scholarly journals Lipogenesis and Redox Balance in Nitrogen-Fixing Pea Bacteroids

2016 ◽  
Vol 198 (20) ◽  
pp. 2864-2875 ◽  
Author(s):  
Jason J. Terpolilli ◽  
Shyam K. Masakapalli ◽  
Ramakrishnan Karunakaran ◽  
Isabel U. C. Webb ◽  
Rob Green ◽  
...  
Keyword(s):  
Direct Reduction ◽  
Root Nodules ◽  
Dicarboxylic Acids ◽  
Tca Cycle ◽  
Redox Potentials ◽  
Acetyl Coa ◽  
Content Type ◽  
The Tca Cycle ◽  
Redox Poise ◽  
Phb Synthase

ABSTRACTWithin legume root nodules, rhizobia differentiate into bacteroids that oxidize host-derived dicarboxylic acids, which is assumed to occur via the tricarboxylic acid (TCA) cycle to generate NAD(P)H for reduction of N2. Metabolic flux analysis of laboratory-grownRhizobium leguminosarumshowed that the flux from [13C]succinate was consistent with respiration of an obligate aerobe growing on a TCA cycle intermediate as the sole carbon source. However, the instability of fragile pea bacteroids prevented their steady-state labeling under N2-fixing conditions. Therefore, comparative metabolomic profiling was used to compare free-livingR. leguminosarumwith pea bacteroids. While the TCA cycle was shown to be essential for maximal rates of N2fixation, levels of pyruvate (5.5-fold reduced), acetyl coenzyme A (acetyl-CoA; 50-fold reduced), free coenzyme A (33-fold reduced), and citrate (4.5-fold reduced) were much lower in bacteroids. Instead of completely oxidizing acetyl-CoA, pea bacteroids channel it into both lipid and the lipid-like polymer poly-β-hydroxybutyrate (PHB), the latter via a type III PHB synthase that is active only in bacteroids. Lipogenesis may be a fundamental requirement of the redox poise of electron donation to N2in all legume nodules. Direct reduction by NAD(P)H of the likely electron donors for nitrogenase, such as ferredoxin, is inconsistent with their redox potentials. Instead, bacteroids must balance the production of NAD(P)H from oxidation of acetyl-CoA in the TCA cycle with its storage in PHB and lipids.IMPORTANCEBiological nitrogen fixation by symbiotic bacteria (rhizobia) in legume root nodules is an energy-expensive process. Within legume root nodules, rhizobia differentiate into bacteroids that oxidize host-derived dicarboxylic acids, which is assumed to occur via the TCA cycle to generate NAD(P)H for reduction of N2. However, direct reduction of the likely electron donors for nitrogenase, such as ferredoxin, is inconsistent with their redox potentials. Instead, bacteroids must balance oxidation of plant-derived dicarboxylates in the TCA cycle with lipid synthesis. Pea bacteroids channel acetyl-CoA into both lipid and the lipid-like polymer poly-β-hydroxybutyrate, the latter via a type II PHB synthase. Lipogenesis is likely to be a fundamental requirement of the redox poise of electron donation to N2in all legume nodules.

scholarly journals Genomic and Transcriptomic Analyses of the Facultative Methanotroph Methylocystis sp. Strain SB2 Grown on Methane or Ethanol

2014 ◽  
Vol 80 (10) ◽  
pp. 3044-3052 ◽  
Author(s):  
Alexey Vorobev ◽  
Sheeja Jagadevan ◽  
Sunit Jain ◽  
Karthik Anantharaman ◽  
Gregory J. Dick ◽  
...  
Keyword(s):  
Methane Oxidation ◽  
Coenzyme A ◽  
Draft Genome ◽  
Carbon Assimilation ◽  
Tca Cycle ◽  
Content Type ◽  
The Tca Cycle ◽  
Conversion Of Methane ◽  
Oxidative Transformations ◽  
Rate Limiting

