Adenosine phosphates and the control of glycolysis and gluconeogenesis in yeast

1. Changes in dry weight, protein, RNA and DNA were measured in yeast during adaptation to glycolytic metabolism. 2. Only RNA increased significantly during the lag phase, but during the exponential phase all these cellular components increased in parallel. 3. The concentrations of ATP, ADP, AMP and glucose 6-phosphate were measured in respiring yeast and during the transition to glycolytic metabolism. 4. In respiring cells the concentration of AMP was at its highest and that of ATP was at its lowest; this relationship was reversed in glycolysing cells. 5. ADP concentration was similar in respiring and glycolysing cells, but glucose 6-phosphate concentration was much higher in the glycolysing cells. 6. A possible reason for mitochondrial repression is suggested. 7. It is concluded that adenosine phosphates do not control the direction of glycolytic flux in yeast and an alternative control of glycolysis and gluconeogenesis by enzyme activation and inactivation is suggested.     

Effect of the growth phase and nutrient medium on the survival of lyophilized cells of Pseudomonas denitrificans]

The influence of the growth phase and cultivation condition of Pseudomonas denitrificans on the survival of the culture after lyophilization was examined. The culture removed for drying in the stationary phase showed the highest resistance to lyophilization. The cells dried at the beginning of exponential phase displayed enhanced sensitivity to lyophilization. Addition to the cultivation medium (meat-peptone broth) of Tween-80 (0.25%) caused an increase of proteins, lipids and polysaccharides in cells and an enhancement of their telerance to lyophilization.     

Effect of protein synthesis inhibitors on the activity of mitochondrial enzymes of Saccharomyces cerevisiae during desiccation

The work deals with the effect of carbon sources, presence of protein synthesis inhibitors (cycloheximide and chloramphenicol) and dehydration regime on the enzyme activity of the dried yeast Saccharomyces cerevisiae. The yeast grown on molasses and dried by aeration demonstrated a notable increase of the NADH-dehydrogenase and succinate dehydrogenase activities as compared with the analogously treated yeast grown on ethanol. The latter showed a notable rise only in the activity of NADH-dehydrogenase during slow drying. Addition of protein synthesis inhibitors into the cultivation medium caused no decrease of activities of the above mentioned enzymes of the dried yeasts in any variant under study.     

Effect of zinc on adenine nucleotide pools in relation to aflatoxin biosynthesis in Aspergillus parasiticus.

The adenylic acid systems of Aspergillus parasiticus were studied in zinc-replete and zinc-deficient media. The adenosine 5'-triphosphate levels of the fungus were high during exponential phase and low during stationary phase in zinc-replete cultures. On the other hand, the levels of adenosine 5'-diphosphate and adenosine 5'-monophosphate were low during exponential phase of growth and high during stationary phase. The adenosine 5'-triphosphate levels during exponential phase may indicate higher primary metabolic activity of the fungus. On the other hand, high adenosine 5'-monophosphate levels during stationary phase may inhibit lipid formation and may enhance aflatoxin levels. The inorganic phosphorus content was low in a zinc-replete medium throughout the growth period, thereby favoring aflatoxin biosynthesis. The energy charge during the exponential phase was high but low during the stationary phase. In general the energy charge values were lower because of high adenosine 5'-monophosphate content.     

Response of the intracellular adenosine triphosphate pool of Saccharomyces cerevisiae to growth inhibition induced by excess L-methionine

Yeast cells accumulate S-adenosyl-l-methionine (S-AM) when cultivated in the presence of l-methionine. Cell growth is inhibited by the addition of high concentrations of l-methionine. A number of investigators have attributed this to the depletion of adenosine triphosphate (ATP) as a consequence of the utilization of that mucleotide for S-AM formation. The cellular ATP pool of Saccharomyces cerevisiae was measured during growth inhibition caused by addition of excess l-methionine. Polyethylenimine thin-layer chromatography and subsequent autoradiography were used to quantitate the extracted ATP. Addition of l-methionine to a level of 5 mg/ml in a culture during exponential growth caused an increase in the doubling time of 40 to 50%. During this period, the cellular ATP level continued increasing normally and, as the cells entered stationary growth, receded to a level characteristic of an uninhibited stationary culture growth. After the addition of methionine, there was never an observed depletion of the ATP pool other than the normal fluctuation which occurs in an uninhibited culture. We have concluded that growth inhibition by excessive methionine does not result from limiting availability of ATP.     

