Carbon Metabolism In Response Research Articles

Metabolic flux reprogramming in Mycobacterium tuberculosis-infected human macrophages.

Metabolic fluxes are at the heart of metabolism and growth in any living system. During tuberculosis (TB) infection, the pathogenic Mycobacterium tuberculosis (Mtb) adapts its nutritional behaviour and metabolic fluxes to survive in human macrophages and cause infection. The infected host cells also undergo metabolic changes. However, our knowledge of the infected host metabolism and identification of the reprogrammed metabolic flux nodes remains limited. In this study, we applied systems-based 13C-metabolic flux analysis (MFA) to measure intracellular carbon metabolic fluxes in Mtb-infected human THP-1 macrophages. We provide a flux map for infected macrophages that quantified significantly increased fluxes through glycolytic fluxes towards pyruvate synthesis and reduced pentose phosphate pathway fluxes when compared to uninfected macrophages. The tri carboxylic acid (TCA) cycle fluxes were relatively low, and amino acid fluxes were reprogrammed upon Mtb infection. The knowledge of host metabolic flux profiles derived from our work expands on how the host cell adapts its carbon metabolism in response to Mtb infection and highlights important nodes that may provide targets for developing new therapeutics to improve TB treatment.

GlMPC activated by GCN4 regulates secondary metabolism under nitrogen limitation conditions in Ganoderma lucidum.

Mitochondrial pyruvate carrier (MPC) is a pyruvate transporter that plays a crucial role in regulating the carbon metabolic flow and is considered an essential mechanism for microorganisms to adapt to environmental changes. However, it remains unclear how MPC responds to environmental stress in organisms. General control non-derepressible 4 (GCN4), a key regulator of nitrogen metabolism, plays a pivotal role in the growth and development of fungi. In this study, we report that GCN4 can directly bind to the promoter region and activate the expression of GlMPC, thereby regulating the tricarboxylic acid cycle and secondary metabolism under nitrogen limitation conditions in Ganoderma lucidum. These findings provide significant insights into the regulation of carbon and nitrogen metabolism in fungi, highlighting the critical role of GCN4 in coordinating metabolic adaptation to environmental stresses.

Response to substrate limitation by a marine sulfate-reducing bacterium.

Sulfate-reducing microorganisms (SRM) in subsurface sediments live under constant substrate and energy limitation, yet little is known about how they adapt to this mode of life. We combined controlled chemostat cultivation and transcriptomics to examine how the marine sulfate reducer, Desulfobacterium autotrophicum, copes with substrate (sulfate or lactate) limitation. The half-saturation uptake constant (Km) for lactate was 1.2 µM, which is the first value reported for a marine SRM, while the Km for sulfate was 3 µM. The measured residual lactate concentration in our experiments matched values observed in situ in marine sediments, supporting a key role of SRM in the control of lactate concentrations. Lactate limitation resulted in complete lactate oxidation via the Wood-Ljungdahl pathway and differential overexpression of genes involved in uptake and metabolism of amino acids as an alternative carbon source. D. autotrophicum switched to incomplete lactate oxidation, rerouting carbon metabolism in response to sulfate limitation. The estimated free energy was significantly lower during sulfate limitation (-28 to -33 kJ mol-1 sulfate), suggesting that the observed metabolic switch is under thermodynamic control. Furthermore, we detected the upregulation of putative sulfate transporters involved in either high or low affinity uptake in response to low or high sulfate concentration.

Cytotoxic T-cells mediate exercise-induced reductions in tumor growth.

Exercise has a wide range of systemic effects. In animal models, repeated exertion reduces malignant tumor progression, and clinically, exercise can improve outcome for cancer patients. The etiology of the effects of exercise on tumor progression are unclear, as are the cellular actors involved. We show here that in mice, exercise-induced reduction in tumor growth is dependent on CD8+ T cells, and that metabolites produced in skeletal muscle and excreted into plasma at high levels during exertion in both mice and humans enhance the effector profile of CD8+ T-cells. We found that activated murine CD8+ T cells alter their central carbon metabolism in response to exertion in vivo, and that immune cells from trained mice are more potent antitumor effector cells when transferred into tumor-bearing untrained animals. These data demonstrate that CD8+ T cells are metabolically altered by exercise in a manner that acts to improve their antitumoral efficacy.

