Characterization of growth and acid formation in a Bacillus subtilis pyruvate kinase mutant

Based on measurements and theoretical analyses, we identified deletion of pyruvate kinase (PYK) activity as a possible route for elimination of acid formation in Bacillus subtilis cultures grown on glucose minimal media. Evidence consistent with the attenuation of PYK flux has come from metabolic flux calculations, metabolic pool and enzymatic activity measurements, and a series of nuclear magnetic resonance experiments, all suggesting a nearly complete inhibition of PYK activity for glucose-citrate fed cultures in which the amount of acid formation was nearly zero. In this paper, we report the construction and characterization of a pyk mutant of B. subtilis. Our results demonstrate an almost complete elimination of acid production in cultures of the pyk mutant in glucose minimal medium. The substantial reduction in acid production is accompanied by increased CO(2) production and a reduced rate of growth. Metabolic analysis indicated a dramatic increase in intracellular pools of phosphoenolpyruvate (PEP) and glucose-6-P in the pyk mutant. The high concentrations of PEP and glucose-6-P could explain the decreased growth rate of the mutant. The substantial accumulation of PEP does not occur in Escherichia coli pyk mutants. The very high concentration of PEP which accumulates in the B. subtilis pyk mutant could be exploited for production of various aromatics.     

Metabolic changes in a pyruvate kinase gene deletion mutant of Corynebacterium glutamicum ATCC 13032

To investigate primary effects of a pyruvate kinase (PYK) defect on glucose metabolism in Corynebacterium glutamicum, a pyk-deleted mutant was derived from wild-type C. glutamicum ATCC13032 using the double-crossover chromosome replacement technique. The mutant was then evaluated under glutamic acid-producing conditions induced by biotin limitation. The mutant showed an increased specific rate of glucose consumption, decreased growth, higher glutamic acid production, and aspartic acid formation during the glutamic acid production phase. A significant increase in phosphoenolpyruvate (PEP) carboxylase activity and a significant decrease in PEP carboxykinase activity occurred in the mutant, which suggested an enhanced overall flux of the anaplerotic pathway from PEP to oxaloacetic acid in the mutant. The enhanced anaplerotic flux may explain both the increased rate of glucose consumption and the higher productivity of glutamic acid in the mutant. Since the pyk-complemented strain had similar metabolic profiles to the wild-type strain, the observed changes represented intrinsic effects of pyk deletion on the physiology of C. glutamicum.     

Metabolic fluxes, pools, and enzyme measurements suggest a tighter coupling of energetics and biosynthetic reactions associated with reduced pyruvate kinase flux.

In this study, it is found that, for Bacillus subtilis, citrate-glucose cometabolism leads to zero acid production over a wide range of growth rates and nearly theoretical carbon yield. Experimental results are presented that point to pyruvate kinase (PYK) as a site of citrate-mediated glycolytic flux attenuation. First, the measured fluxes show that, compared with cultures grown on glucose, the PYK flux drops by more than tenfold when citrate is added. Second, relative to cultures metabolizing glucose, the phosphoenolpyruvate (PEP) pool elevates substantially, whereas the pyruvate pool drops, when citrate is present. Finally, our modeling results indicate that maximizing carbon yield corresponds to nearly eliminating pyruvate kinase (PYK) flux and that the pyruvate supplied by the PEP-consuming glucose transport system can supply the biosynthetic requirements. A literature review suggests some mechanisms for how PYK attenuation by citrate addition can occur. At this juncture, we hypothesize that direct PYK inhibition occurs which, in turn, also leads to phosphofructokinase inhibition via the elevated PEP pool. These two inhibition events combine to throttle glycolytic flux; minimize acid formation; and substantially increase cellular, product, and energetic yields.     

Effects of pyruvate kinase on the growth of Corynebacterium glutamicum and L-serine accumulation

Pyruvate kinase (PYK) is an important enzyme in the intermediary metabolism and has attracted much attention as a target for metabolic engineering of Corynebacterium glutamicum. Genome sequencing revealed that the 308 residue of PYK was mutated from methionine in model strain C. glutamicum ATCC14067 to isoleucine in L-serine-producing strain C. glutamicum SYPS-062. Consequently, a significantly lower PYK activity (77%) was noted in C. glutamicum SYPS-062, when compared with that in C. glutamicum ATCC14067. To confirm the role of this point mutation, pyk in both C. glutamicum SYPS-062 and C. glutamicum SYPS-062-33aΔSSAA was reversely mutated to restore the PYK enzyme activity, which led to a 33.1% and 28.8% decrease in L-serine titer, respectively. This is the first report to show that the (Met-308→Ile) mutation site of pyk is closely associated with its activity and apparently affected L-serine production. Furthermore, pyk was deleted in strain C. glutamicum SYPS-062-33aΔSSAA, and the resulting strain did not show alteration in growth rate and presented a 12% increase in L-serine production.     

