Effect of Slow Growth on Metabolism of Escherichia coli, as Revealed by Global Metabolite Pool (“Metabolome”) Analysis

Escherichia coli growing on glucose in minimal medium controls its metabolite pools in response to environmental conditions. The extent of pool changes was followed through two-dimensional thin-layer chromatography of all ¹⁴C-glucose labelled compounds extracted from bacteria. The patterns of metabolites and spot intensities detected by phosphorimaging were found to reproducibly differ depending on culture conditions. Clear trends were apparent in the pool sizes of several of the 70 most abundant metabolites extracted from bacteria growing in glucose-limited chemostats at different growth rates. The pools of glutamate, aspartate, trehalose, and adenosine as well as UDP-sugars and putrescine changed markedly. The data on pools observed by two-dimensional thin-layer chromatography were confirmed for amino acids by independent analysis. Other unidentified metabolites also displayed different spot intensities under various conditions, with four trend patterns depending on growth rate. As RpoS controls a number of metabolic genes in response to nutrient limitation, an rpoS mutant was also analyzed for metabolite pools. The mutant had altered metabolite profiles, but only some of the changes at slow growth rates were ascribable to the known control of metabolic genes by RpoS. These results indicate that total metabolite pool (“metabolome”) analysis offers a means of revealing novel aspects of cellular metabolism and global regulation.     

Identification of metabolic fluxes in hepatic cells from transient 13C-labeling experiments: Part II. Flux estimation

This contribution addresses the identification of metabolic fluxes and metabolite concentrations in mammalian cells from transient (13)C-labeling experiments. Whilst part I describes experimental set-up and acquisition of required metabolite and (13)C-labeling data, part II focuses on setting up network models and the estimation of intracellular fluxes. Metabolic fluxes were determined in glycolysis, pentose-phosphate pathway (PPP), and citric acid cycle (TCA) in a hepatoma cell line grown in aerobic batch cultures. In glycolytic and PPP metabolite pools isotopic stationarity was observed within 30 min, whereas in the TCA cycle the labeling redistribution did not reach isotopic steady state even within 180 min. In silico labeling dynamics were in accordance with in vivo (13)C-labeling data. Split ratio between glycolysis and PPP was 57%:43%; intracellular glucose concentration was estimated at 101.6 nmol per 10(6) cells. In contrast to isotopic stationary (13)C-flux analysis, transient (13)C-flux analysis can also be applied to industrially relevant mammalian cell fed-batch and batch cultures.     

Identification of metabolic model: Citrate production from glucose by Candida lipolytica

The concept of mass balance was used to analyze the metabolic pathways of citrate production by Candida lipolytica from glucose. Specific rates of glucose consumption, citrate and isocitrate productions, carbon dioxide evolution, and cellular syntheses of protein and carbohydrate were observed in an NH₄⁺-limited chemostat culture. These data permitted one to assess the carbon flux in vivo by solving simultaneous carbon balance equations with respect to intermediary metabolite pools in the steady State. Among the three models considered here, model I (which coordinates the pyruvate carboxylation with the tricarboxylic acid cycle, but disregards the glyoxylate cycle) was considered plausible because the carbon flux calculated so far was acceptable. On the other hand, models II and III (which overlook the pyruvate carboxylation and the 2-oxoglutarate dehydrogenation, respectively) were found to be most unlikely because of the unusual flux assessed from these models.     

The fate of labelled glucose molecules in the rat adipocyte. Dependence on glucose concentration