Transcriptome analysis of Haematococcus pluvialis of multiple defensive systems against nitrogen starvation

Haematococcus pluvialis could accumulate large amounts of triacylglycerol (TAG) and astaxanthin under various environmental stresses. To gain insights into the multiple defensive systems for carbon metabolism against nitrogen starvation, transcriptome analysis was performed. It was found that the genes related to carbon fixation, glycolysis, fatty acid and carotenoid biosynthesis pathways were up-regulated remarkably. Glyceraldehyde 3-phosphate (G3P) biosynthesis was accelerated with the enhanced C3 and C4 pathway. Meanwhile, the pyruvate kinase (PK) and pyruvate dehydrogenase E2 component (aceF) genes were significantly increased 12.9-fold and 13.9-fold, respectively, resulting more pyruvate and acetyl-CoA generation, which were beneficial to carotenoids and fatty acid biosynthesis. Methylerythritol 4-phosphate (MEP) pathway mediated carotenoid precursor isopentenyl diphosphate (IPP) synthesis, as the all eight related genes were up-regulated. The carbon flux toward astaxanthin biosynthesis with the increased astaxanthin pathway genes. The redistribution of carbon was also promoted for TAG accumulation. In addition, the up-regulation of diacylglycerol acyltransferase (DGAT) and phospholipid: diacylglycerol acyltransferase (PDAT) genes indicated that both acyl-CoA dependent and independent pathway regulated TAG accumulation. Therefore, this work reveals the multiple defensive mechanism for carbon metabolism in response to nitrogen starvation, which extended our understanding on the carotenoids, TAG and other important metabolites synthesis.

RNA-seq based transcriptional analysis of Saccharomyces cerevisiae and Lachancea thermotolerans in mixed-culture fermentations under anaerobic conditions

BackgroundIn wine fermentation starter cultures, the blending of non-Saccharomyces yeast with Saccharomyces cerevisiae to improve the complexity of wine has become common practice, but data regarding the impact of co-cultivation on yeast physiology and on genetic and metabolic regulation remain limited. Here we describe a transcriptomic analysis of mixed fermentations of Saccharomyces cerevisiae and Lachancea thermotolerans. The fermentations were carried out in carefully controlled environmental conditions in a bioreactor to reduce transcriptomic responses that would be due to factors other than the presence of the second species.ResultsThe transcriptomic data revealed that both yeast species showed a clear response to the presence of the other. Affected genes primarily belonged to two groups: genes whose expression can be linked to the competition for certain trace elements such as copper and iron, as well as genes required for cell wall structure and integrity. Furthermore, the data revealed divergent transcriptional responses with regard to carbon metabolism in response to anoxic conditions.ConclusionsThe results suggest that the mixed fermentation created a more competitive and stressful environment for the two species than single strain fermentations independently from total biomass, i.e. competition between cells of the same species is less stressful, or may present a different set of challenges, than interspecies competition. The changes in cell wall and adhesion properties encoding genes suggest that the adjustment of physical contact between cells may play a direct role in the response to the presence of competing species.

Copper stress in Staphylococcus aureus leads to adaptive changes in central carbon metabolism.

Copper toxicity has been a long-term selection pressure on bacteria due to its presence in the environment and its use as an antimicrobial agent by grazing protozoa, by phagocytic cells of the immune system, and in man-made medical and commercial products. There is recent evidence that exposure to increased copper stress may have been a key driver in the evolution and spread of methicillin-resistant Staphylococcus aureus, a globally important pathogen that causes significant mortality and morbidity worldwide. Yet it is unclear how S. aureus physiology is affected by copper stress or how it adapts in order to be able to grow in the presence of excess copper. Here, we have determined quantitatively how S. aureus alters its proteome during growth under copper stress conditions, comparing this adaptive response in two different types of growth regime. We found that the adaptive response involves induction of the conserved copper detoxification system as well as induction of enzymes of central carbon metabolism, with only limited induction of proteins involved in the oxidative stress response. Further, we identified a protein that binds copper inside S. aureus cells when stressed by copper excess. This copper-binding enzyme, a glyceraldehyde-3-phosphate dehydrogenase essential for glycolysis, is inhibited by copper in vitro and inside S. aureus cells. Together, our data demonstrate that copper stress leads to the inhibition of glycolysis in S. aureus, and that the bacterium adapts to this stress by altering its central carbon utilisation pathways.