ABSTRACTA minority of methanotrophs are able to utilize multicarbon compounds as growth substrates in addition to methane. The pathways utilized by these microorganisms for assimilation of multicarbon compounds, however, have not been explicitly examined. Here, we report the draft genome of the facultative methanotrophMethylocystissp. strain SB2 and perform a detailed transcriptomic analysis of cultures grown with either methane or ethanol. Evidence for use of the canonical methane oxidation pathway and the serine cycle for carbon assimilation from methane was obtained, as well as for operation of the complete tricarboxylic acid (TCA) cycle and the ethylmalonyl-coenzyme A (EMC) pathway. Experiments withMethylocystissp. strain SB2 grown on methane revealed that genes responsible for the first step of methane oxidation, the conversion of methane to methanol, were expressed at a significantly higher level than those for downstream oxidative transformations, suggesting that this step may be rate limiting for growth of this strain with methane. Further, transcriptomic analyses ofMethylocystissp. strain SB2 grown with ethanol compared to methane revealed that on ethanol (i) expression of the pathway of methane oxidation and the serine cycle was significantly reduced, (ii) expression of the TCA cycle dramatically increased, and (iii) expression of the EMC pathway was similar. Based on these data, it appears thatMethylocystissp. strain SB2 converts ethanol to acetyl-coenzyme A, which is then funneled into the TCA cycle for energy generation or incorporated into biomass via the EMC pathway. This suggests that some methanotrophs have greater metabolic flexibility than previously thought and that operation of multiple pathways in these microorganisms is highly controlled and integrated.

Gluconeogenesis from labeled carbon: estimating isotope dilution

1986 ◽  
Vol 250 (3) ◽  
pp. E296-E305 ◽  
Author(s):  
J. K. Kelleher
Keyword(s):  
Steady State ◽  
Isotope Dilution ◽  
Experimental Tests ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Complex Model ◽  
Acetyl Coa ◽  
The Tca Cycle ◽  
Carbon Compounds ◽  
Mathematical Techniques

To estimate the rate of gluconeogenesis from steady-state incorporation of labeled 3-carbon precursors into glucose, isotope dilution must be considered so that the rate of labeling of glucose can be quantitatively converted to the rate of gluconeogenesis. An expression for the value of this isotope dilution can be derived using mathematical techniques and a model of the tricarboxylic acid (TCA) cycle. The present investigation employs a more complex model than that used in previous studies. This model includes the following pathways that may affect the correction for isotope dilution: 1) flux of 3-carbon precursor to the oxaloacetate pool via acetyl-CoA and the TCA cycle; 2) flux of 4- or 5-carbon compounds into the TCA cycle; 3) reversible flux between oxaloacetate (OAA) and pyruvate and between OAA and fumarate; 4) incomplete equilibrium between OAA pools; and 5) isotope dilution of 3-carbon tracers between the experimentally measured pool and the precursor for the TCA-cycle OAA pool. Experimental tests are outlined which investigators can use to determine whether these pathways are significant in a specific steady-state system. The study indicated that flux through these five pathways can significantly affect the correction for isotope dilution. To correct for the effects of these pathways an alternative method for calculating isotope dilution is proposed using citrate to relate the specific activities of acetyl-CoA and OAA.

Pattern of substrate utilization in vascular smooth muscle using 13C isotopomer analysis of glutamate

1998 ◽  
Vol 275 (6) ◽  
pp. H2227-H2235 ◽  
Author(s):  
Tara M. Allen ◽  
Christopher D. Hardin
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Acetyl Coa ◽  
Contractile State ◽  
The Tca Cycle ◽  
Direct Measurements ◽  
Isotopomer Analysis ◽  
13C Isotopomer Analysis

