Lipophilic Proteins of Mitochondria from Microaerobic and Aerobic Continuous Cultures of Saccharomyces cerevisiae

Products of the mitochondrial protein-synthesizing system have been labeled in vivo in the presence of cycloheximide in microaerobic cells and in cells from glucose-limited and glucose-repressed aerobic continuous cultures of Saccharomyces cerevisiae. Lipophilic proteins were extracted from labeled mitochondrial membranes with aqueous methanol and neutral and acidic chloroform-methanol solvents. In glucose-limited aerobic and microaerobic cells, about half of the total mitochondrial products were soluble in organic solvents; in contrast, almost all of the labeled products were extracted from glucose-repressed mitochondria. Only trace amounts of labeled product were formed in mitochondrial membranes of a petite mutant. Lipophilic proteins were examined by polyacrylamide gel electrophoresis under dissociating conditions. Most of the label was associated with components of apparent molecular weights 12,000, 14,000 and 16,000. The relative proportions of these species in mitochondrial membranes are dependent on the concentrations of oxygen and glucose in which the cells are grown.     

Effects of Oxygen and Glucose on Energy Metabolism and Dimorphism of Mucor genevensis Grown in Continuous Culture: Reversibility of Yeast-Mycelium Conversion

Mucor genevensis was grown in both glucose-limited and glucose-excess continuous cultures over a range of dissolved oxygen concentrations (<0.1 to 25 muM) to determine the effects of glucose and the influence of metabolic mode (fermentative versus oxidative) on dimorphic transformations in this organism. The extent of differentiation between yeast and mycelial phases has been correlated with physiological and biochemical parameters of the cultures. Under glucose limitation, oxidative metabolism increased as the dissolved oxygen concentration increased, and this paralleled the increase in the proportion of the mycelial phase in the cultures. Filamentous growth and oxidative metabolism were both inhibited by glucose even though mitochondrial development was only slightly repressed. However, the presence of chloramphenicol in glucose-limited aerobic cultures inhibited mitochondrial respiratory development but did not induce yeast-like growth, indicating that oxidative metabolism is not essential for mycelial development. Once mycelial cultures had been established under aerobic, glucose-limited conditions, subsequent reversal to anaerobic conditions or treatment with chloramphenicol caused only a limited reversal (<35%) to the yeast-like form. Glucose, however, induced a complete reversion to yeast-like form. It is concluded that glucose is the most important single culture factor determining the morphological status of M. genevensis; mitochondrial development and the functional oxidative capacities of the cell appear to be less important factors in the differentiation process.     

Effect of ethionine on the in vitro synthesis and degradation of mitochondrial translation products in yeast

The effect of ethionine, an amino acid analog of methionine, has been studied in Saccharomyces cerevisiae in relation to cell growth, oxygen consumption, in vitro protein synthesis of mitochondrial translation products (MTPs) and the degradation of those mitoribosomally made proteins by an ATP-dependent process present within the organelle. Ethionine was found to increase the generation time of those cells already committed to cell division and to abolish the initiation of new cell cycles. Oxygen consumption of cultures grown in the presence of the analog was drastically reduced. Ethionine was also found to impair the incorporation of methionine and leucine into mitochondrial translation products, however the synthesis of proteins was not totally blocked and, apparently, mitochondria utilized ethionine as a precursor amino acid. MTPs synthesized by isolated mitochondria in the presence of ethionine were rapidly degraded inside the organelle at a faster rate compared with the normal proteins synthesized under identical conditions in the mitochondria. It is also shown that these in vitro synthesized proteins are degraded by an ATP-stimulated proteolytic system, as has been previously established.     

Effect of oxygen on the metabolism of Zymomonas mobilis

The specific growth rate of the ethanol producing bacterium Zymomonas mobilis was 25–40% lower in the presence of oxygen than under anaerobic conditions, provided the cultures were supplied with a low substrate concentration (20 g glucose/l). However, the molar growth yield of these cultures was not influenced by oxygen. With washed cell suspensions, an oxygen consumption could be initiated by the addition of either glucose, fructose, or ethanol. Cell extracts catalyzed the oxidation of NADH with oxygen at a molar ratio of 2:1. Further experiments showed that this NADH oxidase is located in the cell membrane. The specific oxygen consumption rates of cell suspensions correlated with the intracellular NADH oxidizing activities; both levels decreased with increasing concentrations of the fermentation end-product ethanol. The addition of 5 mM NaCN completely inhibited both the intracellular oxygen reduction and also the oxygen consumption of whole cells. Both catalase and superoxide dismutase were present even in anaerobically grown cells. Aeration seemed to have little effect on the level of catalase, but the superoxide dismutase activity was 5-fold higher in cells grown aerobically. Under aerobic conditions considerable amounts of acetaldehyde and acetic acid were formed in addition to the normal fermentation products, ethanol and carbon dioxide.Dedicated to Professor Dr. H. G. Schlegel on the occasion of his 60th birthday     

Influence of glucose and dissolved oxygen concentrations on yields of Escherichia coli B in dialysis culture.