Enhanced recombinant protein production in pyruvate kinase mutant of Bacillus subtilis

Previous work demonstrated that acetate production was substantially lower in pyruvate kinase (pyk) mutant of Bacillus subtilis. The significantly lower acetate production in the pyk mutant is hypothesized to have positive effect on recombinant protein production either by lifting the inhibitory effect of acetate accumulation in the medium or redirecting the metabolic fluxes beneficial to biomass/protein synthesis. In this study, the impact of the pyk mutation on recombinant protein production was investigated. Green fluorescent protein (GFP+) was selected as a model protein and constitutively expressed in both the wild-type strain and a pyk mutant. In batch cultures, the pyk mutant produced 3-fold higher levels of recombinant protein when grown on glucose as carbon source. Experimental measurements and theoretical analysis show that the higher protein yield of the mutant is not due to removal of an acetate-associated inhibition of expression or gene dosage or protein stability but a much lower acetate production in the mutant allows for a greater fraction of carbon intake to be directed to protein synthesis.     

Regulating expression of pyruvate kinase in Bacillus subtilis for control of growth rate and formation of acidic byproducts

Our prior work has shown that a pyk mutant of Bacillus subtilis exhibited diminished acidic byproduct accumulation, dramatically elevated phosphoenolpyruvate (PEP) pool, and reduced growth rate. To determine if a low acetate-producing but fast-growing strain of B. subtilis could be developed, we placed the expression of the pyk gene under the control of an inducible promoter. Enzyme measurements proved that PYK activity of the inducible PYK mutant (iPYK) increases with the isopropyl-beta-d-thiogalactopyranoside concentration. Batch growth experiments showed that growth rate and acid formation are closely related to the induction level of pyk. Measurements of cell growth rate and acetate formation of the iPYK mutant at different induction levels revealed that a PYK activity of about 12% of wild-type allows for good growth rate (0.4 h(-)(1) versus 0.63 h(-)(1) of wild-type) and low acetate production (0.26 g/L versus 1.05 g/L of wild-type). This is the first report to our knowledge of a metabolically engineered B. subtilis strain that allows good growth rate and low acid production in batch cultures. Finally, it was found that, by varying the pyk induction level, intracellular PEP concentration can be controlled over a wide range. The intracellular PEP concentration is intimately connected to the regulation of the transport of phosphotransferase system (PTS) sugars in the presence of glucose. Because there is no other method for modulating intracellular PEP levels, this finding represents a major advance in one's ability to dissect the function of the PTS and sugar metabolism in bacteria.     

Regulation of pyruvate kinase from Propionibacterium shermanii

Pyruvate kinase from Propionibacterium shermanii was shown to be activated by glucose-6-phosphate (G-6-P) at non-saturating phosphoenol pyruvate (PEP) concentrations but other glycolytic and hexose monophosphate pathway intermediates and AMP were without effect. Half-maximal activation was obtained at 1 mM G-6-P. The presence of G-6-P decreased both the PEP_0.5V and ADP_0.5V values and the slope of the Hill plots for both substrates. The enzyme was strongly inhibited by ATP and inorganic phosphate (P_i) at all PEP concentrations. At non-saturating (0.5 mM) PEP, half-maximal inhibition was obtained at 1.8 mM ATP or 1.4 mM P_i. The inhibition by both P_i and ATP was largely overcome by 4 mM G-6-P. The specific activity of pyruvate kinase was considerably higher in lactate-, glucose- and glycerol-grown cultures than that of the enzyme catalysing the reverse reaction, pyruvate, phosphate dikinase. It is suggested that the activity of pyruvate kinase in vivo is determined by the balance between activators and inhibitors such that it is inhibited during gluconeogenesis while, during glycolysis, the inhibition is relieved by G-6-P.Abbreviations PEP phosphoenolpyruvate - G-6-P glucose-6-phosphate - P_i inorganic phosphate     

Mn2+/Mg2+-dependent pyruvate kinase from a d-lactic acid-producing bacterium Sporolactobacillus inulinus: characterization of a novel Mn2+-mediated allosterically regulated enzyme

