Energy metabolism of Bdellovibrio bacteriovorus. I. Energy production, ATP pool, energy charge.

Bdellovibrio bacteriovorus, strain Bd. 109 Sa, generates ATP mainly by oxidative phosphorylation during electron transport. During exponential growth the ATP pool is constant (9 mmoles/100 mugN) indicating that energy-producing and energy-consuming reactions are well balanced. The ratio of substrate respiration/endogenous respiration is approx. 2.5/1. Energy charge is constant both in endogenous and substrate respiration at values of 0.62 to 0.64. During endogenous respiration (starvation) the ATP pool oscillates at regular intervals. ATP over-production is started after the ATP pool has decreased to a minimum level of 6 nmoles/100 mug N. The alternating over- and under-production of ATP is interpreted as a special regulation which enables the organism to make economic use of its own cellular materials. Addition of substrate (glutamate) to starving cells does not influence the type of ATP pool oscillation as observed in endogenous respiration. The parasitic strain Bd. 109 Pa exhibits the same periodicity of ATP overproduction as does it saprophytic derivative, Bd. 109 Sa. Decrease of viability during starvation is paralleled by a decrease of the ATP pool.     

Energy metabolism of Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus, strain Bd. 109 Sa, generates ATP mainly by oxidative phosphorylation during electron transport. During exponential growth the ATP pool is constant (9 nmoles/100 μg N) indicating that energy-producing and energy-consuming reactions are well balanced. The ratio of substrate respiration/endogenous respiration is approx. 2.5/1. Energy charge is constant both in endogenous and substrate respiration at values of 0.62 to 0.64. During endogenous respiration (starvation) the ATP pool oscillates at regular intervals. ATP over-production is started after the ATP pool has decreased to a minimum level of 6 nmoles/100 μg N. The alternating over- and under-production of ATP is interpreted as a special regulation which enables the organism to make economic use of its own cellular materials. Addition of substrate (glutamate) to starving cells does not influence the type of ATP pool oscillation as observed in endogenous respiration. The parasitic strain Bd. 109 Pa exhibits the same periodicity of ATP overproduction as does its saprophytic derivative, Bd. 109 Sa. Decrease of viability during starvation is paralleled by a decrease of the ATP pool.     

Effect of glycolysis on the metabolism of adenylates in human erythrocytes

The ATP content in human erythrocytes depleted without glucose falls down to half of the initial value within 2-3 hours and reaches practically zero within more than 10 hours. The ADP content increases 2-3-fold during the 1st hour after depletion and then slowly decreases. The AMP content increases 10-fold during several hours, but the rate of this process constantly decreases. The adenylate pool decreases at a constant rate ranging from 0.13 to 0.25 mmol/l cell. h; this is accompanied by accumulation of IMP. Addition of glucose to depleted erythrocytes results in partial recovery of the ATP level within 1-2 hours. The sooner glucose addition after the depletion, the greater the recovery. Simultaneously the ADP and AMP levels drastically decrease to new constant values. The decline of the adenylate pool ceases and the rate of IMP accumulation increases. Normally, the [ATP]/adenylate pool ratio lies within the small interval 0.85-0.94 irrespective of significant individual differences in the absolute values of [ATP]. This ratio is decreased during depletion and restored to the initial value after glucose addition. The mass-action ratio of the adenylate kinase reaction changes greatly during depletion and restoration of erythrocyte ATP.     

