Low Assay Temperatures Research Articles

Regulation of an important glycolytic enzyme, pyruvate kinase, through phosphorylation in the larvae of a species of freeze-tolerant insect, Eurosta solidaginis.

Larvae of the goldenrod gall fly, Eurosta solidaginis, rely on a freeze tolerance strategy to survive the sub-zero temperatures of Canadian winter. Critical to their survival is the accumulation of polyol cryoprotectants and global metabolic rate depression, both of which require the regulation of glycolysis and reorganization of carbohydrate metabolism. This study explored the role that pyruvate kinase (PK) regulation plays in this metabolic reorganization. PK was purified from control (5 °C-acclimated) and frozen (-15 °C-acclimated) larvae and enzyme kinetic properties, structural stability, and post-translational modifications were examined in both enzyme forms. The Km phosphoenolpyruvate (PEP) of frozen PK was 20% higher than that of control PK, whereas the Vmax of frozen PK was up to 50% lower than that of control PK at the lowest assay temperature, suggesting inhibition of the enzyme during the winter. Additionally, the activity and substrate affinity of both forms of PK decreased significantly at low assay temperatures, and both forms were regulated allosterically by a number of metabolites. Pro-Q™ Diamond phosphoprotein staining and immunoblotting experiments demonstrated significantly higher threonine phosphorylation of PK from frozen animals while acetylation and methylation levels remained constant. Together, these results indicate that PK exists in two structurally distinct forms in E. solidaginis. In response to conditions mimicking the transition to winter, PK appears to be regulated to support metabolic rate depression, the accumulation of polyol cryoprotectants, and the need for extended periods of anaerobic carbohydrate metabolism to allow the animal to survive whole-body freezing.

Adjustments in the control of mitochondrial respiratory capacity to tolerate temperature fluctuations.

As the world's climate changes, life faces an evolving thermal environment. Mitochondrial oxidative phosphorylation (OXPHOS) is critical to ensure sufficient cellular energy production, and it is strongly influenced by temperature. The thermally induced changes to the regulation of specific steps within the OXPHOS process are poorly understood. In our study, we used the eurythermal species of planarian Dugesia tigrina to study the thermal sensitivity of the OXPHOS process at 10, 15, 20, 25 and 30°C. We conducted cold acclimation experiments where we measured the adjustment of specific steps in OXPHOS at two assay temperatures (10 and 20°C) following 4weeks of acclimation under normal (22°C) or low (5°C) temperature conditions. At the low temperature, the contribution of the NADH pathway to the maximal OXPHOS capacity, in a combined pathway (NADH and succinate), was reduced. There was partial compensation by an increased contribution of the succinate pathway. As the temperature decreased, OXPHOS became more limited by the capacity of the phosphorylation system. Acclimation to the low temperature resulted in positive adjustments of the NADH pathway capacity due, at least in part, to an increase in complex I activity. The acclimation also resulted in a better match between OXPHOS and phosphorylation system capacities. Both of these adjustments following acclimation were specific to the low assay temperature. We conclude that there is substantial plasticity in the mitochondrial OXPHOS process following thermal acclimation in D. tigrina, and this probably contributes to the wide thermal range of the species.

Temperature and serine phosphorylation regulate glycerol-3-phosphate dehydrogenase in skeletal muscle of hibernating Richardson’s ground squirrels

Glycerol-3-phosphate dehydrogenase (G3PDH) bridges carbohydrate and lipid metabolism by interconverting glycerol-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). This reversible reaction converts G3P derived from triglyceride hydrolysis to DHAP that can then enter glycolysis or gluconeogenesis and, in the reverse reaction, makes G3P for use in triglyceride biosynthesis. Small hibernating mammals rely almost exclusively on triglyceride reserves as their fuel for energy production during torpor and the recovery of glycerol after lipolysis is an important source of carbohydrate over the nonfeeding winter months. G3PDH (∼37 kDa) was purified from skeletal muscle of euthermic and hibernating Richardson's ground squirrels (Urocitellus richardsonii) using three column chromatography steps. Analysis of enzyme kinetic properties revealed that G3PDH from hibernator muscle had higher affinities for G3P and NAD at low (5 °C) assay temperature compared with high (21 or 37 °C) and a greater stability in the presence of denaturing agents (urea, guanidine hydrochloride) or high temperature (50 °C). Immunoblotting showed that hibernator muscle G3PDH had a higher phosphoserine content than the enzyme from euthermic controls and incubation studies showed that enzyme affinity for G3P changed significantly by stimulating endogenous protein kinases or phosphatases. Overall, this study suggests that the properties of ground squirrel muscle G3PDH are modulated by temperature and post-translational phosphorylation to alter enzyme function under euthermic versus hibernating states.