Characteristics of cellular membranes at rehydration of dehydrated yeast Saccharomyces cerevisiae

Summary This paper describes the characteristics of the structural and functional organization of cellular membranes rehydrated after dehydration of the yeast Saccharomyces cerevisiae. It was noted that dehydration and subsequent rehydration of yeast cells causes a considerable increase of cytoplasmic membrane permeability. Addition of CaCl₂, glucose and polyethyleneglycol to the rehydration medium caused a decrease in cell permeability, assessed as the losses of potassium ions, nucleotides, as well as the total losses of intracellular compounds. KCl had a positive effect only at concentrations above 10%. Yeast cells, dried to residual moisture lower than 20%, showed a decrease in membrane permeability as temperatures of the rehydration medium increased up to 38°–43°C. Upon reactivation of viable dehydrated cells in a nutrient medium, a reparation of the structural damages of various intracellular membranes takes place. It was established that at cell dehydration to residual moistures of 8%–12% all the free and a part of bound water is evaporated from cells.     

Changes in the intracellular pool of free amino acids of Streptococcus lactis under different cultivation conditions]

The intracellular pool of free amino of Streptococcus lactis--lysine producer contains a good number of amino acids when cultivated on the corn medium. Glutamic acid, proline, alanine, lysine, leucine, histidine and arginine are in predominance. An almost complete amino acid pool develops at an early exponential phase of Str. lactis growth under stationary cultivation conditions. The content of free amino acids increases 4-fold during the transition from the early exponential phase to the stationary phases under submerged cultivation conditions. This can be attributed to a more intensive amino acid exchange during the medium stirring than during stationary cultivation.     

Development and activation of cyanide-resistant respiration in the yeast Yarrowia lipolytica.

Changes in respiratory activity and in the contents of adenine nucleotides (ATP, ADP, AMP) were studied in cells of the yeast Yarrowia lipolytica during the development of cyanide-resistant respiration. The transition of the yeast from the logarithmic to the stationary growth phase due to exhaustion of glucose was associated with decreased endogenous respiration and with the activation of a cyanide-resistant oxidase. Cyanide activated cell respiration during the stationary growth phase. The cyanide-resistant respiration was inhibited by benzohydroxamic acid (BHA), an inhibitor of the alternative oxidase. In the absence of cyanide, BHA had no effect on the cells which had the cyanide-resistant oxidase. This indicates that the cells do not use the alternative pathway in vivo. The decreased endogenous respiration of the cells was accompanied by decreased contents of adenine nucleotides. Addition of cyanide resulted in a sharp decrease in the content of ATP, in a twofold increase in the content of ADP, and in a fivefold increase in the content of AMP. In the absence of cyanide, BHA had virtually no effect on the contents of adenine nucleotides. The decreased rate of oxygen consumption during the transition of the cells to the stationary growth phase was caused by the decreased activity of the main cytochrome-containing respiratory chain (2,4-dinitrophenol (DNP) stimulated respiration). The alternative oxidase was synthesized in the cell but was inactive. Cyanide stimulated respiration due to activation of the alternative oxidase via the AMP produced. The decrease in the cell content of ATP is suggested to be a factor inducing the synthesis of the alternative oxidase.     

Activation of the alternative oxidase of Yarrowia lipolytica by adenosine 5'-monophosphate

The study of the effect of nucleoside phosphates on the activity of cyanide-resistant oxidase in the mitochondria and the submitochondrial particles of Yarrowia lipolytica showed that adenosine monophosphate (5'-AMP, AMP) did not stimulate the respiration of the intact mitochondria. The incubation of the mitochondria at room temperature (25 degrees C) for 3-5 h or their treatment with ultrasound, phospholipase A, and detergent Triton X-100 at a low temperature inactivated the cyanide-resistant alternative oxidase. The inactivated alternative oxidase could be reactivated by AMP. The reactivating effect of AMP was enhanced by azolectin. Some other nucleoside phosphates also showed reactivating ability in the following descending order. AMP = GMP > GDP > GTP > XMP > IMP. The apparent reaction rate constant Km for AMP upon the reactivation of the alternative oxidase of mitochondria treated with Triton X-100 or incubated at 25 degrees C was 12.5 and 20 microM, respectively. The Km for AMP upon the reactivation of the alternative oxidase of submitochondrial particles was 15 microM. During the incubation of yeast cells under conditions promoting the development of alternative oxidase, the content of adenine nucleotides (AMP, ADP, and ATP) in the cells and their respiration tended to decrease. The subsequent addition of cyanide to the cells activated their respiration, diminished the intracellular content of ATP three times, and augmented the content of AMP five times. These data suggest that the stimulation of cell respiration by cyanide may be due to the activation of alternative oxidase by AMP.     