Yields of Escherichia coli B grown on glucose were determined in dialysis and non-dialysis culture. The molar growth yields were compared under conditions of excess glucose and oxygen as well as glucose- and oxygen-limiting conditions. The molar growth yields on glucose (YG) were determined for different periods during growth in non-dialysis cultures. A rapid decrease of YG was observed and growth ceased even in the presence of high concentrations of glucose and dissolved oxygen in the culture liquid. The decrease in YG was delayed in dialysis cultures where a high YG could be maintained at very high cell concentrations. The inhibition of growth depended on the accumulation of end-products of fermentative degradation of glucose. These products interfered with the oxidative phosphorylation. A large proportion of the glucose was fermented even in the presence of high concentrations of dissolved oxygen in the culture liquid. A decrease in the growth yield per g glucose was also observed.     

Redox driven metabolic tuning: carbon source and aeration affect synthesis of poly(3-hydroxybutyrate) in Escherichia coli

Growth and polymer synthesis were studied in a recombinant E. coli strain carrying phaBAC and phaP of Azotobacter sp. strain FA8 using different carbon sources and oxygen availability conditions. The results obtained with glucose or glycerol were completely different, demonstrating that the metabolic routes leading to the synthesis of the polymer when using glycerol do not respond to environmental conditions such as oxygen availability in the same way as they do when other substrates, such as glucose, are used. When cells were grown in a bioreactor using glucose the amount of polymer accumulated at low aeration was reduced by half when compared to high aeration, while glycerol cultures produced at low aeration almost twice the amount of polymer synthesized at the higher aeration condition. The synthesis of other metabolic products, such as ethanol, lactate, formate and acetate, were also affected by both the carbon source used and aeration conditions. In glucose cultures, lactate and formate production increased in low agitation compared to high agitation, while poly(3-hydroxybutyrate) synthesis decreased. In glycerol cultures, the amount of acids produced also increased when agitation was lowered, but carbon flow was mostly redirected towards ethanol and poly(3-hydroxybutyrate). These results indicated that carbon partitioning differed depending on both carbon source and oxygen availability, and that aeration conditions had different effects on the synthesis of the polymer and other metabolic products when glucose or glycerol were used.     

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.     

Repression of In Vivo Synthesis of the Mitochondrial Elongation Factors T and G in Saccharomyces fragilis

In vivo synthesis of the mitochondrial elongation factors T and G in the yeast Saccharomyces fragilis can be repressed. Enzymatic activity assays and immunochemical titration methods reveal that cells grown in the presence of 8% glucose or in the absence of oxygen contain relatively lower amounts of mitochondrial elongation factors than cells grown in the presence of lactate. In contrast, in vivo production of the cytoplasmic elongation factors 1 and 2 does not respond to such a change of extracellular conditions. The rate of growth does not affect the level of the mitochondrial elongation factors. Production of both enzymes is almost constant during logarithmic growth, but decreases when the stationary phase is reached. Chloramphenicol, an inhibitor of mitochondrial protein synthesis, does not block but, rather, seems to enhance the in vivo synthesis of mitochondrial T or G.     

Specific changes in the synthesis of mitochondrial DNA in chick embryo fibroblasts transformed by Rous sarcoma viruses

In chick-embryo fibroblasts infected with the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup A (wild type), or with a thermosensitive mutant of this virus, T5, the rates of mitochondrial DNA synthesis differ in cells that exhibit normal and malignant phenotypes. In wild type virus-infected cells grown at 36 or 41 degrees C, morphological transformation is expressed, the rate of 2-deoxy-D-[3H]glucose uptake is stimulated, and mitochondrial DNA synthesis in vivo is stimulated three- to fivefold over that in uninfected cells. In T5-infected cells these changes occur only at the permissive temperature (36 degrees C); a shift to the nonpermissive temperature (41 degrees C) causes the reversal of these effects, and the specific activity of purified mitochondrial DNA is characteristic of that from uninfected cells. In contrast, the specific activities of nuclear DNA purified from cells maximally transformed under the permissive conditions do not differ between wild type-infected and uninfected with the T5 virus. In parallel experiments with isolated mitochondria, the rate of mtDNA synthesis in vitro is again greater in mitochondria isolated from transformed cells. In addition, mitochondrial DNA synthesis in vitro in mitochondria from nontransformed and virus-transformed cells exhibits differential sensitivity to inhibition by mercaptoethanol. Furthermore, the ntDNAP polymerase activity in mitochondrial extracts prepared from cells with transformed phenotypes is about sevenfold higher than in extracts from cells with nontransformed phenotypes.     

