Uptake and phosphorylation of glucose and fructose in Daucus carota cell suspensions are differently regulated

Cell suspensions of Daucus carota L. were grown in batch culture on 50 mM sucrose, 100 mM glucose or 100 mM fructose. Sucrose was rapidly converted extra-cellularly into equimolar amounts of glucose and fructose, and glucose was then taken up preferentially. This impaired uptake of fructose could partially be explained by the eight-fold lower affinity of the hexose carrier in the plasmamembrane for fructose compared to glucose. However, cells grown on fructose as the sole carbon source showed a shorter lag phase and showed more biomass production compared to glucose-grown cells, indicating that conversion of glucose and fructose were also differently regulated. Ninety-five % of the glucose phosphorylating activity was membrane-associated and most probably confined to mitochondria; therefore, it might be present in a respiratory ‘compartment’ making glucose a better substrate for respiration than fructose. The soluble fraction contained the majority of the fructokinase activity. This activity was hypothesized to be more or less randomly distributed through the cytosol; in this soluble ‘compartment’ a pool of fructose-6-phosphate is formed. Concomitantly, via glucose-6-phosphate (G-6-P) and glucose-1-phosphate (G-1-P), it is converted into UDPG-glucose, resulting in structural cell components. The observed transient obstruction of the conversion of G-1-P into UDP-glucose in fructose-grown cells, leading to G-1-P accumulation, might be a result of both an altered equilibrium maintained by phosphoglucomutase, interconverting G-6-P and G-1-P and low levels of nucleotide triphosphates. Low nucleotide triphosphate production, connected with a low initial respiration rate, might be caused by the ten-fold lower affinity of the membrane-associated phosphorylating enzymes for fructose compared to glucose. Our results were taken to indicate that two separate pools of glycolytic intermediates exist in D. carota cells: one distributed throughout the cytosol and one surrounding the mitochondria.     

Glycolytic enzymes associate dynamically with mitochondria in response to respiratory demand and support substrate channeling

In Arabidopsis thaliana, enzymes of glycolysis are present on the surface of mitochondria and free in the cytosol. The functional significance of this dual localization has now been established by demonstrating that the extent of mitochondrial association is dependent on respiration rate in both Arabidopsis cells and potato (Solanum tuberosum) tubers. Thus, inhibition of respiration with KCN led to a proportional decrease in the degree of association, whereas stimulation of respiration by uncoupling, tissue ageing, or overexpression of invertase led to increased mitochondrial association. In all treatments, the total activity of the glycolytic enzymes in the cell was unaltered, indicating that the existing pools of each enzyme repartitioned between the cytosol and the mitochondria. Isotope dilution experiments on isolated mitochondria, using (13)C nuclear magnetic resonance spectroscopy to monitor the impact of unlabeled glycolytic intermediates on the production of downstream intermediates derived from (13)C-labeled precursors, provided direct evidence for the occurrence of variable levels of substrate channeling. Pull-down experiments suggest that interaction with the outer mitochondrial membrane protein, VDAC, anchors glycolytic enzymes to the mitochondrial surface. It appears that glycolytic enzymes associate dynamically with mitochondria to support respiration and that substrate channeling restricts the use of intermediates by competing metabolic pathways.     

Isolation of Alcaligenes sp. strain L6 at low oxygen concentrations and degradation of 3-chlorobenzoate via a pathway not involving (chloro)catechols.

Isolations of 3-chlorobenzoate (3CBA)-degrading aerobic bacteria under reduced O2 partial pressures yielded organisms which metabolized 3CBA via the gentisate or the protocatechuate pathway rather than via the catechol route. The 3CBA metabolism of one of these isolates, L6, which was identified as an Alcaligenes species, was studied in more detail. Resting-cell suspensions of L6 pregrown on 3CBA oxidized all known aromatic intermediates of both the gentisate and the protocatechuate pathways. Neither growth on nor respiration of catechol could be detected. Chloride production from 3CBA by L6 was strictly oxygen dependent. Cell-free extracts of 3CBA-grown L6 cells exhibited no catechol dioxygenase activity but possessed protocatechuate 3,4-dioxygenase, gentisate dioxygenase, and maleylpyruvate isomerase activities instead. In continuous culture with 3CBA as the sole growth substrate, strain L6 demonstrated an increased oxygen affinity with decreasing steady-state oxygen concentrations.     

