Phosphorus-31 nuclear magnetic resonance studies of wild-type and glycolytic pathway mutants of Saccharomyces cerevisiae.

High-resolution phosphorus-31 nuclear magnetic resonance (31P NMR) spectra of wild-type and mutant strains of Saccharomyces cerevisiae were observed at a frequency of 145.7 MHz. Levels of various phosphorus metabolites were investigated upon addition of glucose under both aerobic and anaerobic conditions. Three mutant strains were isolated and their biochemical defects characterized: pfk lacked phosphofructokinase activity; pgi lacked phosphoglucose isomerase activity; and cif had no glucose catabolite repression of the fructose bisphosphatase activity. Each mutant strain was found to accumulate characteristic sugar phosphates when glucose was added to the cell suspension. In the case of the phosphofructokinase deficient mutant, the appearance of a pentose shunt metabolite was observed. 31P NMR peak assignments were made by a pH titration of the acid extract of the cells. Separate signals for terminal, penultimate, and central phosphorus atoms in intracellular polyphosphates allowed the estimation of their average molecular weight. Signals for glycero(3)phosphochline, glycero(3)phosphoserine, and glycero(3) phosphoethanolamine as well as three types of nucleotide diphosphate sugars could be observed. The intracellular pH in resting and anaerobic cells was in the range 6.5--6.8 and the level of adenosine 5'-triphosphate (ATP) low. Upon introduction of oxygen, the ATP level increased considerably and the intracellular pH reached a value of pH 7.2--7.3, irrespective of the external medium pH, indicating active proton transport in these cells. A new peak representing the inorganic phosphate of one of the cellular organelles, whose pH differed from the cytoplasmic pH, could be detected under appropriate conditions.     

Regulation of the cAMP level in the yeast Saccharomyces cerevisiae: the glucose-induced cAMP signal is not mediated by a transient drop in the intracellular pH

Addition of glucose to derepressed cells of the yeast Saccharomyces cerevisiae is known to cause a rapid, transient increase in the cAMP level, which lasts for 1-2 min and induces a cAMP-dependent protein phosphorylation cascade. The glucose-induced cAMP signal cannot be explained solely on the basis of an increased ATP level. Transient membrane depolarization and transient intracellular acidification have been suggested as possible triggers for the cAMP peak. Addition of glucose to cells in which the plasma membrane had been depolarized still produced the increase in the cAMP level excluding membrane depolarization as the possible trigger. Using in vivo 31P NMR-spectroscopy we followed phosphate metabolism and the time course of the drop in the intracellular pH after addition of glucose with a time resolution of 15 s. Under aerobic conditions the initial pH and ATP level were high. On addition of glucose, they both showed a rapid, transient drop, which lasted for about 30 s. Under anaerobic conditions, the initial pH and ATP level were low and on addition of glucose they both increased relatively slowly compared to aerobic conditions. Several conditions were found in which the pH drop which occurs under aerobic conditions could be blocked completely without effect on the cAMP signal or without completely preventing it: addition of NH4Cl together with glucose at high extracellular pH and addition of a low concentration of glucose before a high concentration. Also, when glucose was added twice to the same cells no consistent relationship was observed between the pH drop and the cAMP peak. These results appear to exclude transient intracellular acidification as the trigger for the cAMP signal. Hence, we conclude that the effect of glucose cannot be explained on the basis of effects known to be caused by the membrane depolarizing compounds which cause increases in the cAMP level. A new, more specific kind of interaction appears to be involved.     

31P NMR saturation-transfer and 13C NMR kinetic studies of glycolytic regulation during anaerobic and aerobic glycolysis

31P NMR saturation-transfer techniques have been employed in glucose-grown derepressed yeast to determine unidirectional fluxes in the upper part of the Embden-Meyerhof-Parnas pathway. The experiments were performed during anaerobic and aerobic glycolysis by saturating the ATP gamma resonances and monitoring changes in the phosphomonoester signals from glucose 6-phosphate and fructose 1,6-bis-phosphate. These experiments were supplemented with 13C NMR measurements of glucose utilization rates and 13C NMR label distribution studies. Combined with data obtained previously from radioisotope measurements, these 31P and 13C NMR kinetic studies allowed estimation of the net glycolytic flow in addition to relative flows through phosphofructokinase (PFK) and Fru-1,6-P2ase during anaerobic and aerobic glycolysis. The 31P NMR saturation-transfer results are consistent with previous results obtained from measurements of metabolite levels, radioisotope data, and 13C NMR studies [den Hollander, J.A., Ugurbil, K., Brown, T.R., Bednar, M., Redfield, C., & Shulman, R.G. (1986a) Biochemistry 25, 203-211], providing additional support for in vivo measurement of the flows during glycolysis.     