Isolated rat adipocytes were incubated with 15 nM [3-3H]glucose or 100 nM [U-14C]glucose with or without insulin and in the absence or presence of unlabelled glucose. Following a 2 h incubation with 15 nM [3-3H]glucose, about two thirds of the cell-associated 3H-labelled metabolic products were hydrophilic largely anionic intermediates and about one third was lipids. The equivalent values were 40 and 60%, respectively, when using 100 nM [U-14C]glucose. The only 14C-labelled metabolite escaping to the incubation medium was 14CO2, which accounted for about 15% of the rate of metabolism. Therefore, the rate of incorporation of 100 nM [U-14C]glucose into the cell-associated metabolites was quite a good measure of its net influx rate. The conversion of the two tracers to the sum of the metabolic products in cells treated with a maximally stimulating insulin concentration remained constant with glucose concentrations up to about 100 microM and then decreased progressively. The incorporation of radioactivity into the different metabolites varied markedly over the glucose concentration range 0-100 microM, presumably due to the saturation of different metabolic pools at different glucose concentrations. This variation was much less in cells not stimulated with insulin. Consequently, the maximal effect of insulin on the incorporation of the tracers into a given metabolite (e.g., labelled lipids) varied over the entire glucose concentration range. In addition, the apparent sensitivity (ED50) with respect to the incorporation into a given metabolite was also dependent on the glucose concentration.     

The fate of labelled glucose molecules in the rat adipocyte: Dependence on glucose concentration

Isolated rat adipocytes were incubated with 15 nM [3-³H]glucose or 100 nM [U-¹⁴C]glucose with or without insulin and in the absence or presence of unlabelled glucose. Following a 2 h incubation with 15 nM [3-³H]glucose, about two thirds of the cell-associated ³H-labelled metabolic products were hydrophilic largely anionic intermediates and about one third was lipids. The equivalent values were 40 and 60%, respectively, when using 100 nM [U-¹⁴C]glucose. The only ¹⁴C-labelled metabolite escaping to the incubation medium was ¹⁴CO₂, which accounted for about 15% of the rate of metabolism. Therefore, the rate of incorporation of 100 nM [U-¹⁴C]glucose into the cell-associated metabolites was quite a good measure of its net influx rate. The conversion of the two tracers to the sum of the metabolic products in cells treated with a maximally stimulating insulin concentration remained constant with glucose concentrations up to about 100 μM and then decreased progressively. The incorporation of radioactivity into the different metabolites varied markedly over the glucose concentration range 0–100 μM, presumably due to the saturation of different metabolic pools at different glucose concentrations. This variation was much less in cells not stimulated with insulin. Consequently, the maximal effect of insulin on the incorporation of the tracers into a given metabolite (e.g., labelled lipids) varied over the entire glucose concentration range. In addition, the apparent sensitivity (ED₅₀) with respect to the incorporation into a given metabolite was also dependent on the glucose concentration.     

2-Ketoglutarate and the regulation of aconitase and histidase formation in Bacillus subtilis.

In contrast to wild-type cells, the Bacillus subtilis mutant SF109 that lacks the active 2-ketoglutarate dehydrogenase enzymatic complex is unable to increase the specific activity of two enzymes subject to glucose catabolite repression, aconitase and histidase, during limitation of growth by glucose. Examination of the intracellular metabolite pools in the mutant and wild-type cells grown in excess and limiting glucose medium showed that the complete derepression of aconitase and histidase could be correlated with the decrease in the intracellular concentration of 2-ketoglutarate. The complete repression of aconitase that occurred in wild-type and mutant cells could be correlated with a high intracellular concentration of 2-ketoglutarate.     

Constraints on the models of carbohydrate metabolism in the two cell types of Dictyostelium discoideum

The constraints in the parameters in models of the spore and stalk cells in Dictyostelium discoideum have been examined. It was found that the relative sizes of the two cellular glucose pools are not very critical, i.e. they can be varied in the models over a fairly wide range and still allow simulations which are compatible with the data. In contrast, the following model parameters are highly constrained, and must fall within narrow limits: flux through the glycogen cycle; the fraction of glycogen present which actually participates in glycogen turnover; the net rate of glycogen degradation; the concentration of exogenous labelled glucose which actually participates in cellular metabolism; the rates of exchange of this exogenous glucose with the two cellular glucose pools; the concentration of the spore glucose-6-phosphate pool, and the rate of exchange of stalk glucose-1-phosphate and stalk glucose-6-phosphate.     

