Are growth rates of Escherichia coli in batch cultures limited by respiration?

Batch cultures of Escherichia coli were grown in minimal media supplemented with various carbon sources which supported growth at specific growth rates from 0.2 to 1.3/h. The respiration rates of the cultures were measured continuously. With few exceptions, the specific rate of oxygen consumption was about 20 mmol of O2/h per g (dry weight), suggesting that the respiratory capacity was limited at this value. The adenosine triphosphate (ATP) required for the production of cell material from the different carbon sources was calculated on the basis of known ATP requirements in the biochemical pathways and routes of macromolecular synthesis. The calculated ATP requirements, together with the measured growth rates and growth yields on the different carbon sources, were used to calculate the rate of ATP synthesis by oxidative phosphorylation. This rate was closely related to the respiration rate. We suggest that aerobic growth of E. coli in batch cultures is limited by the rate of respiration and the concomitant rate of ATP generation through oxidative phosphorylation.     

Growth and the efficiency of root respiration of Pisum sativum as dependent on the source of nitrogen

Growth and efficiency of root respiration were investigated in Pisum sativum L. cv. Alaska and cv. Rondo. Plants were grown in culture solutions, either in symbiosis with Rhizobium leguminosanm , or with an abundant supply of nitrate or ammonium and completely lacking nodules. In comparison with plants utilizing nitrate or ammonium, Ni-fixing plants showed lower rates of dry matter and nitrogen accumulation, as well as lower rates of total and cytochrome-mediated root respiration. Rates of shoot dry matter accumulation and root respiration in plants utilizing ammonium were lower than in plants utilizing nitrate. The efficiency of root respiration was high in N₂-fixing plants, as indicated by a low activity of the SHAM-sensitive, alternative, non-phosphorylating pathway. In nitrate and ammonium grown plants of cv. Alaska, the efficiency of root respiration was about the same, and in both cases lower than in N₂-fixing plants. The efficiency of root respiration in non-symbiotically grown pea plants was generally higher than in many non-legumes. Comparison of the ATP costs of synthesis of root dry matter for different N-sources was complicated by large differences in relative growth rate of the root and in shoot to root ratio between N-treatments. A quantitative correction of the ATP production during synthesis of root dry matter for differences in shoot to root ratio and root maintenance respiration has been made. It is concluded that ATP costs of root dry matter production are highest in the case of N₂-fixing plants. In plants utilizing ammonium, ATP costs of synthesis of root dry matter were slightly lower than in plants utilizing nitrate. The physiological significance of the alternative pathway in root metabolism is discussed in relation to the assimilation of different sources of nitrogen.     

The synthesis of hippurate from benzoate and glycine by rat liver mitochondria. Submitochondrial localization and kinetics.

1. Rat liver mitochondria make hippurate at up to 4 nmol/min per mg of protein. The rate of synthesis supported by oxidation of glutamate with exogenous Pi present is identical with that supported by ATP plus oligomycin. Lower rates were obtained with other respiratory substrates, and when glutamate was used without Pi. 2. A matrix localization for hippurate synthesis is indicated by the latency of benzoyl-CoA synthetase and glycine N-acyltransferase to their extramitochondrial substrates, failure of exogenous benzoyl-CoA to inhibit incorporation of [14C]hippurate and inhibition of hippurate synthesis supported by ATP, but not glutamate, by carboxyatractyloside. 3. The relative activities of the individual enzymes and the mitochondrial content of benzoyl-CoA in the presence and absence of glycine suggest that hippurate synthesis is rate-limited by formation of benzoyl-CoA. 4. The increases in rates of ATP hydrolysis and of O2 consumption on the addition of benzoate and glycine were in good agreement with those required to support hippurate synthesis. The increase in respiration indicates that State-4 respiration [Chance & Williams (1957) Adv. Enzymol 17, 65-134] is not used, with these conditions, for ATP synthesis.     

Combined effect of propofol and GSNO on oxidative phosphorylation of isolated rat liver mitochondria.

Isolated rat liver mitochondria have been treated with the general anaesthetic propofol (2,6-diisopropylphenol, 200 microM) and the physiological NO donor nitrosoglutathione (GSNO, 200 or 250 microM). The efficiency of the oxidative phosphorylation has been evaluated by measuring the respiration and ATP synthesis rates and the behavior of transmembrane electrical potential. In mitochondria energized by succinate, the simultaneous presence of both propofol and GSNO gives rise to a synergic action in affecting the resting and the ADP-stimulated respiration, the respiratory control ratio, the ATP synthesis, and the formation and utilization of the electrochemical transmembrane potential.     