Glycerol production in relation to the ATP pool and heat production rate of the yeasts Debaryomyces hansenii and Saccharomyces cerevisiae during salt stress

Changes in glycerol production and two parameters related to energy metabolism i. e. the heat production rate and the ATP pool, were assayed during growth of Saccharomyces cerevisiae and Debaryomyces hansenii in 4 mM and 1.35 M NaCl media. For both of the yeasts, the specific ATP pool changed during the growth cycle and reached maximum values around 10 nmol per mg dry weight in both types of media. The levels of glycerol were markedly enhanced by high salinity. In the presence of 1.35 M NaCl, D. hansenii retained most of its glycerol produced intracellularly, while S. cerevisiae extruded most of the glycerol to the environment. The intracellular glycerol level of S. cerevisiae equalled or exceeded that of D. hansenii, however, with values never lower than 3 mol per mg dry weight at all phases of growth. When D. hansenii was grown at this high salinity the intracellular level of glycerol was found to correlate with the specific heat production rate. No such correlation was found for S. cerevisiae. We concluded that during salt stress, D. hansenii possesses the capacity to regulate the metabolism of glycerol to optimize growth, while S. cerevisiae may not be able to regulate when exposed to different demands on the glycerol metabolism.     

Role of adenine nucleotides in the regulation of bacterial energy metabolism: theoretical problems and experimental pitfalls

The effect of growth rate on ATP pool and adenylate energy charge (EC) value of Escherichia coli has been studied in batch culture on different media (mu max varying from 0.1 h-1 to 1.2 h-2) and in continuous culture at dilution rates (D = mu) from 0.045 h-1 to 0.310 h-1. Within the limits of error both ATP pool and EC values did not change with alterations in the relative growth rate of E. coli. The effect of in vivo EC values on experimental errors in ATP, ADP and AMP measurements with the luciferin-luciferase method, and, subsequently, on measurements of different ratios between adenylates, as in the case of adenylate kinase in vivo equilibrium, is discussed.     

Changes in the energy indices of Escherichia coli during exhaustion and renewal of glucose and ammonia supply as a factor responsible for the coupling of energy and constructive types of metabolism

The shift down of glucose in the growth medium lowered the energetic status of cells whereas that of ammonium elevated it, which was indicative of their specific effect on metabolism. The shift up of glucose within the first four seconds promptly increased the intracellular ATP pool, the energy charge and the ATP/ADP ratio up to values characteristic of growth, while the addition of ammonium after its exhaustion resulted in the opposite effect. The described changes are typical of an incomplete coupling between energetic and constructive metabolic types in E. coli.     

A mathematical model for the aerobic growth of Saccharomyces cerevisiae with a saturated respiratory capacity

A mathematical model for the aerobic growth of Saccharomyces cerevisiae in both batch and continuous culture is described. It was based on the experimental observation that the respiratory capacity of organism may become saturated and exhibit a maximum specific oxygen uptake rate after suitable adaptation. This experimental observation led to the possibility that transport into and out of the mitochondrion was of major importance in the overall metabolism of S. cerevisiae and was subject to long-term adaptation. Consistent with this observation a distributed model was proposed which. as its basis, assumed the control of repression or inhibition of the uptake rates of other substrates. No other regulation of fermentation and respiration was assumed. The model provided a suitable structure allowing precise quantification of the changes in rate and stoichiometry of energy production. The model clearly indicated that growth under the wide range of experimental conditions reported could not be predicted using constant values for the maximum specific respiratory rate of constant values of Y_ATP (g biomass/mol ATP) and PO ratio of (mol ATP/atom oxygen). The causes of the variation in the respiratory rate were not determined and it was concluded that a more detailed analysis (reported subsequently) was required. The variation of Y_ATP and PO ratio with specific growth rate implied that the efficiency of ATP generation or ATP utilization decreased with increasing specific growth rate. It was concluded that it was not possible to quantify the individual effect of Y_ATP and PO ratio until independent means for their reliable estimation is available.     

Effect of temperature on the ATP pool and adenylate energy charge in Escherichia coli

The effect of cultivation temperature on the ATP pool and adenylate energy charge (EC) in Escherichia coli has been studied in both batch and continuous cultures. In batch culture, μ_max and the ATP pool increased with increasing growth temperatures between 27–42°C (from 0.26 to 0.62 h⁻¹, and from 5.1 to 8.2 nmol/mg dry wt., respectively). In continuous culture at a constant dilution rate (D = 0.2 h⁻¹), with increasing growth temperatures between 28–43°C, the ATP pool increased about 2-fold (from 4.2 to 8.1 nmol/mg dry wt) and the EC from 0.80 to 0.99.     