Development and application of a membrane cyclone reactor for in vivo NMR spectroscopy with high microbial cell densities

A new bioreactor system has been developed for in vivo NMR spectroscopy of microorganisms under defined physiological conditions. This cyclone reactor with an integrated NMR flow cell is continuously operated in the magnet of a 400-MHz wide-bore NMR spectrometer system. The residence times of medium and cells are decoupled by a circulation-integrated cross-flow microfiltration module to achieve higher cell densities as compared to continuous fermentations without cell retention (increase in cell density up to a factor of 10 in steady state). Volumetric mass transfer coefficients k(L)a of more than 1.0 s(-1) are possible in the membrane cyclone reactor, ensuring adequate oxygen supply [oxygen transfer rate >15,000 mg O(2) .(L h)(-1)] of high cell densities. With the aid of the membrane cyclone reactor we were able to show, using continuous in vivo (31)P NMR spectroscopy of anaerobic glucose fermentation by Zymomonas mobilis, that the NMR signal intensity was directly proportional to the cell concentration in the reactor. The concentration profiles of intracellular inorganic phosphate, NAD(H), NDP, NTP, UDP-sugar, a cyclic pyrophosphate, two sugar phosphate pools, and extracellular inorganic phosphate were recorded after a shift from one steady state to another. The intracellular cyclic pyrophosphate had not been detected before in in vitro measurements of Zymomonas mobilis extracts due to the high instability of this compound. Using continuous in vivo (13)C NMR spectroscopy of aerobic glucose utilization by Corynebacterium glutamicum at a density of 25 g(cell dry weight) . L(-1), the membrane cyclone reactor served to measure the different dynamics of labeling in the carbon atoms of L-lactate, L-glutamate, succinate, and L-lysine with a time resolution of 10 min after impressing a [1-(13)C]-glucose pulse.     

Observations of aerobic, growing escherichia coli metabolism using an on-line nuclear magnetic resonance spectroscopy system

An experimental system has been constructed which enables on-line measurements of phosphorus-31 ((31)P) nuclear magnetic resonance (NMR) spectra for growing bacterial suspensions under anaerobic or aerobic conditions. A sample stream from a laboratory bioreactor is circulated to the NMR sample chamber in a gas exchange system which permits maintenance of aerobic conditions for high-cell-density cultures. (31)P NMR spectra with resolution comparable with those obtained traditionally using dense, concentrated, nongrowing cell suspensions can be obtained at cell densities above 25 g/L with acquisition times ranging from 14 to 3 minutes which decline as cell density increases. This system has been employed to characterize the changes in intracellular state of a stationary phase culture which is subjected to a transition from aerobic to anaerobic conditions. Both intracellular NTP level and cytoplasmic pH are substantially lower under anaerobic conditions. Also, the system has been employed to observe the response of a growing culture to external addition of acetate. Cells are able to maintain pH difference across the cytoplasmic membrane at extracellular acetate concentrations of 5 and 10 g/L. However, acetate concentrations of 20 g/L cause collapse of the transmembrane DeltapH and sharp reduction of the growth rate of the culture. The experimental configuration described should also permit NMR observations of many other types of microbial cultures and of other nuclei. (c) 1993 John Wiley & Sons, Inc.     

Comparative Studies of Glucose Catabolism by Escherichia coli Grown in a Complex Medium under Aerobic and Anaerobic Conditions

Escherichia coli HB101 was grown in complex medium under anaerobic and aerobic conditions. Cells prepared under these two different conditions were characterized by two-dimensional protein gel electrophoresis, by NMR measurements under identical (anaerobic) conditions, and by measuring the kinetics of glucose uptake and catabolite end-product appearance in the medium under identical anaerobic conditions. Specific rates of glucose uptake and end-product formation were significantly greater for the anaerobically grown cells, which also exhibited lower intracellular concentrations of sugar phosphates, nucleoside di-and triphosphates, UDPG, and NAD(H). Two-dimensional gel electrophoretic analyses reveal changes in the intracellular levels of proteins involved in pyruvate catabolism that have been observed previously for E. coli grown in minimal medium under aerobic and anaerobic conditions. Enzymes involved in the TCA cycle were not detected in cells grown aerobically or anaerobically in complex medium.     