The nitrogen-regulated response regulator NrrA is a conserved regulator of glycogen catabolism in β-cyanobacteria.

Cyanobacteria acclimatize to nitrogen deprivation by changing cellular metabolism. The nitrogen-regulated response regulator A (NrrA) is involved in regulation of carbon metabolism in response to nitrogen deprivation. However, it has not been elucidated whether these regulatory functions of NrrA are particular to a few model strains or are general among diverse cyanobacteria. In this study, we showed that regulation and functions of NrrA were highly conserved among β-cyanobacteria, which included physiologically and ecologically diverse strains. All β-cyanobacteria had the nrrA gene, while it was absent in α-cyanobacteria. The canonical NtcA-dependent promoter sequence was found upstream of the nrrA genes in most β-cyanobacteria, and its expression was indeed induced by nitrogen deprivation. Biochemical and physiological analyses of NrrA from phylogenetically distinct cyanobacteria indicated that regulation of NrrA activity and NrrA functions, namely activation of glycogen catabolism, were also common to β-cyanobacteria. These results support the conclusion that NrrA plays an important role in acclimatization to nitrogen deprivation, and that activation of glycogen catabolism is a primitive response to nitrogen deprivation in β-cyanobacteria.

Type-f thioredoxins have a role in the short-term activation of carbon metabolism and their loss affects growth under short-day conditions in Arabidopsis thaliana.

Redox regulation plays a central role in the adaptation of chloroplast metabolism to light. Extensive biochemical analyses in vitro have identified f-type thioredoxins (Trxs) as the most important catalysts for light-dependent reduction and activation of the enzymes of the Calvin-Benson cycle. However, the precise function of type f Trxs in vivo and their impact on plant growth are still poorly known. To address this issue we have generated an Arabidopsis thaliana double knock-out mutant, termed trxf1f2, devoid of both f1 and f2 Trxs. Despite the essential function previously proposed for f-type Trxs, the visible phenotype of the trxf1f2 double mutant was virtually indistinguishable from the wild type when grown under a long-day photoperiod. However, the Trx f-deficient plants showed growth inhibition under a short-day photoperiod which was not rescued at high light intensity. The absence of f-type Trxs led to significantly lower photosynthetic electron transport rates and higher levels of non-photochemical energy quenching. Notably, the Trx f null mutant suffered from a shortage of photosystem I electron acceptors and delayed activation of carbon dioxide fixation following a dark-light transition. Two redox-regulated Calvin-Benson cycle enzymes, fructose 1,6-bisphosphatase (FBPase) and Rubisco activase, showed retarded and incomplete reduction in the double mutant upon illumination, compared with wild-type plants. These results show that the function of f-type Trxs in the rapid activation of carbon metabolism in response to light is not entirely compensated for by additional plastid redox systems, and suggest that these Trxs have an important role in the light adjustment of photosynthetic metabolism.

High hydrostatic pressure upregulate central carbon metabolism genes in a distillery yeast strain

Methods In this study we performed a microarray analysis in a distillery Saccharomyces cerevisiae strain (BT0510) submitted to sublethal pressure treatment of 50 MPa for 30 min at room temperature, followed by incubation for 5, 10 and 15 min at room pressure (0.1 MPa). The transcription of the genes involved in central carbon metabolism in response to HHP was investigated for bioinformatics tools.