The energy budget of Tetrahymena and the material fluxes into and out of the adenylate pool

The material budget of the adenylate pool deals with all processes which physically establish and maintain this pool, while the energy budget is concerned with the intracompartmental ATP recycling. Both budgets were analysed in Tetrahymena thermophila exposed to various energy and material demands. Some of the general conclusions are: at a maximum growth rate the overall ATP consumption during one cell cycle is 10(-10) mol ATP; the contribution of osmoregulation and ciliary motion to the budget is about 1% each; at zero net growth, energy is consumed because of a continuous recycling of matter between the monomer and the polymer compartment. The rate of ATP production is about 1000-fold greater than the rate of adenylate monomer influx. The residence time of adenylate monomers within the pool is about 30 min, but for ATP molecules it is only 2 sec.     

Benzyladenine reversal of abscisic Acid inhibition of growth and RNA synthesis in germinating bean axes

The effect of benzyladenine on growth, ATP pool size and specific radioactivity, and the rate and amount of RNA synthesis in aseptically cultured axes of Phaseolus vulgaris during the first 24 hours of germination were measured in experiments where the duration of benzyladenine application and its concentration were varied. Maximum promotion of growth (25%) occurs at 10(-5)m benzyladenine. Maximum promotion of RNA synthesis (44%) occurs at 10(-5)m benzyladenine. Benzyladenine has little effect on the size or specific radioactivity of the ATP pool. Benzyladenine can completely counteract abscisic acid inhibition of growth and RNA synthesis, and these reversals are measurable in 2 hours. GA(1) and GA(3) do not promote growth or counteract abscisic acid inhibition of growth in germinating bean axes in these experiments.     

Relation between energy production and adenine nucleotide metabolism in human blood platelets

The relation between ATP production and adenine nucleotide metabolism was investigated in human platelets which were starved by incubation in glucose-free, CN^?-containing medium and subsequently incubated with different amounts of glucose. In the absence of mitochondrial energy production (blocked by CN^?) and glycogen catabolism (glycogen almost completely consumed during starvation), lactate production increased proportionally with increasing amounts of glucose. The generated ATP was almost completely consumed in the various ATP-consuming processes in the cell except for a fixed portion (about 7%) that was reserved for restoration of the adenylate energy charge. During the first 10 min after glucose addition, the adenine nucleotide pool remained constant. Thereafter, when the glycolytic flux, measured as lactate formation, was more than 3.5 μmol · min^?1 · 10^?11 cells, the pool increased slightly by resynthesis from hypoxanthine-inosine and then stabilized; at a lower flux the pool decreased and metabolic ATP and energy charge declined to values found during starvation. Between moments of rising and falling adenylate energy charges, periods of about 10 min remained in which the charge was constant and ATP supply and demand had reached equilibrium. This enabled comparison between the adenylate energy charge and ATP regeneration velocity. A linear relation was obtained for charge values between 0.4 and 0.85 and ATP regeneration rates between 0.6 and 3.5 ATP equiv. · min^?1 · 10^?11 cells. These data indicate that in starved platelets ATP regeneration velocity and energy charge are independent and that each appears to be subject to the availability of extracellular substrate.     

The ATP pool in relation to the production of glycerol and heat during growth of the halotolerant yeast Debaryomyces hansenii