In Vitro Characterization of Thermostable CAM Rubisco Activase Reveals a Rubisco Interacting Surface Loop.

To maintain metabolic flux through the Calvin-Benson-Bassham cycle in higher plants, dead-end inhibited complexes of Rubisco must constantly be engaged and remodeled by the molecular chaperone Rubisco activase (Rca). In C3 plants, the thermolability of Rca is responsible for the deactivation of Rubisco and reduction of photosynthesis at moderately elevated temperatures. We reasoned that crassulacean acid metabolism (CAM) plants must possess thermostable Rca to support Calvin-Benson-Bassham cycle flux during the day when stomata are closed. A comparative biochemical characterization of rice (Oryza sativa) and Agave tequilana Rca isoforms demonstrated that the CAM Rca isoforms are approximately10°C more thermostable than the C3 isoforms. Agave Rca also possessed a much higher in vitro biochemical activity, even at low assay temperatures. Mixtures of rice and agave Rca form functional hetero-oligomers in vitro, but only the rice isoforms denature at nonpermissive temperatures. The high thermostability and activity of agave Rca mapped to the N-terminal 244 residues. A Glu-217-Gln amino acid substitution was found to confer high Rca activity to rice Rca Further mutational analysis suggested that Glu-217 restricts the flexibility of the α4-β4 surface loop that interacts with Rubisco via Lys-216. CAM plants thus promise to be a source of highly functional, thermostable Rca candidates for thermal fortification of crop photosynthesis. Careful characterization of their properties will likely reveal further protein-protein interaction motifs to enrich our mechanistic model of Rca function.

Temperature-related differences in mitochondrial function among clones of the cladoceran Daphnia pulex

This study assessed the thermal sensitivity of mitochondrial respiration in the small crustacean Daphnia pulex. More specifically, we wanted to determine if clones that inhabit different latitudes and habitats showed differences in the thermal sensitivity of their mitochondrial function. The experimental design included two clones from temperate environments (Fence from Ontario and Hawrelak from Alberta) and two clones from subarctic environments (A24 from Manitoba and K154 from Quebec). The integrated mitochondrial function was measured with high-resolution respirometry following whole-animal permeabilization. Mitochondrial respiration was performed under six different temperatures (10, 15, 20, 25, 30, and 35°C) in the clone Hawrelak and at two temperatures (10 and 20°C) in the three other clones. In the clone Hawrelak, complexes I and II respiration showed higher sensitivity to temperature variation compared to complex IV respiration. Interestingly, the threshold plot showed no excess capacity of complex IV at 20°C in this clone. The clones showed significant divergence in the ability to oxidize the complex I and complex IV substrates relative to the maximal oxidative phoshorylation capacity of mitochondria. More importantly, some of the clonal divergences were only detected under low assay temperatures, pointing toward the importance of this parameter in comparative studies. Future and more complex studies on clones from wider environmental gradients will help to resolve the link between mitochondrial function and adaptations of organisms to particular conditions, principally temperature.

Regulation of 5'-adenosine monophosphate deaminase in the freeze tolerant wood frog, Rana sylvatica