2-Ketobutyrate: a putative alarmone of Escherichia coli

2-ketobutyrate is synthesized from threonine by threonine deaminase (dehydratase) in E. coli. The effects of 2-ketobutyrate as a regulatory metabolite were studied in vivo. 2-ketobutyrate was shown to inhibit the phosphoenolpyruvate (PEP): sugar phosphotransferase system resulting in aspartate starvation, elevation of ppGpp endogenous pools, and cessation of growth in E. coli grown in glucose and related carbon sources. Accordingly, we propose that 2-ketobutyrate might serve as an alarmone whose concentration precisely governs the shift from anaerobic growth to aerobic growth in E. coli. Such shifts are common phenomena among the Enterobacteriaceae.     

Quantitative analysis of intermediary metabolism in rat hepatocytes incubated in the presence and absence of ethanol with a substrate mixture including ketoleucine.

Hepatocytes isolated from livers of fed rats were incubated with a mixture of glucose (10 mM), ribose (1.0 mM), acetate (1.25 mM), alanine (3.5 mM), glutamate (2.0 mM), aspartate (2.0 mM), 4-methyl-2-oxovaleric acid (ketoleucine) (3.0 mM), and, in paired flasks, 10 mM-ethanol. One substrate was 14C-radiolabelled in any given incubation. Incorporation of 14C into glucose, glycogen, CO2, lactate, alanine, aspartate, glutamate, acetate, urea, lipid glycerol, fatty acids and the 1- and 2,3,4-positions of ketone bodies was measured after 20 and 40 min of incubation under quasi-steady-state conditions. Data were analysed with the aid of a realistic structural metabolic model. In each of the four conditions examined, there were approx. 77 label incorporation measurements and several measurements of changes in metabolite concentrations. The considerable excess of measurements over the 37 independent flux parameters allowed for a stringent test of the model. A satisfactory fit to these data was obtained for each condition. There were large bidirectional fluxes along the gluconeogenic/glycolytic pathways, with net gluconeogenesis. Rates of ureagenesis, oxygen consumption and ketogenesis were high under all four conditions studied. Oxygen utilization was accurately predicted by three of the four models. There was complete equilibration between mitochondrial and cytosolic pools of acetate and of CO2, but for several of the metabolic conditions, two incompletely equilibrated pools of mitochondrial acetyl-CoA and oxaloacetate were required. Ketoleucine was utilized at a rate comparable to that reported by others in perfused liver and entered the mitochondrial pool of acetyl-CoA directly associated with ketone body formation. Ethanol, which was metabolized at rates comparable to those in vivo, caused relatively few changes in overall flux patterns. Several effects related to the increased NADH/NAD+ ratio were observed. Pyruvate dehydrogenase was completely inhibited and the ratio of acetoacetate to 3-hydroxybutyrate was decreased; flux through glutamate dehydrogenase, the citric acid cycle, and ketoleucine dehydrogenase were, however, only slightly inhibited. Net production of ATP occurred in all conditions studied and was increased by ethanol. Futile cycling was quantified at the glucose/glucose 6-phosphate, glycogen/glucose 6-phosphate, fructose 6-phosphate/fructose 1,6-bis-phosphate, and phosphoenolpyruvate/pyruvate/oxaloacetate substrate cycles. Cycling at these four loci consumed about 22% of cellular ATP production in control hepatocytes and 14% in ethanol-treated cells.     

Bacillus subtilis glutamine synthetase mutants pleiotropically altered in glucose catabolite repression.