Growth of aortic vascular smooth muscle cells in lowered oxygen tension

Summary Primary cultures of vascular smooth muscle cells, isolated from rat aorta, were grown under normoxic (20% O₂) and mildly hypoxic (5 % O₂) conditions. Cells from both conditions were compared for growth characteristics, morphology, protein synthesis, lysosomal enzyme activity, and oxygen consumption. In no case was a consistently significant difference observed. These observations indicate that these cells can adapt or are adapted to mildly hypoxic conditions. Moreover, these results may indicate that the culture of vascular smooth muscle cells in mild hypoxia represents a closer approximation of in vivo growth conditions for these cells.Supported by HL19242     

Environmental regulation of carbohydrate metabolism by Streptococcus sanguis NCTC 7865 grown in a chemostat

Carbohydrate metabolism by the oral bacterium Streptococcus sanguis NCTC 7865 was studied using cells grown in a chemostat at pH 7.0 under glucose or amino acid limitation (glucose excess) over a range of growth rates (D = 0.05 h-1-0.4 h-1). A mixed pattern of fermentation products was always produced although higher concentrations of lactate were formed under amino acid limitation. Analysis of culture filtrates showed that arginine was depleted from the medium under all conditions of growth; a further supplement of 10 mM-arginine was also consumed but did not affect cell yields, suggesting that it was not limiting growth. Except at the slowest growth rate (D = 0.05 h-1) under glucose limitation, the activity of the glucose phosphotransferase (PTS) system was insufficient to account for the glucose consumed during growth, emphasizing the importance of an alternative method of hexose transport in the metabolism of oral streptococci. The PTS for a number of sugars was constitutive in S. sanguis NCTC 7865 and, even though the cells were grown in the presence of glucose, the activity of the sucrose-PTS was highest. The glycolytic activity of cells harvested from the chemostat was affected by the substrate, the pH of the environment, and their original conditions of growth. Glucose-limited cells produced more acid than those grown under conditions of glucose excess; at slow growth rates, in particular, greater activities were obtained with sucrose compared with glucose or fructose. Maximum rates of glycolytic activity were obtained at pH 8.0 (except for cells grown at D = 0.4 h-1 where values were highest at pH 7.0), while slow-growing, amino acid-limited cells could not metabolize at pH 5.0. These results are discussed in terms of their possible significance in the ecology of dental plaque and the possible involvement of these bacteria in the initiation but not the clinical progression of a carious lesion.     

The effect of oxygen on the growth and acid production of Streptococcus mutans and Streptococcus sanguis

Abstract Pure cultures of Streptococcus mutans NCTC 10499 and Streptococcus sanguis ATCC10556 were grown in a glucose-limited chemostat under varying concentrations of oxygen in the gas phase. Both streptococci consumed large amounts of oxygen by the partial oxidation of sugars, thus maintaining an anaerobic environment. With increasing oxygen concentrations the degradation products from glucose become more oxidized. Ethanol gradually disappeared from the culture fluid while the acetate concentration increased. In the case of S. sanguis , the products became even more oxidized at higher oxygen concentrations, and carbon dioxide was formed instead of formate. Sudden increase in the oxygen concentration in the gas phase caused elevated oxygen tensions in the cultures, which led to a decrease in the growth rate of the streptococci.     

Formation of the yeast mitochondrial membrane. 3. Accumulation of mitochondrial proteins synthesized in both the cytoplasm and mitochondria in yeast undergoing glucose depression