Exponential growth phase cells of the osmotolerant yeast Debaryomyces hansenii are extremely resistant to dehydration stress

The osmotolerant yeast Debaryomyces hansenii is highly resistant to dehydration stress and this tolerance was more pronounced for cells taken from the exponential growth phase than from the stationary phase. Growth of D. hansenii in medium containing 10% (w v⁻¹) NaCl, resulted in an additional increase in cellular resistance to dehydration, which was most marked for stationary phase cells. It is expected that further investigations of the mechanisms behind this exceptional dehydrational tolerance will reveal new approaches for improvement of the quality of dry yeast.     

Trehalose as cryoprotectant for preservation of yeast strains

Preservation of genetic banks of yeast strains as well as of any kind of eukaryotic cells during dehydration and subsequent rehydration depends upon the maintenance of the integrity of the cell membrane. Trehalose has been successfully used as a non-toxic cryoprotectant for plant cells (Bhandal et al., 1985), as well as for lobster sarcoplasmic vesicles (Rudolph and Crowe, 1985). The hypothesis underlying these observations is that the disaccharide avoids fusion of membranes by replacing water molecules in the bilayer (Crowe et al., 1984). The viability of yeast strains submitted to different drying techniques is reported in this paper. Mutant strains with defects in the regulation of the trehalose-6-phosphate synthase complex were compared. Yeast strains dried in layers at 37°C for 6 h did not lose their viability, however, they died thereafter at 5°C, unless trehalose was used for resuspending the cells before drying. It should be noted that no trehalose accumulation was seen during drying at 37°C under our experimental conditions. In experiments in which cells were frozen at −120°C, addition of 10% trehalose to the suspending buffer had a significant protective effect. On the other hand, a mutant strain with an extremely high trehalose-6-phosphate synthase activity showed an intrinsic capacity for survival which did not depend upon addition of exogenous trehalose. This raises the question of the location of the internal trehalose pool and whether it could replace the externally added cryoprotectant.     

Spray drying as a method for preparing concentrated cultures of Lactobacillus bulgaricus

P. TEIXEIRA, H. CASTRO AND R. KIRBY. 1995. Spray drying and freeze drying as methods for concentration of Lactobacillus bulgaricus starter cultures were compared in terms of viability, lag phase until onset of pH decrease and total acid production. For the experimental conditions used, no significant differences were detected between the methods.
The effect of spray drying on the cell membrane of Lactobacillus bulgaricus was studied. Five separate methods were used to study the theory that spray drying causes cell membrane damage; three relating to leakage of intracellular components from the cell into the surrounding environment (260 and 280 nm absorbing materials, potassium ions and proteins); and two relating to increased cell permeability (increased sensitivity to NaCl and increased permeability to o -nitrophenyl-β-D-galactopyranoside (ONPG). Partial loss of some cytoplasmic material from the damaged cells was observed. The dried cells also became sensitive to NaCl and permeable to ONPG. Heat shock increased the survival of exponential cells as compared to controls but did not result in normal levels found with unshocked stationary phase cells. Heat shock had no effect on stationary phase cells. Different rehydration methods and media were investigated: slow rehydration increased survival.     