Sporolactobacillus inulinus has attracted scientific and commercial interest due to its high efficiency in d-lactic acid production. Pyruvate kinase (PYK) is one of the key regulatory points in glycolysis, and well-activated PYK can improve d-lactic acid production. A novel Mn²⁺/Mg²⁺-dependent PYK from S. inulinus was expressed in Escherichia coli and purified to homogeneity. Kinetic characterization demonstrated that the S. inulinus PYK had drastically higher activity and affinity toward substrates in the presence of Mn²⁺ compared to those of the common PYK cofactor Mg²⁺, and the circular dichroism spectra of the S. inulinus PYK suggested a Mn²⁺-mediated allosteric activation. The S. inulinus PYK was also allosterically regulated by ribose-5-phosphate or AMP activation and inorganic phosphate or ATP inhibition. The inhibition could be marked reduced or fully eliminated in the presence of activators. The result of fermentations by S. inulinus Y2-8 showed that the extracellular-added MnSO₄ and KH₂PO₄ significantly affected glycolysis flux and d-lactic acid production, which is consistent with the allosteric regulation of Mn²⁺ and inorganic phosphate on PYK. The sophisticated regulatory role of PYK would establish the foundation of substantial disturbance or restructuring of cellular metabolism for improving the S. inulinus d-lactic acid production.     

Crassulacean acid metabolism (CAM) in Kalanchoë daigremontiana: Temperature response of phosphoenolpyruvate (PEP)-carboxylase in relation to allosteric effectors

Net CO₂ dark fixation of Kalanchoë daigremontiana varies with night temperature. We found an optimum of fixation at about 15° C; with increasing night temperature fixation decreased. We studied the temperature dependence of the activity of phosphoenolpyruvate (PEP)-carboxylase, the key enzyme for CO₂ dark fixation. We varied the pH, the substrate concentration (PEP), and the L-malate and glucose-6-phosphate (G-6-P) concentration in the assay. Generally, lowering the pH and reducing the amount of substrate resulted in an increase in activation by G-6-P and in an increase in malate inhibition of the enzyme. Furthermore, malate inhibition and G-6-P activation increased with increasing temperature. Activity measurements between 10° C and 45°C at a given concentration of the effectors revealed that the temperature optimum and maximum activities at that optimum varied with the effector applied. Under the influence of 5 mol m^-3 L-malate the temperature optimum and maximum activity dropped drastically, especially when the substrate level was low (at 0.5 mol m^-3 PEP from 32° C to 20° C). G-6-P raised the temperature optimum and maximum activity when the substrate level was low. If both malate and G-6-P were present, intermediate values were measured. We suggest that changes in metabolite levels in K. daigremontiana leaves can alter the temperature features of PEP-carboxylase so that the observed in vivo CO₂ dark fixation can be explained on the basis of PEP-carboxylase activity.Abbreviations PEP-c phosphoenolpyruvate carboxylase - CAM crassulacean acid metabolism - PEP phosphoenolpyruvate - G-6-P glucose-6-phosphate     

Metabolic Flux Responses to Pyruvate Kinase Knockout in Escherichia coli

The intracellular carbon flux distribution in wild-type and pyruvate kinase-deficient Escherichia coli was estimated using biosynthetically directed fractional 13C labeling experiments with [U-13C6]glucose in glucose- or ammonia-limited chemostats, two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, and a comprehensive isotopomer model. The general response to disruption of both pyruvate kinase isoenzymes in E. coli was a local flux rerouting via the combined reactions of phosphoenolpyruvate (PEP) carboxylase and malic enzyme. Responses in the pentose phosphate pathway and the tricarboxylic acid cycle were strongly dependent on the environmental conditions. In addition, high futile cycling activity via the gluconeogenic PEP carboxykinase was identified at a low dilution rate in glucose-limited chemostat culture of pyruvate kinase-deficient E. coli, with a turnover that is comparable to the specific glucose uptake rate. Furthermore, flux analysis in mutant cultures indicates that glucose uptake in E. coli is not catalyzed exclusively by the phosphotransferase system in glucose-limited cultures at a low dilution rate. Reliability of the flux estimates thus obtained was verified by statistical error analysis and by comparison to intracellular carbon flux ratios that were independently calculated from the same NMR data by metabolic flux ratio analysis.     

