Comparative physiology of two protozoan parasites, Leishmania donovani and Trypanosoma brucei, grown in chemostats.

Cultures of the insect stage of the protozoan parasites Leishmania donovani and Trypanosoma brucei were grown in chemostats with glucose as the growth rate-limiting substrate. L. donovani has a maximum specific growth rate (mu max) of 1.96 day-1 and a Ks for glucose of 0.1 mM; the mu max of T. brucei is 1.06 day-1 and the Ks is 0.06 mM. At each steady state (specific growth rate, mu, equals D, the dilution rate), the following parameters were measured: external glucose concentration (Glcout), cell density, dry weight, protein, internal glucose concentration (Glcin), cellular ATP level, and hexokinase activity. L. donovani shows a relationship between mu and yield that allows an estimation of the maintenance requirement (ms) and the yield per mole of ATP (YATP). Both the ms and the YATP are on the higher margin of the range found for prokaryotes grown on glucose in a complex medium. L. donovani maintains the Glcin at a constant level of about 50 mM as long as it is not energy depleted. T. brucei has a decreasing yield with increasing mu, suggesting that it oxidizes its substrate to a lesser extent at higher growth rates. Glucose is not concentrated internally but is taken up by facilitated diffusion, while phosphorylation by hexokinase is probably the rate-limiting step for glucose metabolism. The Ks is constant as long as glucose is the rate-limiting substrate. The results of this study demonstrate that L. donovani and T. brucei have widely different metabolic strategies for dealing with varying external conditions, which reflect the conditions they are likely to encounter in their respective insect hosts.     

Yield and maintenance relations of yeast growth in the chemostat at superoptimal temperatures

A model is proposed that accounts for the decreases in yield which occur in chemostat cultures of mesophilic yeasts at superoptimal growth temperatures. Two yield depressing effects were identified, one due to increased maintenance requirements by the viable fraction of the population, the other due to energy substrate dissipation by the nonviable fraction. The two effects are functions of the dilution rate, as is the fraction of nonviable cells. Experimental results were obtained on the yield, maintenance, and dissipation of energy substrate in a glucose-limited chemostat culture of a respiration-deficient mutant of Saccharomyces cerevisiae at 39°C. The rates of glucose utilization for maintenance and for dissipation constituted, respectively, 33–28% and 15–9% of the total glucose utilization rate over the range of dilution rates tested (0.038–0.064 hr^?1), while the yield varied over this range from 0.066–0.085 g of biomass (dry wt) per gram of glucose.     

Inactivation of membrane transport in Escherichia coli by near-ultraviolet light.

Evidence is presented that near-ultraviolet (near-UV) light can alter galactoside transport in Escherichia coli in several independent ways. It can inactivate the permease system per se, it can interfere with metabolic energy production or transfer, and it can cause an increase in the generalized permeability of the membrane. Earlier publications suggested that near-UV destroys cofactors needed for electron transport and thus places a limitation on energy reserves. In agreement, we found that the active accumulation of [14C]thiomethyl-beta-D-galactopyranoside is decreased after irradiation by a larger factor than that due to action directly on the permease system. The effect on the latter was measured by the decrease in the rate of o-nitrophenyl-beta-D-galactopyranoside (ONPG) transport. As evidence that energy supplies for this "downhill" process did not become rate limiting after irradiation, we found that carbonylcyanide-m-chlorophenyl-hydrazone did not stimulate ONPG transport of irradiated cells. Cells genetically deficient in functional permease or cells treated with formaldehyde still transport ONPG passively, although at much lower rates. With the use of such cells, it was found that high fluences (doses) made the cells leaky. Further evidence that the permease system and the metabolic energy system can be inactivated independently is also presented. It is shown that a photoproduct from the irradiation of chloramphenicol inactivates the permease system much more efficiently than the energy system. In addition, it is shown that thio-beta-D-digalactopyranoside protects the permease system, but not the energy system, both against direct inactivation by near-UV and against photosensitized inactivation in the presence of chloramphenicol.     

Cultivation of Saccharomyces cerevisiae in continuous culture

Summary Glucose limited growth of a respiratory deficient mutant of Saccharomyces cerevisiae was studied in continuous culture under steady state conditions. The maximal growth rate, the Michaelis constant, the cell yield, the maintenance coefficient and the ethanol yield of the growing cell population were determined. The steady state concentrations of cells, glucose and ethanol were measured as functions of the dilution rate and compared with theoretical predictions. A far-reaching agreement between theory and experiment was observed. The decrease of the cell yield in the range of low dilution rates is well explained by introducing the concept of maintenance energy in the general theory of continuous cultures. A deviation of the cell yield from the predicted values, which has been found in the range of high dilution rates, is discussed.     