In a study of the halotolerant yeast Debarymyces hansenii cultured in 4 mM and 2.7 M NaCl the intracellular ATP pool, the heat production, the oxygen uptake, and, in the high culture salinity also, the intracellular glycerol concentration were found to be correlated. The intracellular ATP in the 2.7 M NaCl culture had a constant concentration of 3.5 mM ATP during the second half of the lag phase, while in 4 mM NaCl it rose to a maximum of 3.1 mM during the late log phase. The intracellular glycerol concentration in 2.7 M NaCl was about 1.3M during the entire exponential growth phase. Sine the glycerol concentration of the medium was not more than 0.23 mM, glycerol must contribute to the osmotic balance of the cells in high salinity. The corresponding maximum values for the 4 mM NaCl culture were 0.16 M and 0.08 mM. The experimental enthalpy changes were approximately the same for the two salinities, viz. about-1200 kJ per mole consumed glucose. The Y _m-values for the 4 mM and 2.7 M NaCl cultures were 91 and 59, respectively, the difference being a consequence of the decreased efficiency of growth in high salinity.Abbreviations CFU colony-forming units - PCA perchloric acid - TCA trichloroacetic acid     

Effect of cultural conditions on the concentrations of metabolic intermediates during growth and sporulation of Bacillus licheniformis.

Intracellular concentrations of adenine nucleotides and intermediates of the Embden-Meyerhof pathway and the tricarboxylic acid cycle have been determined during growth and sporulation of Bacillus licheniformis in a variety of different media. The ATP pool was independent of growth rate and nitrogen source, but the use of glucose as a carbon source resulted in a twofold elevation in the ATP pool during exponential growth. The intracellular phosphoenolpyruvate pool was at least twofold higher during gluconeogenesis than during glycolysis. The finding that the use of glutamate as the sole nitrogen source resulted in at least a fivefold elevation of the alpha-ketoglutarate pool suggests a role for alpha-ketoglutarate in the repression of the enzymes of the tricarboxylic acid cycle responsible for alpha-ketoglutarate synthesis. Not one of the metabolites assayed appears to function as a signal of the nutrient deprivation which accompanies the initiation of sporulation.     

The ATP pool in Escherichia coli. I. Measurement of the pool using a modified luciferase assay

A sensitive modification of the luciferase assay for ATP is described. Light output is measured using a conventional liquid-scintillation counter. ATP over the range 2 to 20 μμmoles can be assayed. The sensitivity of the method allows samples to be taken rapidly from growing cultures of bacteria without concentrating them from the medium. Chilling and anaerobiosis of growing cells before extracting with HClO₄ have been shown to cause a reduction in the ATP pool. ATP measurements in growing cultures of Escherichia coli show four different phenomena depending on culture conditions: (a) ATP production is in balance with growth. (b) ATP is consistently over-produced. (c) ATP is consistently under-produced. (d) Cyclic oscillations in ATP production are observed.

Doubling times for growth and ATP production are compared, and the ATP pool in cells grown under carbon-limiting conditions in a chemostat has been measured. Starvation experiments have shown that the ATP pool drops more rapidly anaerobically than aerobically.     

The requirement of low pH for growth of Thiobacillus thiooxidans

Summary Thiobacillus thiooxidans was grown at constant pH. No growth was obtained above pH 4.3. Sulfur was oxidized over the entire range tested (pH 0.9 to 7.0). This was confirmed by pH shift experiments. Carbon dioxide fixation stopped 30 min after shift from low pH to neutral values. On return to high acidity, the rate of CO₂ assimilation increased but not always to the original value. The intracellular binding of glycerol which is passively permeable was also inhibited by raising the pH of the medium. The data suggested that ATP formation may be inhibited at neutral pH. Intracellular ATP pool was reduced by 80% on neutralization of the acid growth medium. These results are compared to recent studies on acid-base transients in T. novellus by Cole and Aleem (1971).     

Adenosine triphosphate pool during the growth cycle in Streptococcus faecalis

Forrest, W. W. (University of Adelaide, Adelaide, South Australia). Adenosine triphosphate pool during the growth cycle in Streptococcus faecalis. J. Bacteriol. 90:1013-1018. 1965.-The adenosine triphosphate (ATP) pool has been studied throughout the growth cycle of Streptococcus faecalis. Normally, the pool is quantitatively related to the concentration of organisms and the growth rate, but deviations from these relationships can occur without affecting the growth rate of the organisms. A critical concentration of ATP seems necessary for exponential growth, and, at lower levels, only linear growth can occur. If no growth can take place, catabolism of added energy source gives rise to a large increase in the pool level. The pool level represents the balance between the demands of the organisms for energy and the supply of energy derived from catabolism of the substrate.     