Effect of the Intravenous Anesthetic 2,6-Diisopropylphenol on Respiration and Energy Production by Rat Brain Synaptosomes

The sensitivity of the mitochondrial energy production system to propofol (DPP) has been investigated in rat brain synaptosomes. DPP at 0.8 mM concentration produced a partial inhibition of coupled respiration, an apparent decrease of the oxygen uptake stimulation induced by CCCP and a full inhibition of the mitochondrial ATP production by synaptosomes. Higher concentrations of DPP (1 mM) fully abolish uncoupler-dependent stimulation and at 1.3 mM DPP also coupled respiration is completely blocked. Similar results were obtained when dinitrophenol replaced CCCP and phenol or propylbenzene replaced DPP. The presence of the alkyl residues seems critical for the DPP effect. In the presence of 30 mM glutamate both respiration and ATP production are enhanced but DPP effects are similar to those obtained in the absence of glutamate.     

Pathway Choice in Glutamate Synthesis in Escherichia coli

Escherichia coli has two primary pathways for glutamate synthesis. The glutamine synthetase-glutamate synthase (GOGAT) pathway is essential for synthesis at low ammonium concentration and for regulation of the glutamine pool. The glutamate dehydrogenase (GDH) pathway is important during glucose-limited growth. It has been hypothesized that GDH is favored when the organism is stressed for energy, because the enzyme does not use ATP as does the GOGAT pathway. The results of competition experiments between the wild-type and a GDH-deficient mutant during glucose-limited growth in the presence of the nonmetabolizable glucose analog α-methylglucoside were consistent with the hypothesis. Enzyme measurements showed that levels of the enzymes of the glutamate pathways dropped as the organism passed from unrestricted to glucose-restricted growth. However, other conditions influencing pathway choice had no substantial effect on enzyme levels. Therefore, substrate availability and/or modulation of enzyme activity are likely to be major determinants of pathway choice in glutamate synthesis.     

Modelling Xanthomonas campestris batch fermentations in a bubble column

Rate and yield expressions relating to biomass and xanthan formation and to nitrogen, glucose, and oxygen consumption were established for Xanthomonas campestris batch fermentations in a bubble column. Microbial growth was described by the logistic rate equation, characterized by a maximum specific growth rate mu(M) = 0.5 h(-1) and a maximum attainable cell concentration provided by nitrogenous compounds. With regard to carbon metabolism, the decrease with time in experimental yields and in the experimental specific rates of xanthan production and glucose assimilation demonstrated the inadequacy of the Luedeking-Piret model. These decreases were connected to the simultaneous drop in dissolved-oxygen tension observed during xanthan synthesis. The knowledge of metabolic pathways and energetic balance were used to establish the relationships between substrate utilization, ATP generation, and xanthan production. The model was structured by assuming the oxygen limitation of both the respiration rate and the efficiency of the oxidative phosphorylation mechanism (P/O ratio). Consequently, the specific rates and yield expressions became dependent on the dissolved-oxygen tension, i.e., of the volumetric oxygen transfer in the fermentor.     

Hyperoxia-induced clonogenic killing of HeLa cells associated with respiratory failure and selective inactivation of Krebs cycle enzymes

Cellular intoxication by elevated concentrations of O2 may be considered as a model for accelerated cellular aging processes resulting from excessive free radical production by normal metabolic pathways. We describe here that exposure of HeLa cell cultures to 80% O2 for 2 days causes progressive growth inhibition and loss of reproductive capacity. This intoxication was correlated with inhibition of cellular O2 consumption and inactivation of 3 mitochondrial flavoproteins, i.e., partial inactivation of NADH and succinate dehydrogenases and total inactivation of alpha-ketoglutarate dehydrogenase. As alpha-ketoglutarate dehydrogenase controls the influx of glutamine/glutamate into the Krebs cycle, which is the major pathway for oxidative ATP generation in HeLa cells, the inactivation of alpha-ketoglutarate dehydrogenase was expectedly correlated with a net fall in glutamine/glutamate utilization. Furthermore, a simultaneous increase in glucose consumption and lactate production was observed, indicating that the cellular response to respiratory failure is to generate more ATP from glycolysis. In spite of this response, extensive depletion of ATP was observed. Thus, hyperoxia-induced growth inhibition and loss of clonogenicity seem to be due primarily to an impairment of mitochondrial energy metabolism resulting from inactivation of SH-group-containing flavoprotein enzymes localized at or near the inner mitochondrial membrane. These observations may be relevant for theories implicating loss of mitochondrial function as a prime factor in the aging process.     