Energetics and kinetics of maltose transport in Saccharomyces cerevisiae: a continuous culture study.

In Saccharomyces cerevisiae, maltose is transported by a proton symport mechanism, whereas glucose transport occurs via facilitated diffusion. The energy requirement for maltose transport was evaluated with a metabolic model based on an experimental value of YATP for growth on glucose and an ATP requirement for maltose transport of 1 mol.mol-1. The predictions of the model were verified experimentally with anaerobic, sugar-limited chemostat cultures growing on a range of maltose-glucose mixtures at a fixed dilution rate of 0.1 h-1. The biomass yield (grams of cells.gram of sugar-1) decreased linearly with increasing amounts of maltose in the mixture. The yield was 25% lower during growth on maltose than during that on glucose, in agreement with the model predictions. During sugar-limited growth, the residual concentrations of maltose and glucose in the culture increased in proportion to their relative concentrations in the medium feed. From the residual maltose concentration, the in situ rates of maltose consumption by cultures, and the Km of the maltose carrier for maltose, it was calculated that the amount of this carrier was proportional to the in situ maltose consumption rate. This was also found for the amount of intracellular maltose. These two maltose-specific enzymes therefore exert high control over the maltose flux in S. cerevisiae in anaerobic, sugar-limited, steady-state cultures.     

Glucose uptake rates of single E. coli cells grown in glucose-limited chemostat cultures

To evaluate the extent to which single-cell glucose uptake rates determine the overall specific growth rate of a culture, dilute chemostat cultures of Escherichia coli BL21 were grown in defined medium under glucose limitation. The glucose uptake dynamics of the cell population was examined at the single-cell level using the fluorescent glucose analog, 2-NBDG. Between dilution rates of 0.12 h(-1) and 0.40 h(-1), mean cellular protein content and steady-state, extracellular glucose concentrations increased with increasing dilution rate. However, the distribution of 2-NBDG uptake rates in the population remained constant over the range of dilution rates studied. This indicates that the growth of cells in continuous culture is not limited by the maximum rate of uptake of glucose but by the availability of glucose for transport. The work represents an example of how quantitative flow cytometry can be applied to gain detailed insight into microbial growth physiology.     

Effect of Decreasing Growth Temperature on Cell Yield of Escherichia coli

Studies of the relationship between yield coefficient and growth rate, as affected by temperature of growth, in Escherichia coli have shown that, over a wide range of temperature, yield is relatively constant until the specific growth rate falls below about 0.2 hr(-1), at which point the yield begins to fall off precipitously. No intermediates of glucose metabolism in a form utilizable at higher temperatures could be found in the medium, and no toxic product was produced which limited growth. At 10 C, 37% of the carbon from glucose-UL-(14)C was assimilated into cellular material, whereas, at 30 C, 53% was assimilated. Cells grown at 10 C contained more carbohydrate than did cells grown at 37 C, and the glycogen-to-protein ratio of cells grown at 10 C was approximately three times higher than that of cells grown at 37 C. Adenosine triphosphatase activities of cells grown at 10 and 35 C were similar. Growth rates on glucose, glycerol, and succinate were quite similar at 10 C, but at 35 C growth was most rapid on glucose and slowest on succinate. The data suggest that the decrease in yield with decrease in temperature is a result of uncoupling of energy production from energy utilization.     

Cultivation of Saccharomyces cerevisiae in continuous culture

Summary Growth of Saccharomyces cerevisiae was investigated under aerobic conditions in a glucose limited chemostat. The steady state concentrations of cells, glucose and ethanol were measured in dependence of the dilution rate. The growth rate showed a biphasic dependence from the glucose concentration. A shift from respiratory to fermentative metabolism (Crabtree-effect) altering heavily the cell yield and the ethanol yield took place in the range of dilution rates between 0.3 h^-1 and 0.5 h^-1. Therefore the classical theory of continuous cultures is not applicable on aerobic growth of Saccharomyces cerevisiae under glucose limitation without introducing further premises. On the other hand the steady state cell concentration as a function of the dilution rate fits well the theoretically calculated curves, if cells are cultivated under conditions where only fermentation or respiration is possible.     

Mutual adjustment of glucose uptake and metabolism in Trypanosoma brucei grown in a chemostat.