Aerobic Fermentation and the Depletion of the Amino Acid Pool in Yeast Cells

The amino acid pool of yeast cells, Saccharomyces cerevisiae, incubated with galactose remains at a constant level for 100 minutes. This is 30 minutes beyond the time at which the oxidative phase of the induced-enzyme formation begins. Washed yeast cells, the pools of which have been depleted 60 per cent by incubation with glucose, do not replenish their pools as do washed cells incubated without a substrate. These facts indicate that the induced enzymes are formed at least partially from pool-replenishing amino acids. The time of onset of pool depletion is the time at which the aerobic fermentation phase of induced-enzyme formation begins for cells incubated with galactose. With 0.1 per cent galactose the respiratory phase begins at 100 minutes but no aerobic fermentation nor pool depletion occurs. The rates of respiration and aerobic fermentation are constant for four glucose concentrations from 0.1 to 1.0 per cent. The amount of aerobicfermentation is proportional to the initial concentration of glucose. Amino acid pool depletion occurs for all concentrations but depletion ceases and is followed by pool replenishment after aerobic fermentation is complete. Ultraviolet radiations, which delay the appearance of the respiratory phase of induced-enzyme formation, completely eliminate both the appearance of aerobic fermentation and pool depletion. The results indicate an intimate association between aerobic fermentation and amino acid pool depletion.     

Plug flow simulating and completely mixed reactors with a premixing tank,in the control of filamentous bulking

Summary A sequence of a substrate rich and substrate starvation phase (feast and famine strategy) is created in both compartmentalized reactors and reactors with premixing tanks. This condition is known to be essential in the control of bulking. With a synthetic waste water containing glucose a reactor with 12 compartments was effective in the control of filamentous bulking. With a dairy industrial waste water, containing a slowly biodegradable COD-fraction, this reactor could not suppress the growth of filamentous bacteria. With dairy and brewery waste water, reactors with premixing tanks were used to create a more pronounced exogenous (substrate rich) phase. Using three or more premixing tanks filamentous bulking could be controlled. During the exogenous phase the floc-forming microorganisms, having a higher substrate uptake rate, can take up the largest amount of substrate and can survive better during the endogenous phase.Substrate concentration and respiration rate profiles were studied and the concentration of the reserve materials was monitored.For each type of waste water the sludge loading and the fraction of readily available COD will determine the system's design.     

Continuous cultures limited by a gaseous substrate: Development of a simple, unstructured mathematical model and experimental verification with Methanobacterium thermoautotrophicum

This article presents a simple, unstructured mathematical model describing microbial growth in continuous culture limited by a gaseous substrate. The model predicts constant gas conversion rates and a decreasing biomass concentration with increasing dilution rate. It has been found that the parameters influencing growth are primarily the gas transfer rate and the dilution rate. Furthermore, it is shown that, for correct simulation of growth, the influence of gaseous substrate consumption on the effective gas flow through the system has to be taken into account.Continuous cultures of Methanobacterium thermoautotrophicum were performed at three different gassing rates. In addition to the measurement of the rates of biomass production, product formation, and substrate consumption, microbial heat dissipation was assessed using a reaction calorimeter. For the on-line measurement of the concentration of the growth-limiting substrate, H(2), a specially developed probe has been used. Experimental data from continuous cultures were in good agreement with the model simulations. An increase in gassing rate enhanced gaseous substrate consumption and methane production rates. However, the biomass yield as well as the specific conversion rates remained constant, irrespective of the gassing rate. It was found that growth performance in continuous culture limited by a gaseous substrate is substantially different from "classic" continuous culture in which the limiting substrate is provided by the liquid feed. In this report, the differences between both continuous culture systems are discussed.     