Although vascular smooth muscle (VSM) derives the majority of its energy from oxidative phosphorylation, controversy exists concerning which substrates are utilized by the tricarboxylic acid (TCA) cycle. We used 13C isotopomer analysis of glutamate to directly measure the entry of exogenous [13C]glucose and acetate and unlabeled endogenous sources into the TCA cycle via acetyl-CoA. Hog carotid artery segments denuded of endothelium were superfused with 5 mM [1-13C]glucose and 0–5 mM [1,2-13C]acetate at 37°C for 3–12 h. We found that both resting and contracting VSM preferentially utilize [1,2-13C]acetate compared with [1-13C]glucose and unlabeled substrates. The entry of glucose into the TCA cycle (30–60% of total entry via acetyl-CoA) exhibited little change despite alterations in contractile state or acetate concentrations ranging from 0 to 5 mM. We conclude that glucose and nonglucose substrates are important oxidative substrates for resting and contracting VSM. These are the first direct measurements of relative substrate entry into the TCA cycle of VSM during activation and may provide a useful method to measure alterations in VSM metabolism under physiological and pathophysiological conditions.

scholarly journals Metabolome Remodeling during the Acidogenic-Solventogenic Transition in Clostridium acetobutylicum

2011 ◽  
Vol 77 (22) ◽  
pp. 7984-7997 ◽  
Author(s):  
Daniel Amador-Noguez ◽  
Ian A. Brasg ◽  
Xiao-Jiang Feng ◽  
Nathaniel Roquet ◽  
Joshua D. Rabinowitz
Keyword(s):  
Amino Acids ◽  
Clostridium Acetobutylicum ◽  
Reducing Power ◽  
Tca Cycle ◽  
Content Type ◽  
Isotope Tracers ◽  
Metabolic Fluxes ◽  
The Tca Cycle ◽  
Concentration Changes ◽  
Almost All

ABSTRACTThe fermentation carried out by the biofuel producerClostridium acetobutylicumis characterized by two distinct phases. Acidogenesis occurs during exponential growth and involves the rapid production of acids (acetate and butyrate). Solventogenesis initiates as cell growth slows down and involves the production of solvents (butanol, acetone, and ethanol). Using metabolomics, isotope tracers, and quantitative flux modeling, we have mapped the metabolic changes associated with the acidogenic-solventogenic transition. We observed a remarkably ordered series of metabolite concentration changes, involving almost all of the 114 measured metabolites, as the fermentation progresses from acidogenesis to solventogenesis. The intracellular levels of highly abundant amino acids and upper glycolytic intermediates decrease sharply during this transition. NAD(P)H and nucleotide triphosphates levels also decrease during solventogenesis, while low-energy nucleotides accumulate. These changes in metabolite concentrations are accompanied by large changes in intracellular metabolic fluxes. During solventogenesis, carbon flux into amino acids, as well as flux from pyruvate (the last metabolite in glycolysis) into oxaloacetate, decreases by more than 10-fold. This redirects carbon into acetyl coenzyme A, which cascades into solventogenesis. In addition, the electron-consuming reductive tricarboxylic acid (TCA) cycle is shutdown, while the electron-producing oxidative (clockwise) right side of the TCA cycle remains active. Thus, the solventogenic transition involves global remodeling of metabolism to redirect resources (carbon and reducing power) from biomass production into solvent production.

scholarly journals N-acetyltaurine and Acetylcarnitine Production for the Mitochondrial Acetyl-CoA Regulation in Skeletal Muscles during Endurance Exercises

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 522
Author(s):  
Teruo Miyazaki ◽  
Yuho Nakamura-Shinya ◽  
Kei Ebina ◽  
Shoichi Komine ◽  
Song-Gyu Ra ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Palmitic Acid ◽  
Skeletal Muscles ◽  
Metabolic Flux ◽  
Human Subjects ◽  
Tca Cycle ◽  
Blood Levels ◽  
Acetyl Coa ◽  
The Tca Cycle ◽  
Human Skeletal Muscle Cells