Metabolic responses to pyruvate kinase deletion in lysine producing Corynebacterium glutamicum

Background

Pyruvate kinase is an important element in flux control of the intermediate metabolism. It catalyzes the irreversible conversion of phosphoenolpyruvate into pyruvate and is under allosteric control. In Corynebacterium glutamicum, this enzyme was regarded as promising target for improved production of lysine, one of the major amino acids in animal nutrition. In pyruvate kinase deficient strains the required equimolar ratio of the two lysine precursors oxaloacetate and pyruvate can be achieved through concerted action of the phosphotransferase system (PTS) and phosphoenolpyruvate carboxylase (PEPC), whereby a reduced amount of carbon may be lost as CO₂ due to reduced flux into the tricarboxylic acid (TCA) cycle. In previous studies, deletion of pyruvate kinase in lysine-producing C. glutamicum, however, did not yield a clear picture and the exact metabolic consequences are not fully understood.

Results

In this work, deletion of the pyk gene, encoding pyruvate kinase, was carried out in the lysine-producing strain C. glutamicum lysC^fbr, expressing a feedback resistant aspartokinase, to investigate the cellular response to deletion of this central glycolytic enzyme. Pyk deletion was achieved by allelic replacement, verified by PCR analysis and the lack of in vitro enzyme activity. The deletion mutant showed an overall growth behavior (specific growth rate, glucose uptake rate, biomass yield) which was very similar to that of the parent strain, but differed in slightly reduced lysine formation, increased formation of the overflow metabolites dihydroxyacetone and glycerol and in metabolic fluxes around the pyruvate node. The latter involved a flux shift from pyruvate carboxylase (PC) to PEPC, by which the cell maintained anaplerotic supply of the TCA cycle. This created a metabolic by-pass from PEP to pyruvate via malic enzyme demonstrating its contribution to metabolic flexibility of C. glutamicum on glucose.

Conclusion

The metabolic flux analysis performed illustrates the high flexibility of the metabolic network of C. glutamicum to compensate for external perturbation. The organism could almost maintain its growth and production performance through a local redirection of the metabolic flux, thereby fulfilling all anabolic and catabolic needs. The formation of the undesired overflow metabolites dihydroxyacetone and glycerol, in the deletion mutant, however, indicates a limiting capacity of the metabolism down-stream of their common precursor glyceraldehyde 3-phosphate and opens possibilities for further strain engineering.     

Banana ripening: implications of changes in glycolytic intermediate concentrations, glycolytic and gluconeogenic carbon flux, and fructose 2,6-bisphosphate concentration

In ripening banana (Musa sp. [AAA group, Cavendish subgroup] cv Valery) fruit, the concentration of glycolytic intermediates increased in response to the rapid conversion of starch to sugars and CO₂. Glucose 6-phosphate (G-6-P), fructose 6-phosphate (Fru 6-P), and pyruvate (Pyr) levels changed in synchrony, increasing to a maximum one day past the peak in ethylene synthesis and declining rapidly thereafter. Fructose 1,6-bisphosphate (Fru 1,6-P₂) and phosphoenolpyruvate (PEP) levels underwent changes dissimilar to those of G 6-P, Fru 6-P, and Pyr, indicating that carbon was regulated at the PEP/Pyr and Fru 6-P/Fru 1,6-P₂ interconversion sites. During the climacteric respiratory rise, gluconeogenic carbon flux increased 50- to 100-fold while glycolytic carbon flux increased only 4- to 5-fold. After the climacteric peak in CO₂ production, gluconeogenic carbon flux dropped dramatically while glycolytic carbon flux remained elevated. The steady-state fructose 2,6-bisphosphate (Fru 2,6-P₂) concentration decreased to ½ that of preclimacteric fruit during the period coinciding with the rapid increase in gluconeogenesis. Fru 2,6-P₂ concentration increased thereafter as glycolytic carbon flux increased relative to gluconeogenic carbon flux. It appears likely that the initial increase in respiration in ripening banana fruit is due to the rapid influx of carbon into the cytosol as starch is degraded. As starch reserves are depleted and the levels of intermediates decline, the continued enhancement of respiration may, in part, be maintained by an increased steady-state Fru 2,6-P₂ concentration acting to promote glycolytic carbon flux at the step responsible for the interconversion of Fru 6-P and Fru 1,6-P₂.     