Metabolic switching of central carbon metabolism in response to nitrate: Application to autofermentative hydrogen production in cyanobacteria

Nitrate removal from culture media is widely used to enhance autofermentative hydrogen production in cyanobacteria during dark anaerobiosis. Here we have performed a systematic inventory of carbon and nitrogen metabolites, redox pools, and excreted product fluxes which show that addition of nitrate to cultures of Synechococcus sp. PCC 7002 has no influence on glycogen catabolic rate, but shifts the distribution of excreted products from predominantly lactate and H2 to predominantly CO2 and nitrite, while increasing the total consumption of intracellular reducing equivalents (mainly glycogen) by 3-fold. Together with LC–MS derived metabolite pool sizes these data show that glycogen catabolism is redirected from the upper-glycolytic (EMP) pathway to the oxidative pentose phosphate (OPP) pathway upon nitrate addition. This metabolic switch in carbon catabolism is shown to temporally correlate with the pyridine nucleotide redox-poise (NAD(P)H/NAD(P)+) and demonstrates the reductant availability controls H2 evolution in cyanobacteria.

Methionine, homocysteine, one carbon metabolism and fetal growth

Methionine and folate are the key components of one carbon metabolism, providing the methyl groups for numerous methyl transferase reactions via the ubiquitous methyl donor, s-adenosyl methionine. Methionine metabolism is responsive to nutrient intake, is regulated by several hormones and requires a number of vitamins (B12, pyridoxine, riboflavin) as co-factors. The critical relationship between perturbations in the mother's methionine metabolism and its impact on fetal growth and development is now becoming evident. The relation of folate intake to fetal teratogenesis has been known for some time. Studies in human pregnancy show a continuous decrease in plasma homocysteine, and an increase in plasma choline concentrations with advancing gestation. A higher rate of transsulfuration of methionine in early gestation and of transmethylation in the 3rd trimester was seen in healthy pregnant women. How these processes are impacted by nutritional, hormonal and other influences in human pregnancy and their effect on fetal growth has not been examined. Isocaloric protein restriction in pregnant rats, resulted in fetal growth restriction and metabolic reprogramming. Isocaloric protein restriction in the non-pregnant rat, resulted in differential expression of a number of genes in the liver, a 50% increase in whole body serine biosynthesis and high rate of transmethylation, suggesting high methylation demands. These responses were associated with a significant decrease in intracellular taurine levels in the liver suggesting a role of cellular osmolarity in the observed metabolic responses. These unique changes in methionine and one carbon metabolism in response to physiological, nutritional and hormonal influences make these processes critical for cellular and organ function and growth.

A systematic investigation of Escherichia coli central carbon metabolism in response to superoxide stress

BackgroundThe cellular responses of bacteria to superoxide stress can be used to model adaptation to severe environmental changes. Superoxide stress promotes the excessive production of reactive oxygen species (ROS) that have detrimental effects on cell metabolic and other physiological activities. To antagonize such effects, the cell needs to regulate a range of metabolic reactions in a coordinated way, so that coherent metabolic responses are generated by the cellular metabolic reaction network as a whole. In the present study, we have used a quantitative metabolic flux analysis approach, together with measurement of gene expression and activity of key enzymes, to investigate changes in central carbon metabolism that occur in Escherichia coli in response to paraquat-induced superoxide stress. The cellular regulatory mechanisms involved in the observed global flux changes are discussed.ResultsFlux analysis based on nuclear magnetic resonance (NMR) and mass spectroscopy (MS) measurements and computation provided quantitative results on the metabolic fluxes redistribution of the E. coli central carbon network under paraquat-induced oxidative stress. The metabolic fluxes of the glycolytic pathway were redirected to the pentose phosphate pathway (PP pathway). The production of acetate increased significantly, the fluxes associated with the TCA cycle decreased, and the fluxes in the glyoxylate shunt increased in response to oxidative stress. These global flux changes resulted in an increased ratio of NADPH:NADH and in the accumulation of α-ketoglutarate.ConclusionsMetabolic flux analysis provided a quantitative and global picture of responses of the E. coli central carbon metabolic network to oxidative stress. Systematic adjustments of cellular physiological state clearly occurred in response to changes in metabolic fluxes induced by oxidative stress. Quantitative flux analysis therefore could reveal the physiological state of the cell at the systems level and is a useful complement to molecular systems approaches, such as proteomics and transcription analyses.