During endurance exercises, a large amount of mitochondrial acetyl-CoA is produced in skeletal muscles from lipids, and the excess acetyl-CoA suppresses the metabolic flux from glycolysis to the TCA cycle. This study evaluated the hypothesis that taurine and carnitine act as a buffer of the acetyl moiety of mitochondrial acetyl-CoA derived from the short- and long-chain fatty acids of skeletal muscles during endurance exercises. In human subjects, the serum concentrations of acetylated forms of taurine (NAT) and carnitine (ACT), which are the metabolites of acetyl-CoA buffering, significantly increased after a full marathon. In the culture medium of primary human skeletal muscle cells, NAT and ACT concentrations significantly increased when they were cultured with taurine and acetate or with carnitine and palmitic acid, respectively. The increase in the mitochondrial acetyl-CoA/free CoA ratio induced by acetate and palmitic acid was suppressed by taurine and carnitine, respectively. Elevations of NAT and ACT in the blood of humans during endurance exercises might serve the buffering of the acetyl-moiety in mitochondria by taurine and carnitine, respectively. The results suggest that blood levels of NAT and ACT indicate energy production status from fatty acids in the skeletal muscles of humans undergoing endurance exercise.

scholarly journals Imaging and modelling of poly(3-hydroxybutyrate) synthesis in Paracoccus denitrificans

2021 ◽  
Author(s):  
Sergio Bordel ◽  
Rob J. M. van Spanning ◽  
Fernando Santos-Beneit
Keyword(s):  
Exponential Growth ◽  
Laser Scanning ◽  
Paracoccus Denitrificans ◽  
Tca Cycle ◽  
Metabolic Model ◽  
Acetyl Coa ◽  
Granule Formation ◽  
The Tca Cycle ◽  
A Genome ◽  
Phb Production

Abstract Poly(3-hydroxybutyrate) (PHB) granule formation in Paracoccus denitrificans Pd1222 was investigated by laser scanning confocal microscopy (LSCM) and gas chromatography analysis. Cells that had been starved for 2 days were free of PHB granules but resynthesized them within 30 minutes of growth in fresh medium with succinate. In most cases, the granules were distributed randomly, although in some cases they appeared in a more organized pattern. The rates of growth and PHB accumulation were analyzed within the frame of a Genome-Scale Metabolic Model (GSMM) containing 781 metabolic genes, 1403 reactions and 1503 metabolites. The model was used to obtain quantitative predictions of biomass yields and PHB synthesis during aerobic growth on succinate as sole carbon and energy sources. The results revealed an initial fast stage of PHB accumulation, during which all of the acetyl-CoA originating from succinate was diverted to PHB production. The next stage was characterized by a tenfold lower PHB production rate and the simultaneous onset of exponential growth, during which acetyl-CoA was predominantly drained into the TCA cycle. Previous research has shown that PHB accumulation correlates with cytosolic acetyl-CoA concentration. It has also been shown that PHB accumulation is not transcriptionally regulated. Our results are consistent with the mentioned findings and suggest that, in absence of cell growth, most of the cellular acetyl-CoA is channeled to PHB synthesis, while during exponential growth, it is drained to the TCA cycle, causing a reduction of the cytosolic acetyl-CoA pool and a concomitant decrease of the synthesis of acetoacetyl-CoA (the precursor of PHB synthesis).

scholarly journals NAD(P)+-Malic Enzyme Mutants of Sinorhizobium sp. Strain NGR234, but Not Azorhizobium caulinodans ORS571, Maintain Symbiotic N2Fixation Capabilities

2012 ◽  
Vol 78 (8) ◽  
pp. 2803-2812 ◽  
Author(s):  
Ye Zhang ◽  
Toshihiro Aono ◽  
Phillip Poole ◽  
Turlough M. Finan
Keyword(s):  
Malic Enzyme ◽  
Sinorhizobium Meliloti ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Rhizobium Leguminosarum ◽  
Dicarboxylic Acids ◽  
Dry Weight ◽  
Tca Cycle ◽  
Broad Host Range ◽  
Azorhizobium Caulinodans ◽  
Content Type

