Inorganic polyphosphate metabolism in Staphylococcus aureus and the action on it of antibiotics]

The culure of Staph. aureus in the exponential growth phase contained 14-18 mg/g of dry orthophosphate biomass and 18-22 mg/g of dry acid insoluble polyphosphate biomass. The extracellular extract of the culture had a phosphohydrolase activity with respect to high molecular polyphosphates, tripolyphosphate and pyrophosphate. Penicillin and bacitracin which inhibited the biosynthesis of the cell wall had no effect on the content of polyphosphates and the phosphohydrolase activity of Staph. aureus. Heliomycin which inhibited the biosynthesis of RNA increased the content of polyphosphates by 1.5 times and decreased the content of ATP by 30 per cent in the cells of Staph. aureus.     

Effect of surface-active agents (tween-21) on indices of energy metabolism in oleandomycin producers

The specific growth rate of Streptomyces antibioticus, a producer of oleandomycin, and the specific rate of the antibiotic accumulation in the culture medium during fermentation were investigated. On the basis of the results obtained the fermentation period was divided into 7 phases of development. The culture treated with the surfactant (Tween-21) is characterized by a higher specific growth rate during the whole fermentation and a higher specific rate of the antibiotic accumulation at the stage of the highest production as compared to the control. The ATP content, the value of the adenylate energy charge and the contents of high-molecular weight polyphosphates in the mycelium were examined. In the phase of the intensive growth St. antibioticus was characterized by a higher ATP level and a higher energy charge. More active accumulation of polyphosphates was observed in the late intensive growth phase. It was also found that after the treatment of the culture with Tween-21 it utilized polyphosphates more actively during the antibiotic biosynthesis.     

Divalent cation mobility throughout exponential growth and sporulation of Bacillus megaterium.

Each of the five elements considered was taken up by Bacillus megaterium during exponential growth. Initial Mg and Mn uptake was rapid and ended by mid-log. For Ca, Fe, and Zn, uptake continued throughout exponential growth. Elements were released from the cells immediately following initial uptake. For Mn, egression continued to t2, with release of 36% of total accumulated. Secondary uptake followed immediately and continued through stage V. Magnesium egression continued to t1 with release of 33% accumulated. Secondary uptake began by t5 (stage IV) and continued slowly through sporulation. Calcium egression ceased by t4 with release of 25% total accumulated. Secondary uptake began by t6 (stage V) and continued until depleted. Zinc egression stopped by t5 with release of 34% accumulated with some secondary uptake by stage V. Iron egression terminated at t4 with release of 59% of total accumulated. This was followed by secondary uptake after t12 (stage VI).     

45Ca2+ uptake in rat brain neurons: absence of sensitivity to the Ca2+ channel ligands nitrendipine and Bay K 8644

K+-stimulated 45Ca2+ uptake into intact rat brain cells was biphasic, consisting of a fast first phase and a slow second phase; the latter was Na+ dependent. Cobalt and cadmium at 10(-4) and 10(-3) M produced 19-97% block of first phase 45Ca2+ uptake, but nitrendipine (to 10(-6) M) and Bay K 8644 (to 10(-6) M) were without effect on uptake and were similarly without effect in cells prepared in the presence of ATP, cAMP, Mg2+, and protease inhibitors. The second phase of K+-stimulated 45Ca2+ uptake was inhibited by 3,4-dichlorobenzamil (IC50, 29.6 microM). Depolarization-induced 45Ca2+ uptake into intact rat brain cells occurs by at least two different mechanisms. The first phase probably represents uptake through 1,4-dihydropyridine-insensitive Ca2+ channels, while the second phase is probably due to Na+-Ca2+ exchange.     