The mutual adjustment of glucose uptake and metabolism in the insect stage of the protozoan parasite Trypanosoma brucei was studied. T. brucei was preadapted in the chemostat to conditions in which either glucose or proline served as the major carbon and energy source. Cells were grown and adapted to either energy or non-energy limitation at a low dilution rate (0.5 day-1) or a high dilution rate (1 day-1). The cells were then used in short- to medium-term uptake experiments with D-[14C]glucose as a tracer. In time course experiments a steady state was reached after 15 min regardless of the preadaptation conditions. This steady-state level increased with increasing glucose availability during preadaptation. The rate of glucose uptake and the hexokinase activity were linearly correlated. In short-term 5- to 90-s) uptake experiments a high transport rate was measured with cultures grown in excess glucose, an intermediate rate was measured with proline-grown cultures, and a low rate was measured in organisms grown under glucose limitation. Glucose metabolism and proline metabolism did not affect each other during the 15-min incubations. Glucose uptake, as a function of the external glucose concentration, did not obey simple Michaelis-Menten kinetics but could be described by a two-step mechanism: (i) transport of glucose by facilitated diffusion and (ii) subsequent metabolism of glucose. The respective rates of the two steps were adjusted to each other. It is concluded that T. brucei is capable of adjusting the different metabolic processes in a way that gives maximum energy efficiency at the cost of short-term flexibility.     

Activity of Pseudomonas aeruginosa in biofilms: Steady state

Aerobic glucose metabolism by Pseudomonas aeruginosa in steady-state biofilms at various substrate loading rates and reactor dilution rates was investigated. Variables monitored were substrate (glucose), biofilm cellular density, biofilm extracellular polymeric substance (EPS) density, and suspended cellular and EPS concentrations. A mathematical model developed to describe the system was compared to experimental data. Intrinsic yield and rate coefficients included in the model were obtained from suspended continuous culture studies of glucose metabolism by P. aeruginosa. Experimental data compared well with the mathematical model, suggesting that P. aeruginosa does not behave differently in steady-state biofilm cultures, where diffusional resistance is negligible, than in suspended cultures. This implies that kinetic and stoichiometric coefficients for P. aeruginosa derived in suspended continuous culture can be used to describe steady-state biofilm processes.     

Effects of periodic change in pressure and dissolved-oxygen concentration on the incubation characteristics of Pseudomonas aeruginosa

Experiments were carried out to identify an efficient procedure for decreasing the amount of excess sludge in deep aeration columns. Incubation characteristics, especially cellular yield, are closely related to excess sludge, maximum specific growth rate, and chemical oxygen demand. They were measured here for the bacterium Pseudomonas aeruginosa, which tends to be predominant in sewage treatment, at a pressure remaining constant or periodically changing in the range of 1-6 atm (abs.). Cellular yields tended to decrease over 1-2 min pressure cycles; in steady operation they decreased considerably with increasing pressure or dissolved oxygen concentration. Maximum specific growth rates were moderately affected by periodic changes in pressure or dissolved oxygen concentration but markedly affected in the higher concentration range under steady operation. Substrate consumption rates on the other hand, were little affected in either case. The values of chemical oxygen demand ranged from 220 to 250 mg/L, and were thus nearly constant.     

Induction and general properties of beta-galactosidase and beta-galactoside permease in Pseudomonas BAL-31.

The hydrolysis of o-nitrophenyl-beta-D-galactopyranoside (ONPG) by BAL-31, a marine Pseudomonas that acts as a host for bacteriophage PM2, was studied with intact cells and with cell-free extracts. A transport system for ONPG in whole cells and a beta-galactosidase activity in extracts were evident for cells grown on lactose minimal medium. It was found that the addition of isopropylthio-beta-D-galactopyranoside (IPTG) to cells growing in rich medium induced an ONPG hydrolytic activity detectable in cell extracts but cryptic in whole cells. The existence of a transport system for IPTG, which remained cryptic for ONPG, became apparent from studies of the rates of induction of beta-galactosidase as a function of cell mass at different concentrations of IPTG. The main properties of beta-galactosidase and the lactose transport system of BAL-31 were studied in terms of how they were affected by pH, temperature, or by the presence of several sugars. IPTG competitively inhibits the hydrolysis of ONPG by cell extracts. In cells pregrown on lactose, IPTG slightly inhibits the transport of ONPG. Glucose, and with less efficiency lactose, also inhibits the hydrolysis of ONPG in cell extracts. The growth of cells on lactose minimal medium was inhibited by the addition of IPTG. A mechanism for this inhibition and for the inhibition of ONPG transport by IPTG is discussed.     