Monitoring Escherichia coli growth in M63 media by ultrasonic noninvasive methods and correlation with spectrophotometric and HPLC techniques

A low-intensity ultrasonic technique (that is noninvasive, nondestructive, and online) has been developed to monitor the growth of Escherichia coli in glucose minimal media under aerobic and anaerobic conditions. Ultrasonic time of flight (TOF) variations were correlated with microorganism growth and the disappearance of nutrients and their subsequent conversion into different metabolites. Spectrophotometric growth data and high-performance liquid chromatography (HPLC) analysis of released and consumed metabolites were compared with the ultrasonic data demonstrating that the ultrasound device presented can provide supplementary real-time noninvasive information about the metabolic processes taking place in the culture. A semiempirical model under aerobic conditions was developed to explain the TOF variations as a function of the glucose concentration and was modified to take into account the growth inhibition due to the initial glucose concentration. The inhibition effect was obtained by fitting HPLC measurements to the logistic function of Boltzmann. Under aerobic and anaerobic culture conditions, the metabolic processes were correlated with TOF experimental variations through a theoretical approach based on ultrasonic propagation in ternary mixtures.     

Fructose metabolism of the purple non-sulfur bacterium Rhodospirillum rubrum: effect of carbon dioxide on growth, and production of bacteriochlorophyll and organic acids

During fermentative metabolism, carbon dioxide fixation plays a key role in many bacteria regarding growth and production of organic acids. The present contribution, dealing with the facultative photosynthetic bacterium Rhodospirillum rubrum, reveals not only the strong influence of ambient carbon dioxide on the fermentative break-down of fructose but also a high impact on aerobic growth with fructose as sole carbon source. Both growth rates and biomass yield increased with increasing carbon dioxide supply in chemoheterotrophic aerobic cultures. Furthermore, intracellular metabolite concentration measurements showed almost negligible concentrations of the tricarboxylic acid cycle intermediates succinate, fumarate and malate under aerobic growth, in contrast to several metabolites of the glycolysis. In addition, we present a dual phase fed-batch process, where an aerobic growth phase is followed by an anaerobic production phase. The biosynthesis of bacteriochlorophyll and the secretion of organic acids were both affected by the carbon dioxide supply, the pH value and by the cell density at the time of switching from aerobic to anaerobic conditions. The formation of pigmented photosynthetic membranes and the amount of bacteriochlorophyll were inversely correlated to the secretion of succinate. Accounting the high biotechnological potential of R. rubrum, optimization of carbon dioxide supply is important because of the favored application of fructose-containing fermentable feedstock solutions in bio-industrial processes.     

Carbon-13 nuclear magnetic resonance studies of myocardial glycogen metabolism in live guinea pigs

Myocardial glycogen metabolism was studied in live guinea pigs by 13C NMR at 20.19 MHz. Open-chest surgery was used to expose the heart, which was then positioned within a solenoidal radio frequency coil for NMR measurements. The time course of myocardial glycogen synthesis during 1-h infusions of 0.5 g of D-[1-13C]glucose (and insulin) into the jugular vein was investigated. The possible turnover of the 13C-labeled glycogen was also studied in vivo by following the labeled glucose infusion with a similar infusion of unlabeled glucose. The degree of 13C enrichment of the C-1 glycogen carbons during these infusions was measured in heart extracts by 1H NMR at 360 MHz. High-quality proton-decoupled 13C NMR spectra of the labeled C-1 carbons of myocardial glycogen in vivo were obtained in 1 min of data accumulation. This time resolution allowed measurement of the time course of glycogenolysis of the 13C-labeled glycogen during anoxia by 13C NMR in vivo. With the solenoidal coil used for 13C NMR, the spin-lattice relaxation time of the labeled C-1 carbons of myocardial glycogen could be measured in vivo. For a comparison, spin-lattice relaxation times of heart glycogen were measured in vitro at 90.55 MHz. Natural abundance 13C NMR studies of the quantitative hydrolysis of extracted heart glycogen in vitro at 90.55 MHz showed that virtually all the carbons in heart glycogen contribute to the 13C NMR signals. The same result was obtained in 13C NMR studies of glycogen hydrolysis in excised guinea pig heart.     