The growth of Saccharomyces cerevisiae CBS 426 on mixtures of glucose and succinic acid: A model

Saccharomyces cerevisiae CBS 426 was grown in continuous culture in a defined medium with a mixture of glucose and succinic acid as the carbon source. Growth on succinic acid was possible after long adaptation periods. The flows of glucose, succinic acid, oxygen, carbon dioxide, and biomass to and from the system were measured. It proved necessary to expand our previous model to accommodate the active transport of succinic acid by the cell. The values found for the efficiency of the oxidative phosphorylation (P/O) and the amount of ATP needed for production of biomass from monomers gave the same values as found for substrate mixtures taken up passively.     

Influence of Temperature on Glucose Utilization by Pseudomonas fluorescens

The influence of temperature on the conversion of glucose into cell material and into energy for maintenance was determined for Pseudomonas fluorescens by a steady-state turbidity method and by a substrate utilization method. Conversion of glucose into cell material was measured as yield; conversion of glucose into energy for maintenance was measured as specific maintenance, the minimum dilution rate in continuous culture below which a steady state is not possible. The values obtained by the two methods were nearly identical; with both, the yield and specific maintenance decreased with decreasing temperature. The specific maintenance consumption rate (milligrams of glucose taken up per milligram of cell dry weight per hour at zero growth) was also calculated by the substrate utilization method and found to decrease with decreasing temperature. However, the amount of glucose consumed per generation for maintenance increased with decreasing temperature. This increased glucose consumption for maintenance may provide a partial explanation for the decrease in yield at low temperatures. Small amounts of glucose were also converted into pigment at all temperatures tested, with the greatest amount formed at 20 C.     

Effects of varying the carbon source limiting growth on yield and maintenance characteristics of Escherichia coli in continuous culture.

The magnitudes of Yo (grams [dry weight] formed per gram of atom O) and mo, the maintenance respiration (milligram-atoms of O per gram [dry weight] per hour), of Escherichia coli B have been determined by growing the organism in aerobic continuous culture limited by a number of different substrates. The value found were as follows: glucose--tyo = 12.5, mo = 0.9; glucose plus 2.7 mM cyclic adenosine 3',5'-monophosphate (cAMP)--Yo = 31.2, mo = 9.3; galactose--Yo = 13.2, mo = 1.8; mannitol--Yo = 20.1, mo = 6.1; L-glutamate--Yo = 25.5, mo = 17.7; glycerol--Yo = 14.9, mo = 10.0; succinate--Yo = 11.2, mo = 12.1; and acetate--Yo = 14.7, mo = 25.4. During growth in anaerobic continuous culture with limiting glucose YATP was found to be 10.3 g (dry weight)/mol of adenosine 5'-triphosphate (ATP) and m ATP was 18.9 mmol of ATP/g (dry weight) per h. The aerobic growth yields of cells growing on glucose, glucose plus cAMP, mannitol, and glutamate were consistent with the hypothesis that carbohydrates partially repress oxidative phosphorylation, but the yields of cells growing on glycerol, succinate, acetate, and galactose were all lower than expected. We conclude that, like the efficiency of oxidative phosphorylation, both the maintenance respiration and the amount of ATP necessary to serve maintenance processes are determined by the identity of the growth substrates. Yields smaller than expected may be explained by the absence of respiratory control exerted by phosphate acceptors.     