Strain SF22, a glutamine-requiring (Gln-) mutant of Bacillus subtilis SMY, is likely to have a mutation in the structural gene for glutamine synthetase, since this strain synthesized 22 to 55% as much glutamine synthetase antigen as did wild-type cells in a 10-min period but had less than 3% of wild-type glutamine synthetase enzymatic activity. The expression of several genes subject to glucose catabolite repression was altered in the Gln- mutant. The induced levels of alpha-glucosidase, histidase, and aconitase were 3.5- to 4-fold higher in SF22 cells than in wild-type cells grown in glucose-glutamine medium, and citrate synthase levels were 8-fold higher in the Gln- mutant than in wild-type cells. The relief of glucose catabolite repression in the Gln- mutant may result from poor utilization of glucose. Examination of the intracellular metabolite pools of cells grown in glucose-glutamine medium showed that the glucose-6-phosphate pool was 2.5-fold lower, the pyruvate pool was 4-fold lower, and the 2-ketoglutarate pool was 2.5-fold lower in the Gln- cells than they were in wild-type cells. Intracellular levels of glutamine were sixfold higher in the Gln- mutant than in wild-type cells. Measurements of enzymes involved in glutamine transport and utilization showed that the elevated pools of glutamine in the Gln- mutant resulted from a threefold increase in glutamine permease and a fivefold decrease in glutamate synthase. The pleiotropic effect of the gln-22 mutation on the expression of several genes suggests that either the glutamine synthetase protein or its enzymatic product, glutamine, is involved in the regulation of several metabolic pathways in B. subtilis.     

Quantitative metabolomics of a xylose-utilizing <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain expressing the <Emphasis Type="Italic">Bacteroides thetaiotaomicron</Emphasis> xylose isomerase on glucose and xylose

The yeast Saccharomyces cerevisiae cannot utilize xylose, but the introduction of a xylose isomerase that functions well in yeast will help overcome the limitations of the fungal oxido-reductive pathway. In this study, a diploid S. cerevisiae S288c[2n YMX12] strain was constructed expressing the Bacteroides thetaiotaomicron xylA (XI) and the Scheffersomyces stipitis xyl3 (XK) and the changes in the metabolite pools monitored over time. Cultivation on xylose generally resulted in gradual changes in metabolite pool size over time, whereas more dramatic fluctuations were observed with cultivation on glucose due to the diauxic growth pattern. The low G6P and F1,6P levels observed with cultivation on xylose resulted in the incomplete activation of the Crabtree effect, whereas the high PEP levels is indicative of carbon starvation. The high UDP-d-glucose levels with cultivation on xylose indicated that the carbon was channeled toward biomass production. The adenylate and guanylate energy charges were tightly regulated by the cultures, while the catabolic and anabolic reduction charges fluctuated between metabolic states. This study helped elucidate the metabolite distribution that takes place under Crabtree-positive and Crabtree-negative conditions when cultivating S. cerevisiae on glucose and xylose, respectively.     

Glycolysis Is Governed by Growth Regime and Simple Enzyme Regulation in Adherent MDCK Cells

Due to its vital importance in the supply of cellular pathways with energy and precursors, glycolysis has been studied for several decades regarding its capacity and regulation. For a systems-level understanding of the Madin-Darby canine kidney (MDCK) cell metabolism, we couple a segregated cell growth model published earlier with a structured model of glycolysis, which is based on relatively simple kinetics for enzymatic reactions of glycolysis, to explain the pathway dynamics under various cultivation conditions. The structured model takes into account in vitro enzyme activities, and links glycolysis with pentose phosphate pathway and glycogenesis. Using a single parameterization, metabolite pool dynamics during cell cultivation, glucose limitation and glucose pulse experiments can be consistently reproduced by considering the cultivation history of the cells. Growth phase-dependent glucose uptake together with cell-specific volume changes generate high intracellular metabolite pools and flux rates to satisfy the cellular demand during growth. Under glucose limitation, the coordinated control of glycolytic enzymes re-adjusts the glycolytic flux to prevent the depletion of glycolytic intermediates. Finally, the model''s predictive power supports the design of more efficient bioprocesses.     