A study of the in-vitro regulation of phosphoenolpyruvate carboxylase from the epidermis of Commelina communis by malate and glucose-6-phosphate

Phosphoenolpyruvate (PEP) carboxylase activity in epidermal extracts of Commelina communis has been compared in the presence of malate and glucose-6-phosphate. The activity of PEP carboxylase was inhibited by increasing malate concentrations at several substrate (PEP) concentrations and changes in both the apparent K _m (PEP) and V _max values in the presence of malate suggested the occurence of mixed-type inhibiton. In the presence of glucose-6-phosphate no increase in enzyme activity was observed, although there was a slight decrease in the K _m (PEP). However, glucose-6-phosphate appeared to alleviate the inhibition caused by malate. The possible implications of these properties in the control of malate production in guard cells is discussed.Abbreviations PEP phosphoenolpyruvate - Glc6P glucose-6-phosphate     

Evidence that ATP exerts control of the rate of glucose utilization in the intact Escherichia coli cell by altering the cellular level of glucose-6-P, an intermediate known to inhibit glucose transport in vitro

The effects of treating nitrogen-starved cultures of $\text{Escherichia coli}$ W4597 (K) with various doses of 2,4-dinitrophenol include increases in the rates of glucose utilization, decreases in ATP and glucose-6-P and maintenance of the level of fructose-1, 6-P₂. A quantitative correlation was observed between the increases in the rates of glucose utilization and decreases in glucose-6-P in agreement with the observation made $\text{in vitro}$ that glucose-6-P inhibits glucose transport in $\text{E. coli}$. A quantitative correlation was also observed between glucose-6-P and ATP indicating that the fall in glucose-6-P is effected by the fall in ATP which indirectly signals increased glucose utilization and increased ATP production.     

Physiological Properties of Saccharomyces cerevisiae from Which Hexokinase II Has Been Deleted

Hexokinase II is an enzyme central to glucose metabolism and glucose repression in the yeast Saccharomyces cerevisiae. Deletion of HXK2, the gene which encodes hexokinase II, dramatically changed the physiology of S. cerevisiae. The hxk2-null mutant strain displayed fully oxidative growth at high glucose concentrations in early exponential batch cultures, resulting in an initial absence of fermentative products such as ethanol, a postponed and shortened diauxic shift, and higher biomass yields. Several intracellular changes were associated with the deletion of hexokinase II. The hxk2 mutant had a higher mitochondrial H⁺-ATPase activity and a lower pyruvate decarboxylase activity, which coincided with an intracellular accumulation of pyruvate in the hxk2 mutant. The concentrations of adenine nucleotides, glucose-6-phosphate, and fructose-6-phosphate are comparable in the wild type and the hxk2 mutant. In contrast, the concentration of fructose-1,6-bisphosphate, an allosteric activator of pyruvate kinase, is clearly lower in the hxk2 mutant than in the wild type. The results suggest a redirection of carbon flux in the hxk2 mutant to the production of biomass as a consequence of reduced glucose repression.     

Identification of essential histidine residues in 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase: analysis by chemical modification with diethyl pyrocarbonate and site-directed mutagenesis

The enzyme 3-deoxy-D-manno-octulosonic acid 8-phosphate (KDO 8-P) synthase from Escherichia coli that catalyzes the aldol-type condensation of D-arabinose 5-phosphate (A 5-P) and phosphoenolpyruvate (PEP) to give KDO 8-P and inorganic phosphate (P(i)) is inactivated by diethyl pyrocarbonate (DEPC). The inactivation is first-order in enzyme and DEPC. A second-order rate constant of 340 M(-1) min(-1) is obtained at pH 7.6 and 4 degrees C. The rate of inactivation is dependent on pH and the pH-inactivation rate data imply the involvement of an amino acid residue with a pK(a) value of 7.3. KDO 8-P synthase activity is not restored to the DEPC-inactivated enzyme following treatment with hydroxylamine. Complete loss of KDO 8-P synthase activity correlates with the ethoxyformylation of three histidine residues by DEPC. KDO 8-P synthase is protected against DEPC inactivation by PEP and partially protected against inactivation by A 5-P. To provide further evidence for the involvement or role of the histidine residues in the aldol-type condensation catalyzed by KDO 8-P synthase, all six histidines were individually mutated to either glycine or alanine. The kinetic constants for the three mutants H40A, H67G, and H246G were unaffected as compared to the wild type enzyme. In contrast, H241G demonstrates a >10-fold increase in K(M) for both PEP and A 5-P and a 4-fold reduction in k(cat), while H97G demonstrates an increase in K(M) for only A 5-P and a 2-fold reduction in k(cat). The activity of the H202G mutant was too low to be measured accurately but the data obtained indicated an approximate 400-fold reduction in k(cat). Circular dichroism measurements of the wild-type and mutant enzymes indicate modest structural changes in only the fully active H67G and H246G mutants. The H241G mutant is protected against DEPC inactivation by PEP and A 5-P to the same extent as the wild-type enzyme, suggesting that the functionally important H241 may not be located in the vicinity of the substrate binding sites. The H97G mutant is protected by PEP against DEPC inactivation to the same degree as the wild-type enzyme but is no longer protected by A 5-P. In the case of the H202G mutant, both A 5-P and PEP protect the mutant against DEPC inactivation but to different extents from those observed for the wild-type enzyme. The catalytic activity of the H97G mutant is partially restored (20% --> 60% of wild-type activity) in the presence of imidazole, while a minor amount of activity is restored to the H202G mutant (<1% --> 4% of wild-type activity) in the presence of imidazole.     