Changes in Arctic marine bacterial carbon metabolism in response to increasing temperature

Arctic areas of deep-water convection have a large potential for export of organic carbon from surface waters into the deep sea and, therefore, are an important part of the global carbon cycle. As the Arctic is reportedly heating up faster than any other part of the planet, temperature-driven changes in the biogeochemical cycling in these areas can be very significant. Here, we study the regulation of bacterial carbon metabolism, which process vast amounts of organic carbon, by temperature and the availability of resources. The response of bacterial production and respiration of natural bacterial assemblages from the Fram Strait was studied by experimental manipulations of temperature and resources in combination. Both bacterial production and respiration were enhanced by temperature so that the total bacterial carbon demand increased sixfold following a temperature increase of 6°C. Respiration responded more strongly than production so that bacterial growth efficiency decreased with increasing temperature. Although neither production nor respiration was limited by resource availability under in situ conditions, the response to temperature was higher in resource-amended treatments, indicative of a substrate-temperature interaction regulating both components of bacterial metabolism. In conclusion, the results show that warming can result in a substantial increase of the carbon flow through bacteria and that most of the carbon consumed would be released as CO2. Moreover, the results suggest that both temperature and availability of resources need to be considered to accurately be able to predict changes in bacterial carbon metabolism in response to climate change.

Crystal Structure of Enzyme I of the Phosphoenolpyruvate Sugar Phosphotransferase System in the Dephosphorylated State

The bacterial phosphoenolpyruvate (PEP) sugar phosphotransferase system mediates sugar uptake and controls the carbon metabolism in response to carbohydrate availability. Enzyme I (EI), the first component of the phosphotransferase system, consists of an N-terminal protein binding domain (EIN) and a C-terminal PEP binding domain (EIC). EI transfers phosphate from PEP by double displacement via a histidine residue on EIN to the general phosphoryl carrier protein HPr. Here we report the 2.4 A crystal structure of the homodimeric EI from Staphylococcus aureus. EIN consists of the helical hairpin HPr binding subdomain and the phosphorylatable betaalpha phospho-histidine (P-His) domain. EIC folds into an (betaalpha)(8) barrel. The dimer interface of EIC buries 1833 A(2) of accessible surface per monomer and contains two Ca(2+) binding sites per dimer. The structures of the S. aureus and Escherichia coli EI domains (Teplyakov, A., Lim, K., Zhu, P. P., Kapadia, G., Chen, C. C., Schwartz, J., Howard, A., Reddy, P. T., Peterkofsky, A., and Herzberg, O. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 16218-16223) are very similar. The orientation of the domains relative to each other, however, is different. In the present structure the P-His domain is docked to the HPr binding domain in an orientation appropriate for in-line transfer of the phosphate to the active site histidine of the acceptor HPr. In the E. coli structure the phospho-His of the P-His domain projects into the PEP binding site of EIC. In the S. aureus structure the crystallographic temperature factors are lower for the HPr binding domain in contact with the P-His domain and higher for EIC. In the E. coli structure it is the reverse.

Proteomic analysis of heterotrophy in Synechocystis sp. PCC 6803

To provide an insight into the heterotrophic metabolism of cyanobacteria, a proteomic approach has been employed with the model organism Synechocystis sp. PCC 6803. The soluble proteins from Synechocystis grown under photoautotrophic and light-activated heterotrophic conditions were separated by 2-DE and identified by MALDI-MS or LC-MS/MS analysis. 2-DE gels made using narrow- and micro-range IPG strips allowed quantitative comparison of more than 900 spots. Out of 67 abundant protein spots identified, 13 spots were increased and 9 decreased under heterotrophy, representing all the major fold changes. Proteomic alterations and activity levels of selected enzymes indicate a shift in the central carbon metabolism in response to trophic change. The significant reduction in light-saturated rate of photosynthesis as well as in the expression levels of rubisco and CO(2)-concentrating mechanism proteins under heterotrophy indicates the down-regulation of the photosynthetic machinery. Alterations in the expression level of proteins involved in carbon utilization pathways refer to enhanced glycolysis, oxidative pentose phosphate pathway as well as tricarboxylic acid cycle under heterotrophy. Proteomic evidences also suggest an enhanced biosynthesis of amino acids such as histidine and serine during heterotrophic growth.