Dehydrating phospholipid vesicles measured in real-time using ATR Fourier transform infrared spectroscopy

According to the water replacement hypothesis, trehalose stabilizes dry membranes by preventing the decrease in spacing between adjacent phopspholipid headgroups during dehydration. Alternatively, the water-entrapment hypothesis postulates that in the dried state sugars trap residual water at the biomolecule sugar interface. In this study, Fourier transform infrared spectroscopy with an attenuated total reflection accessory was used to investigate the influence of trehalose on the dehydration kinetics and residual water content of egg phosphatidylcholine liposomes in real time under controlled relative humidity conditions. In the absence of trehalose, the lipids displayed a transition to a more ordered gel phase upon drying. The membrane conformational disorder in the dried state was found to decrease with decreasing relative humidity. Even at a relative humidity as high as 94% the conformational disorder of the lipid acyl chains decreased after evaporation of the bulk water. The presence of trehalose affects the rate of water removal from the system and the lipid phase behavior. The rate of water removal is decreased and the residual water content is higher, as compared to drying in the absence of trehalose. During drying, the level of hydrogen bonding to the head groups remains constant. In addition, the conformational disorder of the lipid acyl chains in the dried state more closely resembles that of the lipids in the fully hydrated state. We conclude that water entrapment rather than water replacement explains the effect of trehalose on lipid phase behavior of phosphatidylcholine lipid bilayers during the initial phase of drying.     

Effect of growth phase on the content and composition of ceramides of the hydrocarbon-assimilating yeast Candida lipolytica.

Candida lipolytica yeast was grown batchwise on n-hexadecane as the carbon and energy source. Ceramides were quantitatively isolated from total lipids of exponential and stationary phase cells by a combination of column chromatography and preparative high-performance thin-layer chromatography. After acid methanolysis their composition was analyzed by gas-liquid chromatography. The ceramide content of the exponential phase cells was two times higher than the one of the stationary phase cells. The composition of long-chain base moiety of ceramides did not change significantly during the growth. In both growth phases 19-phytosphingosine was the major long-chain base. However, the fatty acid composition of ceramides changed greatly during the growth. In the exponential growth phase, ceramides contained predominantly fatty acids greater than 20 carbon atoms, while fatty acids shorter than 20 atoms predominated in ceramides of the stationary phase, 16:0 being the main one. In the exponential growth phase fatty acid moiety of ceramides was characterized by unusually high degree of unsaturation and relatively high proportion of odd-numbered fatty acids. However, the proportion of both, unsaturated and odd-numbered fatty acid decreased significantly in ceramides of the stationary phase. The unexpected finding was the absence of fatty acid hydroxylation of ceramides in the exponential phase cells and unusually low degree of hydroxylation in the stationary phase.     

Production of reactive oxygen species and loss of viability in yeast mitochondrial mutants: protective effect of Bcl-xL

The capacity of yeast cells to produce reactive oxygen species (ROS), both as a response to manipulation of mitochondrial functions and to growth conditions, was estimated and compared with the viability of the cells. The chronological ageing of yeast cells (growth to late-stationary phase) was accompanied by increased ROS accumulation and a significantly higher loss of viability in the mutants with impaired mitochondrial functions than in the parental strain. Under these conditions, the ectopic expression of mammalian Bcl-x(L), which is an anti-apoptotic protein, allowed cells to survive longer in stationary phase. The protective effect of Bcl-x(L) was more prominent in respiratory-competent cells that contained defects in mitochondrial ADP/ATP translocation, suggesting a model for Bcl-x(L) regulation of chronological ageing at the mitochondria. Yeast can also be triggered into apoptosis-like cell death, at conditions leading to the depletion of the intramitochondrial ATP pool, as a consequence of the parallel inhibition of mitochondrial respiration and ADP/ATP translocation. If respiratory-deficient (rho(0)) cells were used, no correlation between the numbers of ROS-producing cells and the viability loss in the population was observed, indicating that ROS production may be an accompanying event. The protective effect of Bcl-x(L) against death of these cells suggests a mitochondrial mechanism which is different from the antioxidant activity of Bcl-x(L).     