Changes of ATP, polyphosphate and K+ contents in Saccharomyces carlsbergensis during uptake of Mn2+ and glucose

The process of prolonged Mn2+ uptake by the yeast Saccharomyces carlsbergensis in the presence of 100 mM glucose and in the absence of phosphate can be divided into two steps. The first step (0-20 min) of Mn2+ uptake (4.3 mumol/g of wet cells) is characterized by an intense K+ efflux (23.8 mumol/g), synthesis of high molecular weight polyphosphate (HPP) (8.1 mumol/g) and decrease of ATP content (0.06 mumol/g). Simultaneously about 0.6 mumol of glucose is taken up and the level of low molecular weight polyphosphate (LPP) remains practically unchanged. The second step (20-120 min) of Mn2+ uptake (15.6 mumol/g) is characterized by a drop in HPP (16.6 mumol/g) and the synthesis of LPP (19.0 mumol/g). The ATP content decreases by 0.87 mumol/g as compared to the control, while that of K+ increases (5.7 mumol/g). During the first step of Mn2+ uptake the energy of the K+ concentration gradient may be used both for Mn2+ influx (2K+: 1Mn2+) and synthesis of HPP (1P:1.9K+). During the second step the Mn2+ accumulation is apparently driven by HPP conversion into LPP (1:1) and by ATPases serving the Mn2+/H+ exchange.     

Interrelationship of polyphosphate metabolism and levorin biosynthesis in Streptomyces levoris

The effect of inorganic phosphate on biosynthesis of the polyene antibiotic levorin by Streptomyces levoris was studied. At phosphate concentration of 4.0 mM levorin biosynthesis is repressed by 90%, resulting in an increase of ATP and a condensed inorganic polyphosphates content in the producer cells. At phosphate concentration optimal for levorin production (0.04 mM) the level of intracellular ATP sharply falls by the beginning of the steady-state phase of the producer growth and that of polyphosphates decreases 3-6-fold. The inorganic phosphate exerts different effects on polyphosphate metabolism enzymes, such as polyphosphate: D-glucose-6-phosphotransferase, polyphosphate phosphohydrolase, tripolyphosphate phosphohydrolase, pyrophosphate phosphohydrolase, alkaline and acid phosphatase. The strongest effect of phosphate excess is observed in the case of polyphosphate: D-glucose-6-phosphotransferase, whose activity decreases 2-5-fold. The activity of this enzyme was shown to be correlated with the antibiotic accumulation. The data obtained are indicative of interrelationship between the polyphosphate metabolism and levorin biosynthesis.     

Inorganic polyphosphates of different fractions in the mutant yeast Saccharomyces cerevisiae with impaired mitochondrial ATP synthesis

Impaired synthetase function of the mitochondrial ATPase induced by mutation in the ATP22 gene results in decreased accumulation of inorganic polyphosphates in the stationary growth phase of the yeast Saccharomyces cerevisiae grown on glucose. The content of polyphosphates in the mutant strain in this phase is 2.5 times lower than in the parent strain. This difference is most pronounced for the acid-soluble polyP1 fraction and the alkali-soluble polyP3 fraction. Polyphosphate chain length in mutant cells is less than in the parent cells in both the acid-soluble polyP1 and in the salt-soluble polyP2 fractions. The mutation had no effect on polyphosphates content in the mitochondria.     

On-line estimation of viable cells in a hybridoma culture at various DO levels using ATP balancing and redox potential measurement

Two on-line methods for the estimation of viable cell number in hybridoma cultivation were investigated. One used an empirical correlation between redox potential and animal cell density. The other was based on an ATP balance with ATP steady-state assumption. Oxygen uptake rate measurement provided the amount of ATP which was produced by oxidation of NADH. Oxygen uptake rate was measured either by stationary liquid phase balance with surface aeration or by gas balance during bubble aeration with headspace flushing with an inert gas. The amount of ATP produced through the glycolysis was estimated based on the amount of lactate produced. In cultures, in which pH was controlled via manipulation of the gas phase composition, the flow of CO(2) was linearly correlated with the lactate concentration. At constant dissolved oxygen levels, the viable cell density was proportional to the estimated ATP production rate, during exponential growth and during later phases. The estimated specific ATP production rate, however, varied from 2.2 pmol cell(-1) h(-1) at 10% air saturation to 4.5 pmol cell(-1) h(-1) at 100% air saturation. Specific rates of glutamine, glucose, and lactate followed the shape of the specific ATP production rate, whereas the specific oxygen uptake rate was minimal at around 50% air saturation. (c) 1996 John Wiley & Sons, Inc.     