Regulation of carbon metabolism in Chlamydia trachomatis.

The biological significance of glycogen accumulation and how the process is regulated in Chlamydia trachomatis remains poorly defined. C. trachomatis-infected HeLa cells were cultured in medium containing various glucose concentrations (0, 0.1, 1 or 10 mg ml-1) or in the presence of gluconeogenic carbon sources (20 mM glutamate, 20 mM malate, 20 mM alpha-ketoglutarate or 20 mM oxaloacetate), and the effects of these different culture conditions on the production of infectious chlamydial elementary bodies and glycogen accumulation were monitored. When chlamydiae were cultured in glucose concentrations greater than 1 mg ml-1, optimal growth and maximal glycogen accumulation occurred. In contrast to uninfected HeLa cells, which increased their glycogen stores when grown in the presence of high glucose concentrations, chlamydial glycogen accumulation remained essentially constant. When cultured in medium supplemented with either reduced glucose concentrations or any of the gluconeogenic carbon sources, chlamydiae still grew; however, the yield of elementary bodies was substantially decreased, and there was no significant amount of glycogen accumulated by host HeLa cells or C. trachomatis. This suggests that glycogen accumulation may not be essential for chlamydial survival. Reverse transcriptase-polymerase chain reaction (RT-PCR) results indicated that, despite the fact that the source and amount of carbon available in the medium affected chlamydial glycogen accumulation, the expression of genes required for glycogen metabolism was not significantly changed. Similarly, the expression of several genes encoding key enzymes of central metabolism was not affected by alterations in carbon source or availability. Taken together, the data suggest that, unlike most free-living bacteria, chlamydia are unable to alter the expression of genes involved in carbon metabolism in response to changes in environmental conditions.

Oak growth, development and carbon metabolism in response to water stress

Le genre Quercus (Fagaceae) comporte a la fois des especes decidues et des especes sempervirentes adaptees a une large gamme de stations presentant des disponibilites en eau tres diverses. Les chenes ont developpe des adaptations morphologiques et physiologiques pour survivre et pousser dans ces stations. Des adaptations morphologiques dans les feuilles, les tiges et les racines permettent a la fois la tolerance et l'evitement de la secheresse. Les adaptations physiologiques impliquent a la fois le controle et la conductance stomatique du potentiel hydrique foliaire, du degre d'ajustement osmotique, et de la fixation photosynthetique de carbone. L'assimilation de carbone est controlee par des facteurs lies aux stomates ou d'origine non stomatique. La reponse des stomates est sans doute la plus importante reponse de limitation de la fixation photosynthetique de carbone. Les especes les plus tolerantes a la secheresse limitent la fermeture des stomates de maniere a permettre une assimilation substantielle de carbone en situation de contrainte, ce qui leur permet d'ameliorer leur efficience d'utilisation de l'eau; ou alors, elles les rouvrent tres rapidement, des que les reserves hydriques ont ete reconstituees, meme partiellement. Les processus non stomatiques de la photosynthese, tels que la conversion photochimique et les reactions biochimiques des cycles de carboxylation centres sur la Rubisco, semblent particulierement peu sensibles a la secheresse, meme s'il s'avere que des resistances localisees dans le mesophylle et s'opposant a l'influx de CO 2 vers les chloroplastes puissent augmenter. Le rapport sucres solubles/amidon augmente en cours de secheresse, l'expansion foliaire est ralentie, voire bloquee, et le carbone destine initialement au developpement des feuilles est detourne vers la base de la tige et vers les racines, ou il sert a maintenir une croissance minimale, ou au stockage de reserves. De nombreux genotypes de chenes (especes, hybrides, provenances) sont disponibles et peuvent s'adapter a des stations mediocres. L'utilisation de tels genotypes pourrait significativement ameliorer la sylviculture du chene et les plantations d'ornement.