The inhibitors of protein synthesis, chloramphenicol and cycloheximide, were added to cultures of yeast undergoing glucose derepression at different times during the growth cycle. Both inhibitors blocked the increase in activity of coenzyme QH₂-cytochrome c reductase, suggesting that the formation of complex III of the respiratory chain requires products of both mitochondrial and cytoplasmic protein synthesis.The possibility that precursor proteins synthesized by either cytoplasmic or mitochondrial ribosomes may accumulate was investigated by the sequential addition of cycloheximide and chloramphenicol (or the reverse order) to cultures of yeast undergoing glucose derepression. When yeast cells were grown for 3 hr in medium containing cycloheximide and then transferred to medium containing chloramphenicol, the activity of cytochrome oxidase increased at the same rate as the control during the first hour in chloramphenicol. These results suggest that some accumulation of precursor proteins synthesized in the mitochondria had occurred when cytoplasmic protein synthesis was blocked during the growth phase in cycloheximide. In contrast, essentially no products of mitochondrial protein synthesis accumulated as precursors for either oligomycin-sensitive ATPase or complex III of the respiratory chain during growth of the cells in cycloheximide.When yeast were grown for 3 hr in medium containing chloramphenicol followed by 1 hr in cycloheximide, the activities of cytochrome oxidase and succinate-cytochrome c reductase increased at the same rate as the control, while the activities of oligomycin-sensitive ATPase and NADH or coenzyme QH₂-cytochrome c reductase were nearly double that of the control. These data suggest that a significant accumulation of mitochondrial proteins synthesized in the cytoplasm had occurred when the yeast cells were grown in medium containing sufficient chloramphenicol to block mitochondrial protein synthesis. The possibility that proteins synthesized in the cytoplasm may act to control the synthesis of mitochondrial proteins for both oligomycin-sensitive ATPase and complex III of the respiratory chain is discussed.     

Demonstration of anaerobic catalase synthesis in the cz1 mutant of Saccharomyces cerevisiae.

Anaerobic synthesis of catalase T (a typical oxygen-inducible haem containing enzyme) in the cz1 mutant of yeast was demonstrated. The synthesis of catalase T in anaerobically grown mutant cells is stimulated by haemin under carbon-derepressed conditions of growth (galactose as carbon source) but not in glucose repressed cultures. Haem is practically undetectable in the anaerobically grown glucose repressed wild type strain and its level in derepressed cells amounts to 3% of the fully derepressed aerobically grown cells. In the cz1 mutant cultures grown in anoxia both on galactose and glucose the haem level usually exceeds 10% of that in the aerobic control.     

Regulation of synthesis of pyruvate carboxylase in the photosynthetic bacterium Rhodobacter capsulatus.

The synthesis of pyruvate carboxylase (PC) was studied by using quantitative immunoblot analysis with an antibody raised against PC purified from Rhodobacter capsulatus and was found to vary 20-fold depending on the growth conditions. The PC content was high in cells grown on pyruvate or on carbon substrates metabolized via pyruvate (lactate, D-malate, glucose, or fructose) and low in cells grown on tricarboxylic acid (TCA) cycle intermediates or substrates metabolized without intermediate formation of pyruvate (acetate or glutamate). Under dark aerobic growth conditions with lactate as a carbon source, the PC content was approximately twofold higher than that found under light anaerobic growth conditions. The results of incubation experiments demonstrate that PC synthesis is induced by pyruvate and repressed by TCA cycle intermediates, with negative control dominating over positive control. The content of PC in R. capsulatus cells was also directly related to the growth rate in continuous cultures. The analysis of intracellular levels of pyruvate and TCA cycle intermediates in cells grown under different conditions demonstrated that the content of PC is directly proportional to the ratio between pyruvate and C4 dicarboxylates. These results suggest that the regulation of PC synthesis by oxygen and its direct correlation with growth rate may reflect effects on the balance of intracellular pyruvate and C4 dicarboxylates. Thus, this important enzyme is potentially regulated both allosterically and at the level of synthesis.     

Fluxes and enzyme activities in central metabolism of myeloma cells grown in chemostat culture

Activities of enzymes in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, and glutaminolysis have been determined in the mouse myeloma SP2/0.Ag14. Cells were grown on IMDM medium with 5% serum in steady-state chemostat culture at a fixed dilution rate of 0.03 h-1. Three culture conditions, which differed in supply of glucose and oxygen, were chosen so as to change catabolic fluxes in the central metabolism, while keeping anabolic fluxes constant. In the three steady-state situations, the ratio between specific rates of glucose and glutamine consumption differed by more than twentyfold. The specific rates of glucose consumption and lactate production were highest at low oxygen supply, whereas the specific rate of glutamine consumption was highest in the culture fed with low amounts of glucose. Under low oxygen conditions, the specific production of ammonia increased and the consumption pattern of amino acids showed large changes compared with the other two cultures. For the three steady states, activities of key enzymes in glycolysis, the pentose phosphate pathway, glutaminolysis, and the TCA cycle were measured. The differences in the in vivo fluxes were only partially reflected in changes in enzyme levels. The largest differences were observed in the levels of glycolytic enzymes, which were elevated under conditions of low oxygen supply. High activities of phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32) in all cultures suggest an important role for this enzyme as a link between glutaminolysis and glycolysis. For all enzymes, in vitro activities were found that could accommodate the estimated maximum in vivo fluxes. These results show that the regulation of fluxes in central metabolism of mammalian cells occurs mainly through modulation of enzyme activity and, to a much lesser extent, by enzyme synthesis.     