BackgroundThe wood frog, Rana sylvatica, is one of a few vertebrate species that have developed natural freeze tolerance, surviving days or weeks with 65–70% of its total body water frozen in extracellular ice masses. Frozen frogs exhibit no vital signs and their organs must endure multiple stresses, particularly long term anoxia and ischemia. Maintenance of cellular energy supply is critical to viability in the frozen state and in skeletal muscle, AMP deaminase (AMPD) plays a key role in stabilizing cellular energetics. The present study investigated AMPD control in wood frog muscle.ResultsWood frog AMPD was subject to multiple regulatory controls: binding to subcellular structures, protein phosphorylation, and effects of allosteric effectors, cryoprotectants and temperature. The percentage of bound AMPD activity increased from 20 to 35% with the transition to the frozen state. Bound AMPD showed altered kinetic parameters compared with the free enzyme (S0.5 AMP was reduced, Hill coefficient fell to ~1.0) and the transition to the frozen state led to a 3-fold increase in S0.5 AMP of the bound enzyme. AMPD was a target of protein phosphorylation. Bound AMPD from control frogs proved to be a low phosphate form with a low S0.5 AMP and was phosphorylated in incubations that stimulated PKA, PKC, CaMK, or AMPK. Bound AMPD from frozen frogs was a high phosphate form with a high S0.5 AMP that was reduced under incubation conditions that stimulated protein phosphatases. Frog muscle AMPD was activated by Mg·ATP and Mg·ADP and inhibited by Mg·GTP, KCl, NaCl and NH4Cl. The enzyme product, IMP, uniquely inhibited only the bound (phosphorylated) enzyme from muscle of frozen frogs. Activators and inhibitors differentially affected the free versus bound enzyme. S0.5 AMP of bound AMPD was also differentially affected by high versus low assay temperature (25 vs 5°C) and by the presence/absence of the natural cryoprotectant (250 mM glucose) that accumulates during freezing.ConclusionMaintenance of long term viability under the ischemic conditions in frozen muscle requires attention to the control of cellular energetics. Differential regulatory controls on AMPD by mechanisms including binding to muscle proteins, actions allosteric effectors, glucose and temperature effects and reversible phosphorylation adjust enzyme function for an optimal role in controlling cellular adenylate levels in ischemic frozen muscle. Stable modification of AMPD properties via freeze-responsive phosphorylation may contribute both to AMPD control and to coordinating AMPD function with other enzymes of energy metabolism in cold ischemic muscle.

Effects of hibernation on multicatalytic proteinase complex in thirteen-lined ground squirrels, Spermophilus tridecemlineatus

Multicatalytic proteinase complex (MCP) was studied in skeletal muscle of the hibernating ground squirrel, Spermophilus tridecemlineatus. MCP was partially purified using a S-400 gel filtration column and Centricon concentrating devices and assayed fluorometrically using three AMC-labeled substrates. K(m) and V(max) values were determined for each substrate with no significant differences between the enzyme from euthermic versus hibernating animals when assayed at 23 degrees C. However, properties of MCP from euthermic and hibernating ground squirrels were differentially affected by low assay temperature (8-10 degrees C) and also differed from the mouse enzyme, the data indicating that ground squirrel MCP is better suited for low temperature function. MCP preferentially degrades oxidatively-damaged proteins and quantification of protein carbonyl content showed that the level of oxidatively-damaged protein in skeletal muscle decreased by > 75% during hibernation suggesting a continuing role for the MCP in the torpid state.

Protein kinase A: purification and characterization of the enzyme from two cold-hardy goldenrod gall insects

The catalytic subunit of protein kinase A (PKAc) was purified to apparent homogeneity from two species of cold-hardy goldenrod gall insects, Epiblema scudderiana and Eurosta solidaginis. Final specific activity for both enzymes was ∼74.5 nmol of phosphate transferred per minute per milligram protein. Molecular weights were 41 and 40 kDa for E. scudderiana and E. solidaginis PKAc, respectively. K m values at 24°C for the artificial substrate, Kemptide, were 38.1±4.9 and 3.67±0.11 μM for E. scudderiana and E. solidaginis PKAc, respectively, whereas K m Mg-ATP values were 61.1±6.9 and 30.7±4.1 μM. Assay at 4°C lowered the K m for Kemptide of E. scudderiana PKAc by 55% and addition of 1 M glycerol further lowered the K m. Low assay temperature also enhanced holoenzyme dissociation in both species with the K a value for cyclic 3′5′-monophosphate at 4°C lowered to just 13–18% of the value at 24°C. Low temperature did not affect affinity for Mg-ATP or inhibition by PKA inhibitors (PKAi, H7, H89) but increased inhibition by some salts. PKAc from both species showed a break in the Arrhenius relationship at ∼10°C which suggests a conformational change at low temperature; activation energies ( E a) were 2.2–3 fold higher for the lower (<10°C) versus higher (>10°C) range. Addition of naturally occurring polyols, 1 M glycerol or 0.4 M sorbitol, affected E a in some cases. Temperature dependent regulation of holoenzyme dissociation and PKAc kinetic properties may have an role in regulating the enzymes involved in polyol synthesis in cold-hardy insects.