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.     

Oxyconformity in the intertidal worm Sipunculus nudus: the mitochondrial background and energetic consequences

The energetic consequences of strict oxyconformity in the intertidal worm S. nudus were studied by characterizing the Po2 dependence of respiration in mitochondria isolated from the body wall tissue. Mitochondrial respiration rose in a Po2 range between 2.8 and 31.3 kPa from a mean of 56.5 to 223.9 nmol O mg protein(-1) h(-1). Respiration was sensitive to both salicylhydroxamic acid (SHAM) and KCN. Po2 dependence remained unchanged with saturating and non-saturating substrate levels (malate, glutamate and ADP). A concomitant decrease of the ATP/O ratio revealed a lower ATP yield of aerobic metabolism at elevated Po2. Obviously, oxyconforming respiration implies progressive uncoupling of mitochondria. The decrease in ATP/O ratios at higher Po2 was completely reversible. Addition of 90.9 micromol H2O2 l(-1) did not inhibit ATP synthesis. Both observations suggest that oxidative injury did not contribute to oxyconformity. The contribution of the rates of mitochondrial ROS production and proton leakiness to mitochondrial oxygen consumption and uncoupling was investigated by using oligomycin as a specific inhibitor of the ATP synthase. The maximum contribution of oligomycin independent respiration to state 3 respiration remained below 6% and showed a minor, insignificant increase at elevated Po2, at a slope significantly lower than the increment of state 3 respiration. Therefore, Po2 dependent mitochondrial proton leakage or ROS production cannot explain oxyconformity. In conclusion Po2 dependent state 3 respiration likely relates to the progressive contribution of an alternative oxidase (cytochrome o), which is characterized by a low affinity to oxygen and an ATP/O ratio similar to the branched respiratory system of bacteria. The molecular nature of the alternative oxidase in lower invertebrates is still obscure.     

Metabolic control over the oxygen consumption flux in intact skeletal muscle: in silico studies

It has been postulated previously that a direct activation of all oxidative phosphorylation complexes in parallel with the activation of ATP usage and substrate dehydrogenation (the so-called each-step activation) is the main mechanism responsible for adjusting the rate of ATP production by mitochondria to the current energy demand during rest-to-work transition in intact skeletal muscle in vivo. The present in silico study, using a computer model of oxidative phosphorylation developed previously, analyzes the impact of the each-step-activation mechanism on the distribution of control (defined within Metabolic Control Analysis) over the oxygen consumption flux among the components of the bioenergetic system in intact oxidative skeletal muscle at different energy demands. It is demonstrated that in the absence of each-step activation, the oxidative phosphorylation complexes take over from ATP usage most of the control over the respiration rate and oxidative ATP production at higher (but still physiological) energy demands. This leads to a saturation of oxidative phosphorylation, impossibility of a further acceleration of oxidative ATP synthesis, and dramatic drop in the phosphorylation potential. On the other hand, the each-step-activation mechanism allows maintenance of a high degree of the control exerted by ATP usage over the ATP turnover and oxygen consumption flux even at high energy demands and thus enables a potentially very large increase in ATP turnover. It is also shown that low oxygen concentration shifts the metabolic control from ATP usage to cytochrome oxidase and thus limits the oxidative ATP production. respiration rate; parallel activation; oxidative phosphorylation; metabolic control analysis; flux control coefficient; muscle contraction     

Effect of Methionine Sulfoximine and Methionine Sulfone on Glutamate Synthesis in Klebsiella aerogenes

At least two pathways exist in Klebsiella aerogenes for glutamate synthesis. A mutant blocked in one pathway due to the loss of glutamate dehydrogenase (gltD) does not require glutamate and has the same growth characteristics as the parent strain in most media; however, its growth is inhibited by the analogues methionine sulfoximine and methionine sulfone. Wild-type Klebsiella is resistant to 0.1 M methionine sulfoximine or methionine sulfone, whereas the gltD mutant is sensitive to 1 mM concentrations. Either glutamate or glutamine is effective in overcoming this inhibition. Activities of both glutamine synthetase and glutamate synthetase, two enzymes involved in the second pathway of glutamate synthesis, are inhibited by methionine sulfoximine and methionine sulfone. The primary effect of methionine sulfoximine appears to be the prevention of glutamine production necessary for subsequent glutamate synthesis via glutamate synthetase enzyme.     