Endothelial cell and platelet bioenergetics: effect of glucose and nutrient composition

It has been suggested that cells that are independent of insulin for glucose uptake, when exposed to high glucose or other nutrient concentrations, manifest enhanced mitochondrial substrate oxidation with consequent enhanced potential and generation of reactive oxygen species (ROS); a paradigm that could predispose to vascular complications of diabetes. Here we exposed bovine aortic endothelial (BAE) cells and human platelets to variable glucose and fatty acid concentrations. We then examined oxygen consumption and acidification rates using recently available technology in the form of an extracellular oxygen and proton flux analyzer. Acute or overnight exposure of confluent BAE cells to glucose concentrations from 5.5 to 25 mM did not enhance or change the rate of oxygen consumption (OCR) under basal conditions, during ATP synthesis, or under uncoupled conditions. Glucose also did not alter OCR in sub-confluent cells, in cells exposed to low serum, or in cells treated with added pyruvate. Likewise, overnight exposure to fatty acids of varying saturation had no such effects. Overnight exposure of BAE cells to low glucose concentration decreased maximal uncoupled respiration, but not basal or ATP related oxygen consumption. Labeled glucose oxidation to CO(2) increased, but only marginally after high glucose exposure while oleate oxidation to CO(2) decreased. Overnight exposure to linolenic acid, but not oleic or linoleic acid increased extracellular acidification consistent with enhanced glycolytic metabolism. We were unable to detect an increase in production of reactive oxygen species (ROS) from BAE cells exposed to high medium glucose. Like BAE cells, exposure of human platelets to glucose did not increase oxygen consumption. As opposed to BAE cells, platelet mitochondria demonstrate less respiratory reserve capacity (beyond that needed for basal metabolism). Our data do not support the concept that exposure to high glucose or fatty acids accelerates mitochondrial oxidative metabolism in endothelial cells or platelets.     

Uptake of oxygen,release and degradation of hydrogen peroxide by Streptococcus mutans NCTC 10449

Streptococcus mutans NCTC 10499 was cultured under glucose limitation in a chemostat at varying oxygen supply. The rates of oxygen uptake and hydrogen peroxide degradation by cells from the cultures were measured polarographically using a Clark electrode. Oxygenation of the chemostat culture led to adaptation of the organism to oxygen, in that the maximum oxygen uptake rate of the cells was higher when the cells were grown at higher rate of oxygen supply. It is noted that anaerobically grown cells still exhibited significant oxygen uptake. The rate of oxygen uptake followed saturation-type kinetics and Ks values of cells for oxygen were in the micromole range. Hydrogen peroxide accumulation was not observed in aerated chemostat cultures. However, anaerobically grown cells accumulated H2O2 when exposed to oxygen. Cells from aerated cultures did not accumulate hydrogen peroxide. This may be explained by the fact that the rate of hydrogen peroxide degradation was consistently higher than the rate of oxygen uptake.     

Growth condition-dependent sensitivity, photodamage and stress response of Chlamydomonas reinhardtii exposed to high light conditions

Different substrate conditions, such as varying CO(2) concentrations or the presence of acetate, strongly influence the efficiency of photosynthesis in Chlamydomonas reinhardtii. Altered photosynthetic efficiencies affect the susceptibility of algae to the deleterious effects of high light stress, such as the production of reactive oxygen species (ROS) and PSII photodamage. In this study, we investigated the effect of high light on C. reinhardtii grown under photomixotrophy, i.e. in the presence of acetate, as well as under photoautotrophic growth conditions with either low or high CO(2) concentrations. Different parameters such as growth rate, chlorophyll bleaching, singlet oxygen generation, PSII photodamage and the total genomic stress response were analyzed. Although showing a similar degree of PSII photodamage, a much stronger singlet oxygen-specific response and a broader general stress response was observed in acetate and high CO(2)-supplemented cells compared with CO(2)-limited cells. These different photooxidative stress responses were correlated with the individual cellular PSII content and probably directly influenced the ROS production during exposure to high light. In addition, growth of high CO(2)-supplemented cells was more susceptible to high light stress compared with cells grown under CO(2) limitation. The growth of acetate-supplemented cultures, on the other hand, was less affected by high light treatment than cultures grown under high CO(2) concentrations, despite the similar cellular stress. This suggests that the production of ATP by mitochondrial acetate respiration protects the cells from the deleterious effects of high light stress, presumably by providing energy for an effective defense.