Molecular forms and kinetic properties of phosphoenolpyruvate carboxylase from barnyard grass (Echinochloa crus-galli(L.) Beauv.: Poaceae)

The thermal, kinetic, and electrophoretic properties of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) were analyzed in plants from two ecotypes of barnyard grass (Echinochloa crus-galli (L.) Beauv.: Poaceae) originated from sites of contrasting climates in Quebec (QUE) and Mississippi (MISS). The thermostability, cold inactivation, the apparent energy of activation (Ea), the Michaelis-Menten constant (Km), and Vmax/Kmratios for phosphoenolpyruvate (PEP) and Mg2+were analyzed with desalted Sephadex G-25 crude PEPC extracts, with partially purified PEPC from the polyethylene glycol (PEG) 13% fraction and with purified PEPC obtained after elution from DEAE-Sepharose affinity chromatography. PEPC from illuminated leaves from both ecotypes consisted of one isomorph with the same electrophoretic mobility in polyacrylamide gels, similar molecular masses for the native enzyme (400 kDa) and for each subunit of the tetramer (100 kDa), and a same isoelectric point (pI) of 4.95. The only kinetic property for PEPC for which differences of physiological importance among ecotypes were observed at the three levels of purification was Kmfor PEP for which values for QUE plants were significantly lower at low assay temperatures. Differences among ecotypes for thermostability were only observed in assays with crude and partially purified PEPC extracts, while no differences were found for cold inactivation rates, Km(Mg2+) estimates at any level of purification or for Vmax/Kmratios (PEP or Mg2+) from purified PEPC. Significant differences among the two ecotypes were found for catalytic constant (Kcat) estimates obtained with purified PEPC. However, results show higher catalytic efficiency for PEPC from MISS plants at high assay temperatures but no indication of an improved catalytic efficiency for PEPC from QUE plants at low assay temperatures. The lack of ecotypic differences for most thermal and kinetic properties observed with purified PEPC casts doubts about the evolutionary interpretations of results obtained in previous kinetic comparative analyses, which were based on crude or partially purified enzymatic preparations of PEPC extracted from E. crus-galli plants.Key words: phosphoenolpyruvate carboxylase, enzyme kinetics, thermal adaptation, barnyard grass, electrophoresis, Echinochloa crus-galli.

Effect of low temperatures on the activity of oxygen‐scavenging enzymes in two populations of the C4 grass Echinochloa crus‐galli

To discriminate among possible mechanisms responsible for the differential response to cold temperatures among ecotypes of the C4 grass weed species Echinochloa crus‐galli (L.) Beauv., the specific activities of five oxygen‐scavenging enzymes responsible for the elimination or reduction of free radicals and hydrogen peroxide during cold‐induced photoinhibition were determined in 5‐week‐old plants of two populations of the species collected from sites of contrasting climates, Québec (QUE) and Mississippi (MISS). Enzyme activities were measured at temperatures ranging from 5 to 30°C. The specific activities of ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase were significantly higher in cold‐adapted QUE plants at low assay temperatures than in warm‐adapted MISS plants at the same temperature. The specific activities of superoxide dismutase assayed at 5 and 25°C were similar among plants of the two E. crus‐galli populations. Ascorbate concentrations were not different among plants of the two populations, suggesting that the observed differences in the specific activities of ascorbate peroxidase assayed at 5°C, truly reflect a better capacity of the QUE enzyme to reduce H2O2 to water at temperature conditions associated with the photoinhibitory process. The enhanced specific activity of four of the five oxygen‐scavenging enzymes measured in the cold‐adapted QUE population at low assay temperatures correlates with the syndrome of cold‐adapted features reported for plants of this population in earlier studies.

The effects of temperature and chorionic gonadotropin on the functional properties of lactate dehydrogenase from oocytes of loach Misgurnus fossilis

Acclimation of loach to relatively low and high temperatures gives rise to changes in the properties of LDH from oocytes. The minimum of apparent K m values for pyruvate of LDH from immature oocytes of fish acclimated to low temperature was registered at low assay temperatures, whereas adaptation to high temperatures leads to the enzyme showing a minimum at high temperatures. In thermal acclimation of fish the K m minimum value drifts during 15 days. During oocyte maturation, the K m minimum drifts during 40 hr. The differences in the kinetic features of LDH from oocytes both at thermal acclimation and during oocyte maturation are not associated with the appearance of new isozymes.