Influence of chronological aging on the survival and nucleotide content of Saccharomyces cerevisiae cells grown in different conditions: occurrence of a high concentration of UDP-N-acetylglucosamine in stationary cells grown in 2% glucose

Saccharomyces cerevisiae cells (strain W303) grown in a minimal medium (containing 2% or 0.1% glucose) until exponential or stationary phase, were subjected to chronological aging in water, and yeast viability and nucleotide content were analyzed along several days of nutrient starvation. Cells collected in exponential phase (whether grown in the presence of 0.1% or 2% glucose) were viable up to five days and thereafter the viability decreased linearly with a half-survival rate of around eight days. ATP and other nucleoside triphosphates decreased similarly in both cases. Cells collected in stationary phase, and transferred to water, behaved differently whether grown in 0.1% or in 2% glucose, with a half-survival life of around nine and 28 days respectively. A double mutant in glycogen synthase (gsy1delta gsy2delta) and its isogenic wild-type strain, grown to stationary phase in 2% glucose, presented a similar half-survival life of around eight days. The W303 cells grown to stationary phase in the presence of 2% glucose showed a 7-fold increase of UDP-N-acetylglucosamine (UDP-GlcNAc) as compared with the level present in the cells grown in any of the other three metabolic situations. The nature of UDP-GlcNAc was established by MALDI-TOF ionization analysis. It is also worth noting that the rate of decay of NAD+ was lower than that of ATP in any of the situations here considered.     

Osmotically induced intracellular trehalose, but not glycine betaine accumulation promotes desiccation tolerance in Escherichia coli

Trehalose considerably increased the tolerance of Escherichia coli to air drying, whether added as an excipient prior to drying or accumulated as a compatible solute in response to osmotic stress. The protective effect of exogenously added trehalose was concentration dependent, up to a threshold value of 350 mM. However, trehalose alone cannot explain the intrinsically greater desiccation tolerance of stationary compared to exponential phase E. coli cells, although their tolerance was also enhanced by exogenous or endogenously accumulated trehalose. In contrast, glycine betaine whether added as an excipient or accumulated intracellularly had no influence on desiccation tolerance. These data demonstrate that the protection provided by compatible solutes to cells subjected to desiccation differs from that during osmotic stress, due to the much greater reduction in available cell water. The protective effects of trehalose during desiccation appear to be due to its stabilising influence on membrane structure, its chemically inert nature and the propensity of trehalose solutions to form glasses upon drying, properties which are not shared by glycine betaine.     

Mitochondrial respiratory thresholds regulate yeast chronological life span and its extension by caloric restriction

We have explored the role of mitochondrial function in aging by genetically and pharmacologically modifying yeast cellular respiration production during the exponential and/or stationary growth phases and determining how this affects chronological life span (CLS). Our results demonstrate that respiration is essential during both growth phases for standard CLS, but that yeast have a large respiratory capacity, and only deficiencies below a threshold (~40% of wild-type) significantly curtail CLS. Extension of CLS by caloric restriction also required respiration above a similar threshold during exponential growth and completely alleviated the need for respiration in the stationary phase. Finally, we show that supplementation of media with 1% trehalose, a storage carbohydrate, restores wild-type CLS to respiratory-null cells. We conclude that mitochondrial respiratory thresholds regulate yeast CLS and its extension by caloric restriction by increasing stress resistance, an important component of which is the optimal accumulation and mobilization of nutrient stores.     

The ultrastructure of the cell surface and plasma membrane of exponential and stationary phase cells of Schizosaccharomyces pombe,grown in different media

In order to study the ultrastructure of the cell surface and plasma membrane of Schizosaccharomyces pombe as a function of growth conditions we investigated exponential and stationary phase cells grown in rich and minimal medium.Electron microscopic preparation techniques based on rapid cryofixation (without cryoprotectants) were used. The intramembraneous aspects of the plasma membrane were described by freeze fracturing. For the first time the dynamic surface structures could be directly analyzed by freeze drying in the scanning electron microscope and in thin section of freeze substituted samples. This preparation techniques reveal hair-like structures on the surface of yeast cells. The hairs of cells grown in the rich medium are longer than those grown in the minimal medium. A mutant defective in the structure of a cell surface galactomannoprotein (acid phosphatase) reveals (under conditions of maximal acid phosphatase expression) a cell surface structure that differs from the wild type. It is likely that the hairs represent the peripheral galactomannan layer or part of it.On the membrane fracture faces the number, shape, distribution and state of aggregation of the intramembraneous particles are different between membranes of growing and non-growing cells and between cells grown under different physiological conditions. In the minimal medium corresponding periodical structures on the plasmic and exoplasmic fracture faces were observed, which clearly differ between exponential and stationary phase cells. The number, length and depth of plasma membrane invaginations increase as the cells go from the exponential phase to the stationary phase. Short and flattened invaginations are filled with thin periodic structures.