Manganese oxidation by an intracellular protein of a Pseudomonas species.

Cultures of a Pseudomonas sp. strain MnB 1 produce an intracellular, manganese oxidizing protein (abbrev. as Mn ox. protein) during the stationary phase of growth. This protein is heat labile, can be inactivated by protease and has a pH-optimum for manganese oxidation at pH 7.0. Mn2+ is oxidized only at concentrations below 3-10(-5) M. The occurrence of the protein is not dependent on the presence of Mn2+, but is clearly related to the cessation of growth after the end of the exponential growth phase. Oxygen, coenzymes, and low molecular weight components of the cell extract seem not to be involved in the reaction as electron acceptors for the oxidation of Mn2+. Continued manganese oxidation by Mn ox. protein results in a progressive decrease in activity which corresponds to the amount of formed manganese oxide.     

Cell growth and quabain-sensitive 86Rg+ uptake and (Na++K+)-ATPase activity in 3T3 and SV40 transformed 3T3 fibroblasts.

The uptake of ouabain-sensitive 86Rb+ uptake measured at 5 min and the uptake measured at 60 min was 4.5- and 2.7-fold greater respectively for SV40 transformed 3T3 cells compared to 3T3 cells during the late log phase of growth. This uptake, however, varied markedly with cell growth. Ouabain-sensitive 86Rb+ uptake was found to be a sensitive indicator of protein synthesis as measured by total protein content. Cessation of cell growth as measured by total protein content was associated with a decline in ouabain-sensitive 86Rb+ uptake in both cell types. This increase ouabain-sensitive cation transport was reflected in increased levels of (Na++K)-ATPase activity for SV40 3T3 cells, which showed a 2.5-fold increase V but the same Km as 3T3 cells. These results are compared with the results of related work. Possible mechanisms for these effects are discussed and how changes in cation transport might be related to alterations in cell growth.     

Bacterial acetone carboxylase is a manganese-dependent metalloenzyme

Bacterial acetone carboxylase catalyzes the ATP-dependent carboxylation of acetone to acetoacetate with the concomitant production of AMP and two inorganic phosphates. The importance of manganese in Rhodobacter capsulatus acetone carboxylase has been established through a combination of physiological, biochemical, and spectroscopic studies. Depletion of manganese from the R. capsulatus growth medium resulted in inhibition of acetone-dependent but not malate-dependent cell growth. Under normal growth conditions (0.5 microm Mn2+ in medium), growth with acetone as the carbon source resulted in a 4-fold increase in intracellular protein-bound manganese over malate-grown cells and the appearance of a Mn2+ EPR signal centered at g = 2 that was absent in malate-grown cells. Acetone carboxylase purified from cells grown with 50 microm Mn2+ had a 1.6-fold higher specific activity and 1.9-fold higher manganese content than cells grown with 0.5 microm Mn2+, consistently yielding a stoichiometry of 1.9 manganese/alpha2beta2gamma2 multimer, or 0.95 manganese/alphabetagamma protomer. Manganese in acetone carboxylase was tightly bound and not removed upon dialysis against various metal ion chelators. The addition of acetone to malate-grown cells grown in medium depleted of manganese resulted in the high level synthesis of acetone carboxylase (15-20% soluble protein), which, upon purification, exhibited 7% of the activity and 6% of the manganese content of the enzyme purified from acetone-grown cells. EPR analysis of purified acetone carboxylase indicates the presence of a mononuclear Mn2+ center, with possible spin coupling of two mononuclear sites. The addition of Mg.ATP or Mg.AMP resulted in EPR spectral changes, whereas the addition of acetone, CO2, inorganic phosphate, and acetoacetate did not perturb the EPR. These studies demonstrate that manganese is essential for acetone carboxylation and suggest a role for manganese in nucleotide binding and activation.     