Thermal Kinetics of Glutathione Reductase and their Relation to Thermotolerance in Diverse Cultivars of Maize

The characteristics of glutathione reductase from a number of maize cultivars with contrasting thermotolerance have been investigated. The apparent Km (Michaelis constant) for oxidised glutathione (GSSG) was measured between 10 and 45°C at constant pH. The enzyme from highland cultivars adapted to cool environments had a slightly lower apparent Km for GSSG than that from lowland tropical cultivars at low assay temperatures. Similarly the enzyme from lowland tropical cultivars had a lower apparent Km for GSSG at high assay temperatures. However these effects were small and regression lines plotted through the data were not significantly different in slope or intercept. There was a strong correlation (r = 0·939) between apparent Km and Vmax (maximum initial velocity) as assay temperature was varied. The interpretation of apparent Km/temperature relationships is discussed with hypothetical examples of the effects of temperature on enzyme activity/substrate concentration plots. It is demonstrated that an increase in apparent Km at higher assay temperatures need not necessarily reflect any temperature-dependent impairment of enzyme function. The apparent Km for GSSG of glutathione reductase from maize increased over four-fold when the temperature was raised from 10 to 45°C, but it is concluded that in vivo rates of reaction are likely to be increased rather than decreased by this same change in temperature. Glutathione reductase would thus appear to be equally well adapted to function at all these temperatures. This suggests that the potential of enzyme thermal kinetics to predict thermotolerance may be limited.

Effects of soil temperature and nitrogen status on kinetics of 15 NO3 - uptake by roots of field-grown Agropyron desertorum (Fisch. ex Link) Schult.

Plant NO8 - acquisition is largely determined by root uptake capacity. Although root uptake capacity has been shown to be sensitive to both root temperature and previous nitrogen (N) supply in hydroponic systems, the uptake capacity response to similar environmental factors under field conditions has not been investigated. Using 15 NO3 - , root uptake capacities were determined in excised roots of Agropyron desertorum (Fisch. ex Link) Schult grown in the field at two soil temperatures and two N fertilization treatments. Variation in soil and root temperatures was achieved by application of clear plastic film or insulating mulch to the soil immediately around the target plants. Uptake rates were measured at six different assay solution concentrations (from 1 to 1000 μM external 15 NO3 - concentration range). Two months after the imposition of soil N and temperature treatments, a biphasic transport system (a high-affinity) saturable phase and a low-affinity transport phase) was apparent in low N-treated plants. Nitrate uptake capacity in the low-concentration range (1-500μM) was significantly reduced in N-fertilized plants compared with unfertilized control plants and the effect was more pronounced at high (27 °C) than low (17 °C) soil and assay temperatures. Furthermore, high soil N status inhibited the expression of a low-affinity NO3 - transport system which was clearly apparent at external NO3 - concentration ranges between 500 and 1000 mM in plants grown at low soil N. Prior soil N and temperature history may ultimately determine root ability to exploit NO3 - flushes which can result from changes in soil environmental conditions.

Temperature mediated processes in teleost immunity: Differential abilities of channel catfish T and B lymphocytes to cap membrane antigen

1. 1. The effect of both in vivo acclimation temperature and in vitro assay temperatures on channel catfish T and B lymphocyte membrane antigen (mAg) capping were investigated to determine if capping might be the temperature sensitive step involved in the low temperature immunosuppression of channel catfish T cell responses. 2. 2. Flow cytometry was used to monitor the kinetics of capping induced by a mouse monoclonal antibody (mAb 11G3) specific for a common antigenic determinant present on channel catfish T and B cells. Results indicated that the kinetics of mAg capping were dependent on in vitro assay and in vivo acclimation temperatures and the length of time of in vivo acclimation. 3. 3. T cells from fish appropriately acclimated to 27°C cap mAg more efficiently at low assay temperatures than do B cells. 4. 4. Activation energies were 32 and 47 kcal/mol for B and T cells, respectively, from fish acclimated to 17°C for 3 weeks, but were significantly lower (14 and 22 kcal/mol, respectively) after acclimation for 5 weeks. 5. 5. In summary, it appears that after appropriate in vivo acclimation, channel catfish T cells are better able to cap mAg at low assay temperatures than are B cells. These results suggest that mAg capping is not the low temperature sensitive step involved in T cell immunosuppression in channel catfish.