Galactosamine inhibition of protein synthesis in Bacteroides thetaiotaomicron

Galactosamine does not support growth of Bacteroides thetaiotaomicron. Despite this, galactosamine was more effective than utilizable carbohydrates such as glucose in preventing synthesis of the inducible enzymes alpha-glucosidase and chondroitin lyase. Galactosamine also stopped overall protein synthesis. By contrast glucose and other utilizable carbohydrates increased the rate of protein synthesis. Addition of glucose to bacteria which had been treated with galactosamine restored the ability of the bacteria to synthesize protein and to produce inducible enzymes. Moreover, when B. thetaiotaomicron was incubated with [1-14C]galactosamine for 30 min at 37 degrees C, about one-third of the label which was taken up by the cells comigrated with glucosamine-6-phosphate on a thin-layer chromatogram. Thus galactosamine appears to be phosphorylated by the bacteria. After 2 h incubation of the bacteria with [1-14C]galactosamine, there was a significant increase in the amount of label which could be extracted from acidified extracellular fluid with diethyl ether. This indicates that galactosamine can be metabolized to the level of volatile fatty acids. The rate of uptake of galactosamine and the amount of labeled fatty acids produced from galactosamine were both much lower than the values obtained when glucosamine was the substrate. Thus, although some metabolism of galactosamine occurs, the rate is apparently too slow to enable galactosamine to support growth of B. thetaiotaomicron.     

Effects of growth temperature on yield and maintenance during glucose-limited continuous culture of Escherichia coli.

The effects of growth temperature on the aerobic growth yield with respect to oxygen consumption (Y0-grams [dry weight] per gram-atom of O) and the rate of maintenance respiration (m0-milligram-atoms of O/gram [dry weight] per hour) are reported for Escherichia coli B cultivated continuously in the presence of oxygen with limiting glucose. During anaerobic continuous culture, YATP(max) (grams [dry weight] per mole of ATP corrected for maintenance) increases from 10.3 to 12.7 as the growth temperature is lowered from 37 to 25 C. Over this same range, Y0(max) (Y0 corrected for maintenance respiration) rises from 12.5 to 28.8 and remains at the higher value down to 17.5 C. From 37 to 32 C, m0 increases from 0.9 to 4.4 but then falls to 1.5 as the temperature is lowered to 17.5 C. The value of m0 sharply rises some 13-fold as the temperature is raised to 42 C without a significant change in the value of Y0(max). Changes of Y0(max) are consistent with a temperature-sensitive doubling of the efficiency of oxidative phosphorylation, but the reasons for the changes of the rate of maintenance respiration are not known.     

Monitoring hybridoma metabolism in continuous suspension culture at the intracellular level

A model mouse hybridoma cell line was grown in continuous culture experiments in a serum-free low-protein lipid-free medium. The steady-state responses of cell numbers, extra- and intracellular metabolite concentrations, substrate and (by) product consumption/production rates, and yield coefficients were investigated as a function of step changes in the glutamine concentration of the feed medium. In addition to the commonly performed analysis of metabolites in culture supernatants, we prepared perchloric acid extracts of cells and determined the amount and the composition of intracellular amino acids and organic acids. Significant differences were found with respect to intracellular metabolite pools for cells growing at nearly identical specific growth rates. To our knowledge this is the first time that data on the intracellular concentrations (pools) of amino acids and Krebs cycle intermediates are reported in the literature that were obtained under carefully defined culture conditions such as those attained in continuous culture experiments.     

Role of Alcoholic Intermediates in Formation of Isomeric Ketones From n-Hexadecane by a Soil Arthrobacter

A soil Arthrobacter species isolated from an Oregon soil was capable of transforming n-hexadecane to a series of ketonic products, the 2-,3-, and 4-hexadecanones, with evidence for accumulation of 2- and 3-hexadecanols as oxidative intermediates when yeast extract or peptone was used as a growth substrate. The accumulation and participation of internal alcohols in this type of hydrocarbon transformation has not been previously reported. In the absence of yeast extract or peptone, growth from low-level inocula was not observed when n-hexadecane or two oxidation products, 2-hexadecanol and 3-hexadecanone, were used as substrates. However, washed resting cell suspensions of the organism transformed 2-hexadecanol, or a mixture of 2-,3-, and 4-hexadecanols, to the corresponding ketones without lag, indicating the possible constitutive nature of the alcohol dehydrogenase enzyme(s) carrying out this reaction. The addition of glucose to these resting cells stimulated transformation of n-hexadecane to alcoholic and ketonic oxidation products. Formation of isomeric internal alcohols appears to be a limiting step in ketone formation by this Arthrobacter isolate.     