Comparative studies of Escherichia coli strains using different glucose uptake systems: Metabolism and energetics

Modifying substrate uptake systems is a potentially powerful tool in metabolic engineering. This research investigates energetic and metabolic changes brought about by the genetic modification of the glucose uptake and phosphorylation system of Escherichia coli. The engineered strain PPA316, which lacks the E. coli phosphotransferase system (PTS) and uses instead the galactose-proton symport system for glucose uptake, exhibited significantly altered metabolic patterns relative to the parent strain PPA305 which retains PTS activity. Replacement of a PTS uptake system by the galactose-proton symport system is expected to lower the carbon flux to pyruvate in both aerobic and anaerobic cultivations. The extra energy cost in substrate uptake for the non-PTS strain PPA 316 had a greater effect on anaerobic specific growth rate, which was reduced by a factor of five relative to PPA 305, while PPA 316 reached a specific growth rate of 60% of that of the PTS strain under aerobic conditions. The maximal cell densities obtained with PPA 316 were approximately 8% higher than those of the PTS strain under aerobic conditions and 14% lower under anaerobic conditions. In vivo NMR results showed that the non-PTS strain possesses a dramatically different intracellular environment, as evidenced by lower levels of total sugar phosphate, NAD(H), nucleoside triphosphates and phosphoenolpyruvate, and higher levels of nucleoside diphosphates. The sugar phosphate compositions, as measured by extract NMR, were considerably different between these two strains. Data suggest that limitations in the rates of steps catalyzed by glucokinase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, and pyruvate kinase may be responsible for the low overall rate of glucose metabolism in PPA316. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 583-590, 1997.     

Glucose Metabolism and Kinetics of Phosphorus Removal by the Fermentative Bacterium Microlunatus phosphovorus

Phosphorus and carbon metabolism in Microlunatus phosphovorus was investigated by using a batch reactor to study the kinetics of uptake and release of extracellular compounds, in combination with ³¹P and ¹³C nuclear magnetic resonance (NMR) to characterize intracellular pools and to trace the fate of carbon substrates through the anaerobic and aerobic cycles. The organism was subjected to repetitive anaerobic and aerobic cycles to induce phosphorus release and uptake in a sequencial batch reactor; an ultrafiltration membrane module was required since cell suspensions did not sediment. M. phosphovorus fermented glucose to acetate via an Embden-Meyerhof pathway but was unable to grow under anaerobic conditions. A remarkable time shift was observed between the uptake of glucose and excretion of acetate, resulting in an intracellular accumulation of acetate. The acetate produced was oxidized in the subsequent aerobic stage. Very high phosphorus release and uptake rates were measured, 3.34 mmol g of cell⁻¹ h⁻¹ and 1.56 mmol g of cell⁻¹ h⁻¹, respectively, values only comparable with those determined in activated sludge. In the aerobic period, growth was strictly dependent on the availability of external phosphate. Natural abundance ¹³C NMR showed the presence of reserves of glutamate and trehalose in cell suspensions. Unexpectedly, [1-¹³C]glucose was not significantly channeled to the synthesis of internal reserves in the anaerobic phase, and acetate was not during the aerobic stage, although the glutamate pool became labeled via the exchange with intermediates of the tricarboxylic acid cycle at the level of glutamate dehydrogenase. The intracellular pool of glutamate increased under anaerobic conditions and decreased during the aerobic period. The contribution of M. phosphovorus for phosphorus removal in wastewater treatment plants is discussed on the basis of the metabolic features disclosed by this study.     