Synaptic mitochondria are critical for mobilization of reserve pool vesicles at Drosophila neuromuscular junctions

In a forward screen for genes affecting neurotransmission in Drosophila, we identified mutations in dynamin-related protein (drp1). DRP1 is required for proper cellular distribution of mitochondria, and in mutant neurons, mitochondria are largely absent from synapses, thus providing a genetic tool to assess the role of mitochondria at synapses. Although resting Ca2+ is elevated at drp1 NMJs, basal synaptic properties are barely affected. However, during intense stimulation, mutants fail to maintain normal neurotransmission. Surprisingly, FM1-43 labeling indicates normal exo- and endocytosis, but a specific inability to mobilize reserve pool vesicles, which is partially rescued by exogenous ATP. Using a variety of drugs, we provide evidence that reserve pool recruitment depends on mitochondrial ATP production downstream of PKA signaling and that mitochondrial ATP limits myosin-propelled mobilization of reserve pool vesicles. Our data suggest a specific role for mitochondria in regulating synaptic strength.     

Dinucleoside tetraphosphate variations in cultured tumor cells during their cell cycle and growth

Asynchronous and synchronized cultures of A549 and HTC cells were used to detect possible, cell cycle or cell density specific variations in the intracellular pools of dinucleoside tetraphosphates (Ap4X). No important variations of the nucleotide pools were observed during cell growth. When HTC cells were released from mitotic arrest, a decrease by a factor of N3 Ap4X and ATP levels was observed when the cells entered the G1 phase. This decrease is essentially due to cell doubling. When A549 cells were released from an arrest at the G1/S boundary, the nucleotide pool size increased slightly during the G2 phase just before mitosis. This result is in agreement with both earlier data from our laboratory and the observed decrease in Ap4X pool after release from mitotic-arrested HTC cells. These results suggest that the Ap4X and ATP pools are only subjected to very small variations during the cell cycle, essentially in the G2 phase and after mitosis.     

ATP pool and bioluminescence in psychrophilic bacteria Photobacterium phosphoreum

Bioluminescence activity and ATP pool were investigated in the cells of psychrophilic bacteria Photobacterium phosphoreum collected from the exponential and stationary growth phases and immobilized in polyvinyl alcohol (PVA) cryogel. In liquid culture, ATP pool remained at an almost constant level throughout the luminescence cycle (over 100 h). The ATP pool in the stationary-phase and PVA-immobilized cells remained constant throughout their incubation in the medium (over 200 h) and in 3% NaCl solution (over 100 h). Quantitative assessment of integral photon yield and ATP pool indicated that bioluminescence decay in growing or stationary cells was not caused by limitation from the energy substrates of the luciferase reaction. Kinetic and quantitative parameters of emission activity and ATP pool excluded the possibility of formation of the aldehyde substrate for luciferase via reduction of the relevant fatty acids in NADPH and ATP-dependent reductase reaction and its oxidation in the monooxygenase reaction. Our results indicate that the aliphatic aldehyde is not utilized in the process of light emission.     

Energy aspects of the growth of Escherichia coli synchronized by starvation

The quantitative determination of adenyl nucleotides based on the separation of their dansyl derivatives by thin layer chromatography has made it possible to study the dynamics of changes in the pool of ATP, ADP and AMP in Escherichia coli K-12 during its synchronous growth after glucose starvation. The energy parameters (the adenylate pool, energy charge, teh ATP/ADP ratio, the rates of oxygen uptake and ATP generation, the economic coefficients of oxygen and ATP utilization) were compared with changes in the growth characteristics (the rate of growth and biomass concentration). This comparison allowed the authors to draw the conclusion about the uncoupled constructive and energy metabolism and about the possible regulatory role of energy parameters in the synchronised culture growth.