Elucidation and structural analysis of conserved pools for genome-scale metabolic reconstructions

In this article, we introduce metabolite concentration coupling analysis (MCCA) to study conservation relationships for metabolite concentrations in genome-scale metabolic networks. The analysis allows the global identification of subsets of metabolites whose concentrations are always coupled within common conserved pools. Also, the minimal conserved pool identification (MCPI) procedure is developed for elucidating conserved pools for targeted metabolites without computing the entire basis conservation relationships. The approaches are demonstrated on genome-scale metabolic reconstructions of Helicobacter pylori, Escherichia coli, and Saccharomyces cerevisiae. Despite significant differences in the size and complexity of the examined organism's models, we find that the concentrations of nearly all metabolites are coupled within a relatively small number of subsets. These correspond to the overall exchange of carbon molecules into and out of the networks, interconversion of energy and redox cofactors, and the transfer of nitrogen, sulfur, phosphate, coenzyme A, and acyl carrier protein moieties among metabolites. The presence of large conserved pools can be viewed as global biophysical barriers protecting cellular systems from stresses, maintaining coordinated interconversions between key metabolites, and providing an additional mode of global metabolic regulation. The developed approaches thus provide novel and versatile tools for elucidating coupling relationships between metabolite concentrations with implications in biotechnological and medical applications.     

Reduced renal cortical ribonucleoside triphosphate pools in three different hypophosphatemic animal models

Renal cortical metabolite quantitation in three different models of hypophosphatemia demonstrates that ribonucleoside triphosphate depletion is a common manifestation of reduced plasma phosphate concentration. Total tissue inorganic phosphate is not necessarily altered as the result of hypophosphatemia. Compensatory increases in mono- and diphosphate pools maintain the total ribonucleotide pool but cause reductions in the energy charge for the adenine, guanine, and uridine nucleotides. The coordinate regulation of all ribonucleoside triphosphate pools suggests cellular dysfunction accompanying hypophosphatemia may be the consequence of a generalized derangement in energy and nucleotide metabolism.     

Photosystem II regulation of macromolecule synthesis in the blue-green alga Aphanocapsa 6714.

Polymers synthesized by heterotrophically growing (glucose as carbon source) cultures of Aphanocapsa 6714 were compared with polymers synthesized in photosynthetically grown cultures. Loss of photosystem II by dark incubation, or inhibition of light-grown cells with the photosystem II-specific inhibitor dichlorophenylmethylurea, caused an 80 to 90% reduction in the rate of lipid and total ribonucleic acid synthesis, and more than a 90% reduction in the rate of protein synthesis. In contrast, glycogen synthesis was reduced only about 50% in dark cells and less than 30% in dichlorphenylmethylurea-inhibited cells. After longer heterotrophic growth, glycogen became the major component, whereas in photosynthetically grown cultures protein was the major constituent. 14C (from 14CO2 and/or [14C]glucose) assimilated into protein by heterotrophically grown cells was found in amino acids in nearly the same proportions as in photosynthetically grown cells. Thus, routes of biosynthesis available to autotropic cells were also available to heterotrophic cultures, but the supply of carbon precursors to those pathways was greatly reduced. The limited biosynthesis in heterotrophic cells was not due to a limitation for cellular energy. The adenylates were maintained at nearly the same concentrations (and hence the energy charge also) as in photosynthetic cells. The concentration of reduced nicotinamide adenine dinucleotide phosphate was higher in heterotrophic (dark) cells than in photosynthetic cells. From rates of CO2 fixation and/or glycogen biosynthesis it was determined that stationary-phase cells expended approximately 835, 165, and less than 42 nmol of adenosine 5'-triphosphate per mg (dry weight) of algae per 30 min during photosynthetic, photoheterotrophic, and chemoheterotrophic metabolism, respectively. Analysis of the soluble metabolite pools in dark heterotrophic cultures by double-labeling experiments revealed rapid equilibration of 14C through the monophosphate pools, but much slower movement of label into the diphosphate pools of fructose-1,6-diphosphate and sedoheptulose-1,7-diphosphate. Carbon did flow into 3-phosphoglycerate in the dark; however, the initial rate was low and the concentration of this metabolite soon fell to an undetectable level. In photosynthetic cells, 14C quickly equilibrated throughout all the intermediates of the reductive pentose cycle, in particular, into 3-phosphoglycerate. Analysis of glucose-6-phosphate dehydrogenase in cell extracts showed that the enzyme was very sensitive to product inhibition by reduced nicotinamide adenine dinucleotide.     