Phosphorus-31 nuclear magnetic resonance studies of bioenergetics in wild-type and adenosinetriphosphatase(1-) Escherichia coli cells

By use of 31P NMR, the transmembrane pH gradient (delta pH) and the intracellular levels of phosphorylated metabolites were measured in aerobic suspensions of wild-type Escherichia coli cells in the presence and absence of the adenosinetriphosphatase (ATPase) inhibitor dicyclohexylcarbodiimide (DCCD); the same parameters were also determined in E. coli mutants deficient in ATPase activity under both anaerobic and aerobic conditions. A method is described by which dense suspensions of E. coli cells (approximately 3 X 10(11) cells/mL) were oxygenated so that steady-state O2 levels in the suspensions were far greater than the Km for O2 consumption. Under these conditions, in wild-type MRE600 cells, the intracellular concentrations of PI, NTP, and NDP were measured to be 3.0 +/- 1.5, 8 +/- 1, and 1.2 +/- 1 mM, respectively, while the intracellular pH was approximately 7.5 over the external pH range studied (6 to approximately 7.0). Upon treatment with DCCD, the intracellular NTP level was drastically reduced and intracellular Pi concentration increased in respiring wild-type cells; in the same cells, however, DCCD did not affect the intracellular pH and the delta pH. During respiration in the presence of lactate, ATPase- cells established a delta pH but failed to synthesize any detectable levels of NTP. Conversely, ATPase- cells accumulated high levels of NTP but did not generate a delta pH during glycolysis under anaerobic conditions. These results are in complete agreement with the generally accepted chemiosmotic hypothesis. 31P NMR data on intact ATPase- NR70 cells were in agreement with the previously proposed [Rosen, B. P., Brey, R., & Hasan, S. (1978) J. Bacteriol. 134, 1030] existence of a proton leak in this strain which is sealed by DCCD or by spontaneous mutation into strain NR71. However, the NMR data also indicated that other major differences exist between NR71 and NR70 cells.     

Comparison of glucose fermentation by suspended and gel-entrapped yeast cells: An in vivo nuclear magnetic resonance study

Phosphorus-31 nuclear magnetic resonance ((31)P NMR) was used to compare the anaerobic metabolism of glucose by suspended and gel-entrapped Saccharomyces bayanus cells. The fermentation of glucose was carried out in a reaction system with continuous circulation through the NMR sample tube. The intracellular pH and the levels of some phosphorylated compounds were the levels of some phosphorylated compounds were noninvasively monitored by (31)P NMR while glucose, fermentation products, and biomass were determined by analytic techniques comparisons showed that no significant differences are observed in the relative concentrations in the spectra, but distinct profiles for the variation of both intracellular and extracellular pH are found. The internal pH of immobilized cells is maintained at a constant value throughout the fermentation as opposed to freely suspended cells for which a steady decrease in the internal pH occurs. A faster and stronger acidification is also observed in the external medium of the assays with suspended cells. Furthermore, higher yields for ethanol and biomass production and lower yields of fermentation by-products are obtained with immobilized cells. It is concluded that the higher intracellular pH achieved in the presence of the gel matrix had a regulatory effect on the metabolism which favored the ethanol production pathway. (c) 1993 John Wiley & Sons, Inc.     

31P and 13C NMR studies of intermediates of aerobic and anaerobic glycolysis in Saccharomyces cerevisiae

The levels of intermediates of aerobic and anaerobic glycolysis were determined in perchloric acid extracts prepared from glycolyzing suspensions of Saccharomyces cerevisiae by 31P and 13C NMR spectroscopy. From 31P NMR measurements a small increase in the level of nucleoside triphosphates was found in derepressed cells upon oxygenation, while the ratio of nucleoside diphosphates to nucleoside triphosphates was a factor of 3 lower aerobically. Combined with the previous observation that the level of intracellular Pi is lower by a factor of 3 aerobically, this leads to the conclusion that the phosphate potential [NTP]/([NDP][Pi]) is lower by an order of magnitude during anaerobic glycolysis than during aerobic glycolysis. There was no correlation between the level of glucose 6-phosphate and the rate of glucose utilization. We used 13C NMR to determine the scrambling of the 13C label from C1 to C6 in fructose 1,6-bisphosphate (Fru-P2). There was more scrambling of the label during aerobic than during anaerobic glycolysis. Since the level of Fru-P2 did not change much upon oxygenation, this suggests that in aerobic glycolysis there is control of at least one enzyme in the lower part of the Embden-Meyerhof-Parnas pathway, below Fru-P2, which gives the 13C level more time to equilibrate between C1 and C6 of Fru-P2. Previous 13C NMR measurements of glucose utilization rates had shown a 2-fold reduction upon oxygenation, reflecting control in the early stages of the pathway.     