Energetics of Microbacterium thermosphactum in glucose-limited continuous culture.

Microbacterium thermosphactum was grown at 25 degrees C in glucose-limited continuous culture under aerobic (greater than 120 microM oxygen) and anaerobic (less than 0.2 microM oxygen) conditions. The end products of the anaerobic metabolism of glucose were identified as L-lactate and ethanol. Together these compounds accounted for between 85 and 90% of the glucose utilized over the full range of growth rates studied. In addition, 4% of the glucose utilized was incorporated into cellular material. Under anaerobic conditions the molar growth yield was 40 g (dry weight) of cells per mol of glucose utilized, and the maintenance energy coefficient was 0.4 mmol of glucose utilized per g (dry weight) of cells per h. For cells grown under aerobic conditions in the corresponding values were 73 g/mol and 0.2 mmol/g per h, respectively. The molar growth yield with respect to adenosine 5'-triphosphate varied with the growth rate of the culture, and the true molar growth yield with respect to adenosine 5'-triphosphate was found to be 20 g/mol of adenosine 5'-triphosphate.     

Energetics of Microbacterium thermosphactum in glucose-limited continuous culture.

Microbacterium thermosphactum was grown at 25 degrees C in glucose-limited continuous culture under aerobic (greater than 120 microM oxygen) and anaerobic (less than 0.2 microM oxygen) conditions. The end products of the anaerobic metabolism of glucose were identified as L-lactate and ethanol. Together these compounds accounted for between 85 and 90% of the glucose utilized over the full range of growth rates studied. In addition, 4% of the glucose utilized was incorporated into cellular material. Under anaerobic conditions the molar growth yield was 40 g (dry weight) of cells per mol of glucose utilized, and the maintenance energy coefficient was 0.4 mmol of glucose utilized per g (dry weight) of cells per h. For cells grown under aerobic conditions in the corresponding values were 73 g/mol and 0.2 mmol/g per h, respectively. The molar growth yield with respect to adenosine 5'-triphosphate varied with the growth rate of the culture, and the true molar growth yield with respect to adenosine 5'-triphosphate was found to be 20 g/mol of adenosine 5'-triphosphate.     

Implications of the simultaneous occurrence of hepatic glycolysis from glucose and gluconeogenesis from glycerol.

Glycolysis from [6-(3)H]glucose and gluconeogenesis from [U-(14)C]glycerol were examined in isolated hepatocytes from fasted rats. A 5 mm bolus of glycerol inhibited phosphorylation of 40 mm glucose by 50% and glycolysis by more than 60%, and caused cellular ATP depletion and glycerol 3-phosphate accumulation. Gluconeogenesis from 5 mm glycerol was unaffected by the presence of 40 mm glucose. When nonsaturating concentrations of glycerol (< 200 microm) were maintained in the medium by infusion of glycerol, cellular ATP concentrations remained normal. The rate of uptake of infused glycerol was unaffected by 40 mm glucose, but carbohydrate synthesis from glycerol was inhibited 25%, a corresponding amount of glycerol being diverted to glycolytic products, whereas 10 mm glucose had no inhibitory effect on conversion of infused glycerol into carbohydrate. Glycerol infusion depressed glycolysis from 10 mm and 40 mm glucose by 15 and 25%, respectively; however, the overall rates of glycolysis were unchanged because of a concomitant increase in glycolysis from the infused glycerol. These studies show that exposure of hepatocytes to glucose and low quasi-steady-state concentrations of glycerol result in the simultaneous occurrence, at substantial rates, of glycolysis from glucose and gluconeogenesis from the added glycerol. We interpret our results as demonstrating that, in hepatocytes from normal rats, segments of the pathways of glycolysis from glucose and gluconeogenesis from glycerol are compartmentalized and that this segregation prevents substantial cross-over of phosphorylated intermediates from one pathway to the other. The competition between glucose and glycerol implies that glycolysis and phosphorylation of glycerol take place in the same cells, and that the occurrence of simultaneous glycolysis and gluconeogenesis may indicate channelling within the cytoplasm of individual hepatocytes.     