Exposure to high glutamate concentration activates aerobic glycolysis but inhibits ATP‐linked respiration in cultured cortical astrocytes

Astrocytes play a key role in removing the synaptically released glutamate from the extracellular space and maintaining the glutamate below neurotoxic level in the brain. However, high concentration of glutamate leads to toxicity in astrocytes, and the underlying mechanisms are unclear. The purpose of this study was to investigate whether energy metabolism disorder, especially impairment of mitochondrial respiration, is involved in the glutamate‐induced gliotoxicity. Exposure to 10‐mM glutamate for 48 h stimulated glycolysis and respiration in astrocytes. However, the increased oxygen consumption was used for proton leak and non‐mitochondrial respiration, but not for oxidative phosphorylation and ATP generation. When the exposure time extended to 72 h, glycolysis was still activated for ATP generation, but the mitochondrial ATP‐linked respiration of astrocytes was reduced. The glutamate‐induced astrocyte damage can be mimicked by the non‐metabolized substrate d ‐aspartate but reversed by the non‐selective glutamate transporter inhibitor TBOA. In addition, the glutamate toxicity can be partially reversed by vitamin E. These findings demonstrate that changes of bioenergetic profile occur in cultured cortical astrocytes exposed to high concentration of glutamate and highlight the role of mitochondria respiration in glutamate‐induced gliotoxicity in cortical astrocytes. Copyright © 2014 John Wiley & Sons, Ltd.     

Glutamine metabolism in lymphocytes of the rat.

The metabolism of glutamine in resting and concanavalin-A-stimulated lymphocytes was investigated. In incubated lymphocytes isolated from rat mesenteric lymph nodes, the rates of oxygen and glutamine utilization and that of aspartate production were approximately linear with respect to time for 60 min, and the concentrations of adenine nucleotides plus the ATP/ADP or ATP/AMP concentration ratios remained approximately constant for 90 min. The major end products of glutamine metabolism were glutamate, aspartate and ammonia: the carbon from glutamine may contribute about 30% to respiration. When both glucose and glutamine were presented to the cells, the rates of utilization of both substances increased. Evidence was obtained that the stimulation of glycolysis by glutamine could be due, in part, to an activation of 6-phosphofructokinase. Starvation of the donor animal increased the rate of glutamine utilization. The phosphoenolpyruvate carboxykinase inhibitor mercaptopicolinate decreased the rate of glutamine utilization by 28%; the rates of accumulation of glutamate and ammonia were decreased, whereas those of lactate, aspartate and malate were increased. The mitogen concanavalin A increased the rate of glutamine utilization (by about 51%). The rate of [3H]thymidine incorporation into DNA caused by concanavalin A in cultured lymphocytes was very low in the absence of glutamine; it was increased about 4-fold at 1 microM-glutamine and was maximal at 0.3 mM-glutamine; neither other amino acids nor ammonia could replace glutamine.     

Relationships Among ATP Synthesis, K+ Gradients, and Neurotransmitter Amino Acid Levels in Isolated Rat Brain Synaptosomes

Correlations were made among ATP synthesis, transmembrane K+ gradients, and leakage of three amino acid neurotransmitters, gamma-aminobutyric acid (GABA), aspartate, and glutamate, in rat brain synaptosomes incubated under normoxic and respiration-limited conditions. Even under normoxic conditions, a substantial proportion of total ATP synthesis (8%) was provided by glycolysis. Limitation of respiration by approximately 30% through addition of amobarbital (Amytal) caused a twofold decrease in the creatine phosphate/creatine ([CrP]/[Cr]) ratio, and consequently the [ATP]/[ADP] ratio, and a threefold increase in lactate production. There was a detectable decrease in intracellular [K+] and small rises in external GABA, aspartate, and glutamate concentrations. More severe limitations in ATP synthesis caused larger declines in the [CrP]/[Cr] ratio and progressive leakage of K+ and neurotransmitter amino acids. A comparison of delta GATP and delta GNa, K showed the former to be larger by 6 kcal, which indicates that the plasma membrane Na+/K+ pump operates at far from equilibrium. Under respiration-limited conditions, even when total ATP synthesis decreased by approximately 80% and [ATP] declined to less than 0.4 mM, delta GATP was still larger than delta GNa,K. It is suggested that during hypoxia and ischemia, the activity of the plasma membrane Na+/K+ pump in brain becomes limited by [ATP], which falls below the Km value for the low-affinity regulatory site on the enzyme. This failure of the pump and consequent collapse of the ion gradients may contribute to the leakage of neurotransmitter amino acids that occurs in these pathological states.     