Application of a competition model to the growth of Streptococcus mutans and Streptococcus sanguis in binary continuous culture

Streptococcus mutans 6715-15 and Streptococcus sanguis 10558 were grown together in continuous culture with glucose as the limiting carbon source. The relationship of growth rate to substrate concentration was determined for pure cultures of each organism in continuous and batch cultures. A model based on competition for a growth-limiting substrate (glucose) was used to predict the proportions of each organism when grown in binary cultures. The results indicate that interactions other than competition for glucose carbon exist between S. mutans and S. sanguis grown under these conditions.     

Application of a competition model to the growth of Streptococcus mutans and Streptococcus sanguis in binary continuous culture.

Streptococcus mutans 6715-15 and Streptococcus sanguis 10558 were grown together in continuous culture with glucose as the limiting carbon source. The relationship of growth rate to substrate concentration was determined for pure cultures of each organism in continuous and batch cultures. A model based on competition for a growth-limiting substrate (glucose) was used to predict the proportions of each organism when grown in binary cultures. The results indicate that interactions other than competition for glucose carbon exist between S. mutans and S. sanguis grown under these conditions.     

The effects of substrate composition, quantity, and diversity on microbial activity

Variation in organic matter inputs caused by differences in plant community composition has been shown to affect microbial activity, although the mechanisms controlling these effects are not entirely understood. In this study we determine the effects of variation in substrate composition, quantity, and diversity on soil extracellular enzyme activity and respiration in laboratory microcosms. Microbial respiration responded predictably to substrate composition and quantity and was maximized by the addition of labile substrates and greater substrate quantity. However, there was no effect of substrate diversity on respiration. Substrate composition significantly affected enzyme activity. Phosphatase activity was maximized with addition of C and N together, supporting the common notion that addition of limiting resources increases investment in enzymes to acquire other limiting nutrients. Chitinase activity was maximized with the addition of chitin, suggesting that some enzymes may be stimulated by the addition of the substrate they degrade. In contrast, activities of glucosidase and peptidase were maximized by the addition of the products of these enzymes, glucose and alanine, respectively, for reasons that are unclear. Substrate diversity and quantity also stimulated enzyme activity for three and four of the six enzymes assayed, respectively. We found evidence of complementary (i.e., non-additive) effects of additions of different substrates on activity for three of the six enzymes assayed; for the remaining enzymes, effects of adding a greater diversity of substrates appeared to arise from the substrate-specific effects of those substrates included in the high-diversity treatment. Finally, in a comparison of measures of microbial respiration and enzyme activity, we found that labile C and nutrient-acquiring enzymes, not those involved in the degradation of recalcitrant compounds, were the best predictors of respiration rates. These results suggest that while composition, quantity, and diversity of inputs to microbial communities all affect microbial enzyme activity, the mechanisms controlling these relationships are unique for each particular enzyme.     

Characterization of 3-chlorobenzoate degrading aerobic bacteria isolated under various environmental conditions

The rates of bacterial growth in nature are often restricted by low concentrations of oxygen or carbon substrates. In the present study the metabolic properties of 24 isolates that had been isolated using various concentrations of 3-chlorobenzoate, benzoate and oxygen as well as using continuous culture at high and low growth rates were determined to investigate the effects of these parameters on the metabolism of monoaromatic compounds. Bacteria were enriched from different sampling sites and subsequently isolated. In batch culture this was done both under low oxygen (2% O(2)) and air-saturated concentrations. Chemostat enrichments were performed under either oxygen or 3-chlorobenzoate limiting conditions. Bacteria metabolizing aromatics with gentisate or protocatechuate as intermediates (gp bacteria) as well as bacteria metabolizing aromatic compounds via catechols (cat bacteria) were isolated from batch cultures when either benzoate or 3CBA were used as C sources, regardless of the enrichment conditions applied. In contrast, enrichments performed in chemostats at low dilution rates resulted in gp-type organisms only, whereas at high dilution rates cat-type organisms were enriched, irrespective of the oxygen and 3-chlorobenzoate concentration used during enrichment. It is noteworthy that the gp-type of bacteria possessed relatively low μ(max) values on 3CBA and benzoate along with relatively high substrate and oxygen affinities for these compounds. This is in contrast with cat-type of bacteria, which seemed to be characterized by high maximum specific growth rates on the aromatic substrates and relatively high apparent half saturation constants. In contrast, bacteria degrading chlorobenzoate via gentisate or protocatechuate may possibly be better adapted to conditions leading to growth at reduced rates such as low oxygen and low substrate concentrations.     