The effect of glucose on the control of carbohydrate metabolism in ripening bananas

The effect of exogenous glucose on the major fluxes of carbohydrate metabolism in cores of climacteric fruit of banana (Musa cavendishii Lamb ex Paxton) was determined with the intention of using the effects in the application of top-down metabolic control analysis. Hands of bananas, untreated with ethylene, were allowed to ripen in the dark at 21 °C. Cores were removed from climacteric fruit and incubated in 100 or 200 mM glucose for 4 or 6 h. The rates of starch breakdown, sucrose and fructose accumulation and CO₂ production were measured. The steady-state contents of hexose monophosphates, adenylates and pyruvate were determined. In addition, the detailed distribution of label was determined after supply of the following: [U-¹⁴C]-, [1-¹⁴C]-, [3,4¹⁴C]and [6-¹⁴C]glucose, and [U-¹⁴C]glycerol. The data were used to estimate the major fluxes of carbohydrate metabolism. Supply of exogenous glucose led to increases in the size of the hexose-monophosphate pools. There was a small stimulation of the rate of sugar synthesis and a major increase in the rate of starch synthesis. Starch breakdown was inhibited. Respiration responded to the demand for ATP by sugar synthesis. The effect of glucose on fluxes and metabolite pools is discussed in relation to our understanding of the control and regulation of carbohydrate metabolism in ripening fruit.Abbreviations Glc6P glucose-6-phosphate - Glc1P glucose-1-phosphate - Fru6P fructose-6-phosphate - AEC adenylate energy charge We thank Geest Foods Group, Great Dunmow, Essex, UK for giving us the bananas. SAH thanks the managers of the Broodbank Fund for a fellowship.     

GC-EI-TOF-MS analysis of in vivo carbon-partitioning into soluble metabolite pools of higher plants by monitoring isotope dilution after 13CO2 labelling

The established GC-EI-TOF-MS method for the profiling of soluble polar metabolites from plant tissue was employed for the kinetic metabolic phenotyping of higher plants. Approximately 100 typical GC-EI-MS mass fragments of trimethylsilylated and methoxyaminated metabolite derivatives were structurally interpreted for mass isotopomer analysis, thus enabling the kinetic study of identified metabolites as well as the so-called functional group monitoring of yet non-identified metabolites. The monitoring of isotope dilution after (13)CO(2) labelling was optimized using Arabidopsis thaliana Col-0 or Oryza sativa IR57111 plants, which were maximally labelled with (13)C. Carbon isotope dilution was evaluated for short (2h) and long-term (3 days) kinetic measurements of metabolite pools in root and shoots. Both approaches were shown to enable the characterization of metabolite specific partitioning processes and kinetics. Simplifying data reduction schemes comprising calculation of (13)C-enrichment from mass isotopomer distributions and of initial (13)C-dilution rates were employed. Metabolites exhibited a highly diverse range of metabolite and organ specific half-life of (13)C-label in their respective pools ((13)C-half-life). This observation implied the setting of metabolite specific periods for optimal kinetic monitoring. A current experimental design for the kinetic metabolic phenotyping of higher plants is proposed.     

Monitoring novel metabolic pathways using metabolomics and machine learning: induction of the phosphoketolase pathway in Aspergillus nidulans cultivations

The physiological phenotype of Aspergillus nidulans was determined under different environmental conditions through the quantification of the intracellular and extracellular metabolite pools and clear evidence of the presence of a novel fungal metabolic pathway, the phosphoketolase pathway, was obtained. Induction of the phosphoketolase pathway resulted after blocking the EMP pathway through the deactivation of glyceraldehyde-3-P dehydrogenase (G3PD). Deactivation of G3PD in cultivations of A. nidulans on glucose and xylose led to a 10-fold decrease in the specific growth rate; however, growth could still be sustained solely through the phosphoketolase pathway. Metabolomics and machine learning tools were successfully used to monitor the alteration caused by the inhibition of G3PD in the metabolism of A. nidulans grown on glucose, xylose, acetate as well as mixtures of glucose or xylose with acetate. This is the first study that demonstrates in vivo that the fungal central carbon metabolism includes an active phosphoketolase pathway.