A COMPARATIVE STUDY OF DIHYDROXYLATED PHENOTHIAZINE DERIVATIVES AS MITOCHONDRIAL INHIBITORS

Abstract— —The effects of several dihydroxylated phenothiazine derivatives on brain mitochondrial calcium retention, respiration and ATP synthesis were studied and compared to those of 7,8-dihydroxy-chlorpromazine. This compound has previously been shown to disrupt retention of calcium accumulated in the presence of ATP, to inhibit state III and state IV respiration with glutamate as substrate and to inhibit ATP synthesis. It was found that 2,3-dihydroxypromazine has little effect on mitochondrial calcium retention, but is a potent inhibitor of oxidative phosphorylation. 3,7-Dihydroxychlorpromazine and 3,8-dihydroxychlorpromazine produced a relatively slow and incomplete efflux of pre-accumulated calcium and caused a limited inhibition of respiration. 7,8-Dihydroxyperphenazine and 7,8-dihydroxyprochlorperazine promoted an efflux of mitochondrial calcium and inhibited mitochondrial respiration as was seen with 7,8-dihydroxychlorpromazine. The effects seen with 7,8-dioxochlorpromazine are also similar to those seen with 7,8-dihydroxychlorpromazine, suggesting that oxidation products may contribute to the inhibitory effects observed.     

Effect of corticosterone treatment on energy metabolism in rat liver mitochondria.

1. Effect of in vivo treatment (40 mg/kg body wt) with corticosterone on energy metabolism in rat liver mitochondria was examined under acute and chronic conditions in 20-, 35- and 60-day-old rats. 2. Acute treatment did not affect body or liver weight. However, chronic treatment caused increased liver weight in the former two age groups; in the 60-day-old animals the liver weight decreased. 3. Acute treatment resulted in a generalized decrease in state 3 respiration rates and state 4 respiration rates without having any significant effect on ADP/O ratios with glutamate, succinate and ascorbate + TMPD as substrates. However, rates of ATP synthesis decreased significantly. The effect was age-dependent, older animals showed increased resistance. 4. Chronic treatment resulted in uncoupling of oxidative phosphorylation without having significant effects on respiration rates. Once again, the effects were age-dependent. Consequently, the ATP synthesis rates were significantly lowered. However, it was apparent that the underlying mechanisms were entirely different. 5. With succinate as the substrate the state 3 respiration rates increased with age to reach adult values by day 60. The coupling efficiency was also exhibited via maturational changes.     

High efficiency versus maximal performance — The cause of oxidative stress in eukaryotes: A hypothesis

Degenerative diseases are in part based on elevated production of ROS (reactive oxygen species) in mitochondria, mainly during stress and excessive work under stress (strenuous exercise). The production of ROS increases with increasing mitochondrial membrane potential (ΔΨ_m). A mechanism is described which is suggested to keep ΔΨ_m at low values under normal conditions thus preventing ROS formation, but is switched off under stress and excessive work to maximize the rate of ATP synthesis, accompanied by decreased efficiency. Low ΔΨ_m and low ROS production are suggested to occur by inhibition of respiration at high [ATP]/[ADP] ratios. The nucleotides interact with phosphorylated cytochrome c oxidase (COX), representing the step with the highest flux-control coefficient of mitochondrial respiration. At stress and excessive work neural signals are suggested to dephosphorylate the enzyme and abolish the control of COX activity (respiration) by the [ATP]/[ADP] ratio with consequent increase of ΔΨ_m and ROS production. The control of COX by the [ATP]/[ADP] ratio, in addition, is proposed to increase the efficiency of ATP production via a third proton pumping pathway, identified in eukaryotic but not in prokaryotic COX. We conclude that ‘oxidative stress’ occurs when the control of COX activity by the [ATP]/[ADP] ratio is switched off via neural signals.