ABSTRACTC4-dicarboxylic acids appear to be metabolized via the tricarboxylic acid (TCA) cycle in N2-fixing bacteria (bacteroids) within legume nodules. InSinorhizobium melilotibacteroids from alfalfa, NAD+-malic enzyme (DME) is required for N2fixation, and this activity is thought to be required for the anaplerotic synthesis of pyruvate. In contrast, in the pea symbiontRhizobium leguminosarum, pyruvate synthesis occurs via either DME or a pathway catalyzed by phosphoenolpyruvate carboxykinase (PCK) and pyruvate kinase (PYK). Here we report thatdmemutants of the broad-host-rangeSinorhizobiumsp. strain NGR234 formed nodules whose level of N2fixation varied from 27 to 83% (plant dry weight) of the wild-type level, depending on the host plant inoculated. NGR234 bacteroids had significant PCK activity, and while singlepckAand singledmemutants fixed N2at reduced rates, apckA dmedouble mutant had no N2-fixing activity (Fix−). Thus, NGR234 bacteroids appear to synthesize pyruvate from TCA cycle intermediates via DME or PCK pathways. These NGR234 data, together with other reports, suggested that the completely Fix−phenotype ofS. meliloti dmemutants may be specific to the alfalfa-S. melilotisymbiosis. We therefore examined the ME-like genesazc3656andazc0119fromAzorhizobium caulinodans, asazc3656mutants were previously shown to form Fix−nodules on the tropical legumeSesbania rostrata. We found that purified AZC3656 protein is an NAD(P)+-malic enzyme whose activity is inhibited by acetyl-coenzyme A (acetyl-CoA) and stimulated by succinate and fumarate. Thus, whereas DME is required for symbiotic N2fixation inA. caulinodansandS. meliloti, in other rhizobia this activity can be bypassed via another pathway(s).

scholarly journals Use of a Bacterial Luciferase Monitoring System To Estimate Real-Time Dynamics of Intracellular Metabolism in Escherichia coli

2016 ◽  
Vol 82 (19) ◽  
pp. 5960-5968 ◽  
Author(s):  
Tomohiro Shimada ◽  
Kan Tanaka
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Monitoring System ◽  
Carbon Sources ◽  
Tca Cycle ◽  
Bacterial Cells ◽  
Bacterial Luciferase ◽  
Content Type ◽  
Time Dynamics ◽  
The Tca Cycle

ABSTRACTRegulation of central carbon metabolism has long been an important research subject in every organism. While the dynamics of metabolic flows during changes in available carbon sources have been estimated based on changes in metabolism-related gene expression, as well as on changes in the metabolome, the flux change itself has scarcely been measured because of technical difficulty, which has made conclusions elusive in many cases. Here, we used a monitoring system employingVibrio fischeriluciferase to probe the intracellular metabolic condition inEscherichia coli. Using a batch culture provided with a limited amount of glucose, we performed a time course analysis, where the predominant carbon source shifts from glucose to acetate, and identified a series of sequential peaks in the luciferase activity (peaks 1 to 4). Two major peaks, peaks 1 and 3, were considered to correspond to the glucose and acetate consuming phases, respectively, based on the glucose, acetate, and dissolved oxygen concentrations in the medium. The pattern of these peaks was changed by the addition of a different carbon source or by an increasing concentration of glucose, which was consistent with the present model. Genetically, mutations involved in glycolysis or the tricarboxylic acid (TCA) cycle/gluconeogenesis specifically affected peak 1 or peak 3, respectively, as expected from the corresponding metabolic phase. Intriguingly, mutants for the acetate excretion pathway showed a phenotype of extended peak 2 and delayed transition to the TCA cycle/gluconeogenesis phase, which suggests that peak 2 represents the metabolic transition phase. These results indicate that the bacterial luciferase monitoring system is useful to understand the real-time dynamics of metabolism in living bacterial cells.IMPORTANCEIntracellular metabolic flows dynamically change during shifts in available carbon sources. However, because of technical difficulty, the flux change has scarcely been measured in living cells. Here, we used aVibrio fischeriluciferase monitoring system to probe the intracellular metabolic condition inEscherichia coli. Using a limited amount of glucose batch culture, a series of sequential peaks (peaks 1 to 4) in the luciferase activity was observed. Changes in the pattern of these peaks by the addition of extra carbon sources and in mutant strains involved in glycolysis or the TCA cycle/gluconeogenesis gene assigned the metabolic phase corresponding to peak 1 as the glycolysis phase and peak 3 as the TCA cycle/gluconeogenesis phase. Intriguingly, the acetate excretion pathway engaged in peak 2 represents the metabolic transition phase. These results indicate that the bacterial luciferase monitoring system is useful to understand the real-time dynamics of metabolism in living bacterial cells.