Kinetic Properties of Phosphoenolpyruvate Carboxylase in Peperomia camptotricha

Kinetic characteristics of phosphoenolpyruvate carboxylase (PEPC) from the epiphytic C₃ or C₄: CAM intermediate plant, Peperomia camptotricha, were investigated. Few day versus night differences in V_max,K_m(PEP)), or malate inhibition were observed, even in extracts from water-stressed plants which characteristically perform CAM, regardless of efforts to stabilize day/night forms. The PEPC extracted from plants during the light period remained stable, without much of an increase or decrease in activity for at least 22 hours at 0 to 4°C. Extracts from mature, fully developed leaves had slightly greater PEPC activity than from very young, developing leaves. Generally, however, the kinetic properties of PEPC extracted from mature leaves of plants grown under short day (SD), long day (LD), or 1-week water-stress conditions, as well as from young, developing leaves, were similar. The PEPC inhibitor, l-malate, decreased the V_max and increased the K_m(PEP) for all treatments. Under specific conditions, malate did not inhibit PEPC rates in the dark extracts as much as the light. The PEPC activator, glucose-6-phosphate (G-6-P), lowered the K_m(PEP) for all treatments. At saturating PEP concentrations, PEPC activity was independent of pH in the range of 7.5 to 9.0. At subsaturating PEP concentrations, the pH optimum was 7.8. The rates of PEPC activity were lower in the light period extracts than the dark, at pH 7.1, but day/night PEPC was equally active at pH 7.8. At pH 7.5 and a subsaturating PEP concentration, G-6-P significantly activated PEPC. At pH 8, however, only slight activation by G-6-P was observed. The lower pH of 7.5 combined with l-malate addition, greatly inhibited PEPC, particularly in extracts from young, developing leaves which were completely inhibited at an l-malate concentration of 1 millimolar. However, malate did not further inhibit PEPC activity in mature leaves when assayed at pH 7.1. The fairly constant day/night kinetic and regulatory properties of PEPC from P. camptotricha are unlike those of PEPC from CAM or C₄ species studied, and are consistent with the photosynthetic metabolism of this plant.     

Glycolytic Flux Is Adjusted to Nitrogenase Activity in Nodules of Detopped and Argon-Treated Alfalfa Plants

To investigate the short-term (30–240 min) interactions among nitrogenase activity, NH₄⁺ assimilation, and plant glycolysis, we measured the concentrations of selected C and N metabolites in alfalfa (Medicago sativa L.) root nodules after detopping and during continuous exposure of the nodulated roots to Ar:O₂ (80:20, v/v). Both treatments caused an increase in the ratios of glucose-6-phosphate to fructose-1,6-bisphosphate, fructose-6-phosphate to fructose-1,6-bisphosphate, phosphoenolpyruvate (PEP) to pyruvate, and PEP to malate. This suggested that glycolytic flux was inhibited at the steps catalyzed by phosphofructokinase, pyruvate kinase, and PEP carboxylase. In the Ar:O₂-treated plants the apparent inhibition of glycolytic flux was reversible, whereas in the detopped plants it was not. In both groups of plants the apparent inhibition of glycolytic flux was delayed relative to the decline in nitrogenase activity. The decline in nitrogenase activity was followed by a dramatic increase in the nodular glutamate to glutamine ratio. In the detopped plants this was coincident with the apparent inhibition of glycolytic flux, whereas in the Ar:O₂-treated plants it preceded the apparent inhibition of glycolytic flux. We propose that the increase in the nodular glutamate to glutamine ratio, which occurs as a result of the decline in nitrogenase activity, may act as a signal to decrease plant glycolytic flux in legume root nodules.