Cyanide-resistant respiration in Torulopsis candida

The effect of cyanide and the inhibitors of cyanide-resistant oxidase--hydroxamic acids on endogenous respiration and oxidation of a number of substrates by Torulopsis candida resting cells taken at different stages of growth on glucose and hexadecane was studied and made it possible to arrive at the following conclusions. 1. The effect of cyanide on endogenous respiration of T. candida differs during its growth on glucose and hexadecane. On hexadecane, irrespective of the growth phage, cyanide inhibits endogenous respiration by 70--75%. On glucose, cyanide inhibits endogenous respiration not more than by 35% and only at the exponential growth phase whereas it stimulates endogenous respiration in the course of other growth stages. 2. The effect of cyanide on respiration of the resting cells of T. candida which oxidize glucose, hexadecane, primary alcohols and tetradecanoic acid hardly depends on the growth stage. It is determined mainly by the nature of a substrate to be oxidized. 3. Hydroxamic acid have no effect on the cell respiration in the absence of cyanide. However, in its presence, they entirely inhibit both endogenous respiration and oxidation of the aforementioned substrates. 4. Under almost all above experimental conditions, the sensitivity of cell respiration to cyanide changes only slightly at different stages of growth on either glucose or hexadecane. This feature markedly distinguishes T. candida among other cyanide-resistant yeasts.     

Induction of cytochrome formation and stimulation of oxidative dissimilation by hemin inStreptococcus lactis andLeuconostoc mesenteroides

Aerobic glucose dissimilation of washed cells ofStreptococcus lactis grown in peptone-glucose-yeast extract medium is characterized by the formation of large amounts of lactic acid, a small amount of acetic acid, and traces of acetoin: a corresponding amount of oxygen is taken up. Aerobic metabolism by washed cells ofS. lactis andLeuconostoc mesenteroides is far more oxidative when the cells have been grown on peptone-glucose-yeast extract agar supplied with 10 ppm of hemin than when they have been grown in the absence of hemin. In the former case respiration is strongly inhibited by KCN and only slightly by bis(tributylgermanium) oxide, (Bu₃Ge)₂O. Respiration of cells grown without hemin, on the other hand, is strongly inhibited by (Bu₃Ge)₂O but only moderately by KCN. In cells grown in the presence of hemin, spectra of ana ₂- andb-type cytochrome were recognized but not in cells grown without hemin. The NADH-oxidase activity of such cells is not affected by KCN.Our results strongly suggest that by growth in the presence of hemin a cytochrome-mediated respiration system is induced which replaces, in part, the NADH-oxidase-mediated respiration. Whereas the latter is sensitive to (Bu₃Ge)₂O, the former apparently is little or not. However it is quite sensitive to KCN.When hemin is added to washed cells ofS. lactis grown without hemin the rate of oxygen uptake increases immediately though no cytochromes are present and respiration remains sensitive to (Bu₃Ge)₂O. Possibly hemin stimulates the NADH-oxidase activity of these cells.The author gratefully acknowledges the skilful technical assistance of Mrs. A. J. M. Dekkers-van der Mark and the able performance of gas chromatographic determinations by Miss E. Ch. Th. Gevers and Mrs. G. G. Versluisde Haan.