagenomic approach to investigate the microbial population structure and its metabolic function in the equatorial EIO. Our results show that Cyanobacterial Prochlorococcus made up the majority of the population. Interestingly, there were fewer contributions from clades SAR11 (Alphaproteobacteria) and SAR86 (Gammaproteobacteria) to microbial communities than contributions from Prochlorococcus. Based on functional gene analysis, functional genes rbcL, narB, and nasA were relatively abundant among the relevant genes. The abundance of Prochlorococcus implies its typically ecological adaptation in the local ecosystem. The microbial metabolic potential shows that in addition to the main carbon fixation pathway Calvin cycle, the rTCA cycle and the 3-HP/4-HB cycle have potential alternative carbon fixation contributions to local ecosystems. For the nitrogen cycle, the assimilatory nitrate and nitrite reduction pathway is potentially the crucial form of nitrogen utilization; unexpectedly, nitrogen fixation activity was relatively weak. This study extends our knowledge of the roles of microbes in energy and resource cycling in the EIO and provides a foundation for revealing profound biogeochemical processes driven by the microbial community in the ocean.