THE OXIDATIVE METABOLISM OF RHODOTORULA GRACILIS

1958 ◽  
Vol 4 (3) ◽  
pp. 205-213 ◽  
Author(s):  
John H. Litchfield ◽  
Z. John Ordal
Keyword(s):  
Oxidative Metabolism ◽  
Dicarboxylic Acids ◽  
Tca Cycle ◽  
Pentose Phosphate ◽  
Cell Suspensions ◽  
Resting Cell ◽  
The Tca Cycle ◽  
Rhodotorula Gracilis ◽  
Oxidation Of Glucose

The oxidative metabolism of Rhodolorula gracilis NRRL Y-1091 was investigated. Data was presented showing the oxidation of glucose, acetate, pyruvate, xylose, D- and L-arabinose, by both glucose- and xylose-grown cells. Gluconate was oxidized by the glucose-grown cells, while ribose was oxidized by the xylose-grown cells. Cell-free extracts of glucose-grown cells oxidized glucose, gluconate, glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, and ribose-5-phosphate. Pentose phosphate was demonstrated as a product of glucose-6-phosphate oxidation. Resting-cell suspensions were unable to oxidize citrate, but they oxidized the dicarboxylic acids of the TCA cycle. Citrate was detected as a product of the oxidation of acetate, pyruvate, and malate in the presence of fluoroacetate. Cell-free extracts of glucose-grown cells oxidized citrate, isocitrate, and the dicarboxylic acids of the TCA cycle.

scholarly journals Imaging and modelling of poly(3-hydroxybutyrate) synthesis in Paracoccus denitrificans

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sergio Bordel ◽  
Rob J. M. van Spanning ◽  
Fernando Santos-Beneit
Keyword(s):  
Exponential Growth ◽  
Laser Scanning ◽  
Paracoccus Denitrificans ◽  
Tca Cycle ◽  
Metabolic Model ◽  
Acetyl Coa ◽  
Granule Formation ◽  
The Tca Cycle ◽  
A Genome ◽  
Phb Production

AbstractPoly(3-hydroxybutyrate) (PHB) granule formation in Paracoccus denitrificans Pd1222 was investigated by laser scanning confocal microscopy (LSCM) and gas chromatography analysis. Cells that had been starved for 2 days were free of PHB granules but resynthesized them within 30 min of growth in fresh medium with succinate. In most cases, the granules were distributed randomly, although in some cases they appeared in a more organized pattern. The rates of growth and PHB accumulation were analyzed within the frame of a Genome-Scale Metabolic Model (GSMM) containing 781 metabolic genes, 1403 reactions and 1503 metabolites. The model was used to obtain quantitative predictions of biomass yields and PHB synthesis during aerobic growth on succinate as sole carbon and energy sources. The results revealed an initial fast stage of PHB accumulation, during which all of the acetyl-CoA originating from succinate was diverted to PHB production. The next stage was characterized by a tenfold lower PHB production rate and the simultaneous onset of exponential growth, during which acetyl-CoA was predominantly drained into the TCA cycle. Previous research has shown that PHB accumulation correlates with cytosolic acetyl-CoA concentration. It has also been shown that PHB accumulation is not transcriptionally regulated. Our results are consistent with the mentioned findings and suggest that, in absence of cell growth, most of the cellular acetyl-CoA is channeled to PHB synthesis, while during exponential growth, it is drained to the TCA cycle, causing a reduction of the cytosolic acetyl-CoA pool and a concomitant decrease of the synthesis of acetoacetyl-CoA (the precursor of PHB synthesis).

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