Short-term metabolome dynamics and carbon, electron, and ATP balances in chemostat-grown Saccharomyces cerevisiae CEN.PK 113-7D following a glucose pulse

The in vivo kinetics in Saccharomyces cerevisiae CEN.PK 113-7D was evaluated during a 300-second transient period after applying a glucose pulse to an aerobic, carbon-limited chemostat culture. We quantified the responses of extracellular metabolites, intracellular intermediates in primary metabolism, intracellular free amino acids, and in vivo rates of O(2) uptake and CO(2) evolution. With these measurements, dynamic carbon, electron, and ATP balances were set up to identify major carbon, electron, and energy sinks during the postpulse period. There were three distinct metabolic phases during this time. In phase I (0 to 50 seconds after the pulse), the carbon/electron balances closed up to 85%. The accumulation of glycolytic and storage compounds accounted for 60% of the consumed glucose, caused an energy depletion, and may have led to a temporary decrease in the anabolic flux. In phase II (50 to 150 seconds), the fermentative metabolism gradually became the most important carbon/electron sink. In phase III (150 to 300 seconds), 29% of the carbon uptake was not identified in the measurements, and the ATP balance had a large surplus. These results indicate an increase in the anabolic flux, which is consistent with macroscopic balances of extracellular fluxes and the observed increase in CO(2) evolution associated with nonfermentative metabolism. The identified metabolic processes involving major carbon, electron, and energy sinks must be taken into account in in vivo kinetic models based on short-term dynamic metabolome responses.     

In vivo 31P NMR study of early cellular responses to hyperosmotic shock in cultured glioma cells.

Cell volume regulation in the face of osmotic stress is a fundamental homeostatic activity, and is most critical in brain, which is spatially constrained. Despite the importance of this phenomenon, little is known about volume regulation in the brain, primarily because of the cellular heterogeneity in the tissue. We describe here simultaneous in vivo 31P nuclear magnetic resonance (NMR) measurements of cell volume, intracellular pH and phosphate metabolites during early responses to hyperosmotic stress in C6 glioma cells perfused in NMR-compatible bioreactors. Cell volume was measured using dimethyl methylphosphonate (DMMP) as a probe which has an intracellular NMR resonance shifted upfield from the extracellular resonance. The sensitivity of these measurements allowed 31P NMR spectra to be collected every 30 s. Following an increase in osmolarity from 320 to 480 mOsm by addition of NaCl to the perfusate, C6 glioma cells shrank to 67% of their original volume. We also observed a simultaneous increase of intracellular pH coincident with the decrease in cell volume. The signals from ATP decreased by 10%, but those from phosphocreatine (PCr) increased by 31% after hyperosmotic shock. However, correcting the ATP signals for the decrease in cell volume indicated that its intracellular concentrations increased after treatment. Signals from glycerophosphorylcholine (GPC) and glycerophosphorylethanolamine (GPE) were not changed significantly. This is the first in vivo report of early cellular responses monitored by NMR spectroscopy following hyperosmotic shock in cultured cells.     

Short-Term Metabolome Dynamics and Carbon, Electron, and ATP Balances in Chemostat-Grown Saccharomyces cerevisiae CEN.PK 113-7D following a Glucose Pulse

The in vivo kinetics in Saccharomyces cerevisiae CEN.PK 113-7D was evaluated during a 300-second transient period after applying a glucose pulse to an aerobic, carbon-limited chemostat culture. We quantified the responses of extracellular metabolites, intracellular intermediates in primary metabolism, intracellular free amino acids, and in vivo rates of O₂ uptake and CO₂ evolution. With these measurements, dynamic carbon, electron, and ATP balances were set up to identify major carbon, electron, and energy sinks during the postpulse period. There were three distinct metabolic phases during this time. In phase I (0 to 50 seconds after the pulse), the carbon/electron balances closed up to 85%. The accumulation of glycolytic and storage compounds accounted for 60% of the consumed glucose, caused an energy depletion, and may have led to a temporary decrease in the anabolic flux. In phase II (50 to 150 seconds), the fermentative metabolism gradually became the most important carbon/electron sink. In phase III (150 to 300 seconds), 29% of the carbon uptake was not identified in the measurements, and the ATP balance had a large surplus. These results indicate an increase in the anabolic flux, which is consistent with macroscopic balances of extracellular fluxes and the observed increase in CO₂ evolution associated with nonfermentative metabolism. The identified metabolic processes involving major carbon, electron, and energy sinks must be taken into account in in vivo kinetic models based on short-term dynamic metabolome responses.     