D-Glucose Transport in Cultured Cells of Neural Origin: The Membrane as Possible Control Point of Glucose Utilization

Abstract: The function of plasma membrane as control point of glucose metabolism has been studied in confluent monolayer of C1300 neuroblastoma (N2A) and glioma (C6) cells. In neuroblastoma, steady state intracellular glucose concentration reached the extracellular levels, while intracellular contents in C6 glioma cells remained very low. In C6 glial cells the amount of glycogen as source of energy was much higher than that found in C1300 neuroblastoma cells. Influx rates of D-glucose in C6 glioma cells were only half those found in neuroblastoma cells. During the influx period (0-40 s) the transport of glucose in these cells did not exceed the phosphorylation rate, whereas a steady, time-dependent increase in glucose content was observed in neuroblastoma cells. While glucose uptake in neuroblastoma cells seems to be regulated at the level of phosphorylating enzymes, the control point in C6 glioma is believed to be membrane transport.     

Hexose transport derepressed and refractory to purine regulation in NAD(H)-depleted Nil cells

Nil hamster fibroblasts depleted of NAD(H) by growth in medium devoid of nicotinamide (NAm^?MEM) exhibit up to 2-3-fold higher rates of glucose transport. Derepression of glucose transport is observed only when Nil cells have become severely depleted of both intracellular NAD(H) and ATP, despite the continued presence of 5.5 mM D-glucose in the growth medium. Neither the initial rate of transport, approximated from 3-O-methylglucose uptake, nor accumulation of D-glucose itself is repressed upon restoring nicotinamide to the medium. Exposure of the cells to NAD⁺ (10^?5 M), however, leads to a sharp curtailment of transport within 2 to 3 hours. The purines, hypoxanthine and guanine, that sharply reduce glucose transport capacity of normal cells, have no significant effect upon transport activity of NAD(H)-depleted cells.     

Application of the euglycaemic clamp technique to bioassay of insulin analogues.

The euglycaemic clamp method may offer a precise and clinically valid approach to assess the in vivo potency of new insulin analogues or derivatives relative to a human insulin standard. The proposed protocol was designed to overcome problems due to differences in pharmacokinetics between the test and standard preparations. An analogue of human insulin, GlyA21+ArgB27+ThrB30-NH2, which is absorbed very slowly after subcutaneous injection, and human insulin were compared in intravenous clamp experiments in pigs. Both insulins were infused for 4 h to achieve steady state glucose metabolism. The infusion rate ranged from 2.5-8 pmol min-1 kg-1. Parallel dose response curves were obtained with the mean glucose infusion rate from 180-240 min as the response and the logarithm of the insulin infusion rate as the dose. Standard bioassay analysis showed that the molar potency of the analogue relative to human insulin was 95.2% with a 95% confidence interval of 82.3-111.2%. To assess the clinical validity of the method a similar euglycaemic clamp study was carried out in human volunteers. The insulin infusion rates were 3 and 6 pmol min-1 kg-1, and the mean glucose infusion rate over the final 180-240 min period of the clamp was used as response. The statistical analysis showed, as in the pig clamp bioassay, no significant deviations from steady state or from the assumption of parallelism. The resulting molar potency of the analogue relative to human insulin was 85.5% with a 95% confidence interval of 49.5-128.4%. This was in agreement with the result of the pig clamp bioassay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Limitation of Photosynthesis by Carbon Metabolism : I. Evidence for Excess Electron Transport Capacity in Leaves Carrying Out Photosynthesis in Saturating Light and CO(2)

It has been investigated how far electron transport or carbon metabolism limit the maximal rates of photosynthesis achieved by spinach leaves in saturating light and CO₂. Leaf discs were illuminated with high light until a steady state rate of O₂ evolution was attained, and then subjected to a 30 second interruption in low light, to generate an increased demand for the products of electron transport. Upon returning to high light there is a temporary enhancement of photosynthesis which lasts 15 to 30 seconds, and can be up to 50% above the steady state rate of O₂ evolution. This temporary enhancement is only found when saturating light intensities are used for the steady state illumination, is increased when low light rather than darkness is used during the interruption, and is maximal following a 30 to 60 seconds interruption in low light. Decreasing the temperature over the 10 to 30°C range led to the transient enhancement becoming larger. The temporary enhancement is associated with an increased ATP/ADP ratio, a decreased level of 3-phosphoglycerate, and increased levels of triose phosphate and ribulose 1,5-bisphosphate. Since electron transport can occur at higher rates than in steady state conditions, and generate a higher energy status, it is concluded that leaves have a surplus electron transport capacity in saturating light and CO₂. From the alterations of metabolites, it can be calculated that the enhanced O₂ evolution must be accompanied by an increased rate of ribulose 1,5-bisphosphate regeneration and carboxylation. It is suggested that the capacity for sucrose synthesis ultimately limits the maximal rates of photosynthesis, by restricting the rate at which inorganic phosphate can be recycled to support electron transport and carbon fixation in the chloroplast.