Adaptation and Acclimation of Higher Plants at the Enzyme Level: Kinetic Properties of NAD Malate Dehydrogenase in Three Species of Viola

SUMMARY (1) Kinetic properties of NAD malate dehydrogenase (MDH) were investigated in three species of Viola: V. blanda, V.fimbriatula and V. sororia. Plants from three clones of each species, collected over the latitudinal range of each species in eastern North America, were acclimated under controlled conditions at three different thermoperiods: 7/15 IC, 15/22 IC and 22/28 OC. (2) The apparent Michaelis constant (Km.) for oxalacetic acid increased with rising assay temperatures, and cold acclimation reduced Km values at low assay temperatures in plants of the cold-adapted Quebec clones of V. blanda and V. sororia acclimated at 7/15 OC and 15/22 OC. (3) The Vm.ax/Km ratios for MDH were higher at assay temperatures closely related to growth conditions although this trend was expressed differentially depending on the species or the clone analysed. (4) Differential trends were observed in between-species comparisons and can be correlated with the microclimate in the habitat of the species.

Katalytische Aktivität der Serumlipase im kontinuierlichen titrimetrischen Test in Abhängigkeit von der Temperatur

The catalytic activity of the pancreatic lipase in serum was measured between 20 degrees C and 37 degrees C by the continuous titrimetric assay. As the temperature was increased, the time course of substrate hydrolysis showed an increasing tendency to become non-linear. The degree of non-linearity and its time of onset depended on the sample, the volume of serum in the assay, and the composition of the triglyceride substrate mixture. Twenty seven series of investigations between 20 degrees C and 32 degrees C (maximal) showed a Q10 value of 1.45 (s = 0.03) and an activation energy of mu = 27.42 +/- 0.565 kJ/mol (6550 +/- 135 cal/mol; mean +/- s). At low assay temperature, the addition of colipase caused a slight deterioration of the reaction kinetics, whereas at higher temperatures it caused a marked improvement in the time course of substrate hydrolysis. We were unable, however, to identify a fixed colipase concentration that would promote a linear reaction irrespective of sample origin and volume. These results show that the assay should be performed at 25 degrees C, and that the test conditions described in 1969 and 1982 are valid without modification. If these conditions are observed, there is no experimental basis for the addition of colipase of the quality commercially available at present.

Effect of temperature on the uptake of glucose, gluconate, and 2-ketogluconate by Pseudomonas fluorescens.

The effect of temperature on the uptake systems involved with glucose catabolism was studied in a psychrotrophic strain of Pseudomonas fluorescens. The Km values for glucose and gluconate uptake were approximately 1 and 30 μM respectively whether uptake was assayed at 30 or 5 °C. The Km for 2-ketogluconate (2-KG) uptake was approximately 40 μM assayed at 30 °C and 20 μM assayed at 5 °C. Little or no induction of 2-KG uptake was detected in cells grown at 30 °C with glucose or gluconate. High induced levels of 2-KG uptake were observed in cells grown at 5 °C. The induced level of glucose uptake in cells grown at 5 °C with glucose was only one-half the induced level in cells grown at 30 °C with glucose (when activities were compared at the same assay temperature). The effect of low-assay temperature on the activities of these uptake systems was most pronounced for gluconate. When the assay temperature was decreased from 30 to 5 °C, uptake activity decreased 12-fold, 6-fold, and 5-fold for gluconate, glucose, and 2-KG, respectively. Because of the differential effect of temperature on the induction of glucose uptake and on the activity of glucose, gluconate, and 2-KG uptake, the major uptake activity measured in cells grown at 5 °C with glucose or gluconate was through 2-KG. Glucose and 2-KG appeared to inhibit significantly gluconate uptake and (or) catabolism which might promote the continued oxidation of gluconate to 2-KG at low growth temperatures. The results confirm the lack of glucose or gluconate catabolism by the direct oxidative non-phosphorylated pathway through 2-KG at higher growth temperatures and the major function of this pathway at lower growth temperatures.