In vivo 31P and multilabel 13C NMR measurements for evaluation of plant metabolic pathways

Reliable measurements of intracellular metabolites are useful for effective plant metabolic engineering. This study explored the application of in situ 31P and 13C NMR spectroscopy for long-term measurements of intracellular pH and concentrations of several metabolites in glycolysis, glucan synthesis, and central carbon metabolic pathways in plant tissues. An NMR perfusion reactor system was designed to allow Catharanthus roseus hairy root cultures to grow for 3-6 weeks, during which time NMR spectroscopy was performed. Constant cytoplasmic pH (7.40+/-0.06), observed during the entire experiment, indicated adequate oxygenation. 13C NMR spectroscopy was performed on hairy root cultures grown in solutions containing 1-13C-, 2-13C-, and 3-13C-labeled glucose in separate experiments and the flow of label was monitored. Activities of pentose phosphate pathways, nonphotosynthetic CO2 fixation, and glucan synthesis pathways were evident from the experimental results. Scrambling of label in glucans also indicated recycling of triose phosphate and their subsequent conversion to hexose phosphates.     

Stoichiometric flux balance models quantitatively predict growth and metabolic by-product secretion in wild-type Escherichia coli W3110.

Flux balance models of metabolism use stoichiometry of metabolic pathways, metabolic demands of growth, and optimality principles to predict metabolic flux distribution and cellular growth under specified environmental conditions. These models have provided a mechanistic interpretation of systemic metabolic physiology, and they are also useful as a quantitative tool for metabolic pathway design. Quantitative predictions of cell growth and metabolic by-product secretion that are experimentally testable can be obtained from these models. In the present report, we used independent measurements to determine the model parameters for the wild-type Escherichia coli strain W3110. We experimentally determined the maximum oxygen utilization rate (15 mmol of O2 per g [dry weight] per h), the maximum aerobic glucose utilization rate (10.5 mmol of Glc per g [dry weight] per h), the maximum anaerobic glucose utilization rate (18.5 mmol of Glc per g [dry weight] per h), the non-growth-associated maintenance requirements (7.6 mmol of ATP per g [dry weight] per h), and the growth-associated maintenance requirements (13 mmol of ATP per g of biomass). The flux balance model specified by these parameters was found to quantitatively predict glucose and oxygen uptake rates as well as acetate secretion rates observed in chemostat experiments. We have formulated a predictive algorithm in order to apply the flux balance model to describe unsteady-state growth and by-product secretion in aerobic batch, fed-batch, and anaerobic batch cultures. In aerobic experiments we observed acetate secretion, accumulation in the culture medium, and reutilization from the culture medium. In fed-batch cultures acetate is cometabolized with glucose during the later part of the culture period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of the cAMP level in the yeast Saccharomyces cerevisiae: intracellular pH and the effect of membrane depolarizing compounds

Addition of plasma membrane depolarizing agents, such as dinitrophenol (DNP) and azide, to cells of Saccharomyces cerevisiae under aerobic conditions, is known to cause an increase in the cAMP level within 15 s. We found that both compounds lowered the intracellular pH (measured by in vivo 32P-NMR) drastically within the same time period. Plasma membrane depolarization, however, was much slower: DNP and azide had no effect on the membrane potential during, respectively, the first 2 min and the first 10 min after addition. Apparently, the intracellular pH of yeast is much more sensitive to perturbation than the membrane potential. The effect of both compounds on the cAMP level was highly dependent on the extracellular pH: when the latter was raised, the effect disappeared completely between pH 6 and 7. A similar dependence on the extracellular pH was observed for the lowering of intracellular pH. Addition of organic acids, such as acetate and butyrate, at low pH and under aerobic conditions, also caused an immediate increase in the cAMP level and an immediate drop in the intracellular pH. These results suggest that agents such as DNP and azide do not raise the cAMP level in yeast cells because of their membrane depolarizing properties but because they lower the intracellular pH. Under anaerobic conditions, DNP, azide and organic acids were much less effective in increasing the cAMP level. Addition of a small amount of glucose, however, restored their capacity to enhance the cAMP level. This suggests that under anaerobic conditions and in the absence of glucose the ATP level is a limiting factor for cAMP synthesis.