The effects of “Cell age” upon the lethal effects of physical and chemical mutagens in the yeast, Saccharomyces cerevisiae

Summary SummaryYeast cultures progressing from the exponential to the stationary phase of growth showed changes in cell sensitivity to physical agents such as UV light, heat shock at 52° C and the chemical mutagens ethyl methane sulphonate, nitrous acid and mitomycin C.Exponential phase cells showed maximum resistance to UV light and minimum resistance to heat shock and the three chemicals. The increased resistance of exponential phase cells to UV light was shown to be dependent upon the functional integrity of the RAD ₅₀ gene.Treatment of growing yeast cultures with radioactively labelled ethyl methane sulphonate indicated the preferential uptake of radioactivity during the sensitive exponential stage of growth. The results indicated that the differential uptake of the chemical mutagens was responsible for at least a fraction of the variations in cell sensitivity observed in yeast cultures at different phases of growth.     

Levels of adenylic system components (ATP, ADP, AMP) and cAMP during development of Blastocladiella emersonii

Concentrations of ATP, ADP, AMP, cAMP as well as pyruvate and glucose-6-phosphate were measured in B. lastocladiella emersonii cells developing via RS morphogenetic pathway. They varied significantly in the course of development (1.3-14.8 mumole/g dry weight for the sum of ATP+ADP+AMP; 0.012-5.3 nmole for cAMP; 0.47-1.9 mumole for pyruvate; 0.36-4.78 mumole for glucose-6-phosphate). At the same time the adenylate energy charge remained essentially unchanged (about 0.8) from the middle of exponential growth till the end of the stationary phase. At the late stages of RS-sporangia formation the concentration of all the above compounds decreased by about 10 times, and the adenylate energy charge only by 30%. Positive correlation between the levels of ATP and cAMP in RS cells was demonstrated. The concentration of adenylic nucleotides and cAMP showed the most noticable changes at the end of exponential growth; transition of the point of no return was not accompanied by significant changes in the pools of adenylic system, cAMP or energy charge.     

Intracellular levels of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) in cultures of Streptomyces griseus producing streptomycin.

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) were identified in the vegative mycelium of Streptomyces griseus. Adenosine 5'-diphosphate 3'-diphosphate (ppApp) and adenosine 5'-triphosphate 3'-diphosphate (pppApp) were not present but several other phosphorus-containing compounds which may have been inorganic polyphosphates were detected. During exponential growth of S. griseus the concentrations of ppGpp and pppGpp were several times higher than in the stationary stage. They fell sharply when exponential growth ended and then remained at an almost constant basal level. For the tetraphosphate the maximum concentration was about 50, and for the basal level about 10, pmol per millilitre of a culture with an optical density of 1.0. Production of streptomycin started several hours after exponential growth had ended and the concentrations of ppGpp and pppGpp had fallen. Streptomycin synthesis was delayed if the cells were resuspended just before production started in fresh medium lacking phosphate, but it was not delayed by glucose starvation. Both cultures, as well as cultures transferred to nitrogen-free medium, showed an immediate increase in ppGpp content to about four-fold the basal level. The results suggest that the guanosine polyphosphates do not directly control initiation of streptomycin production in S. griseus. Twelve additional species of Streptomyces examined all contained ppGpp and pppGpp.     

High-molecular polyphosphates and pyrophosphate in cyclosporin- producing Tolypocladium sp. and their role in the processes of growth and antibiotic synthesis

The contents of high-energy phosphorous compounds, i.e. three fractions of polyphosphates, pyrophosphate, and ATP were determined in isogenic strains of Tolypocladium sp. differing in cyclosporine production levels. The content of polyphosphates was 1 to 2 orders of magnitude greater than that of pyrophosphate or ATP and did not depend on the strain productivity. During the period of the mycelial intensive growth and at the beginning of antibiotic synthesis, the level of polyphosphates lowered 2-3-fold and the content of pyrophosphate markedly decreased as well. The activities of polyphosphatase and pyrophosphatase during the culture growth and cyclosporine biosynthesis was higher in the highly productive strain.     

State of adenosine phosphates during dehydration of yeast

Summary The effect of cultivation and dehydration conditions on the adenosine phosphate content of yeast cells has been studied. Irrespective of the cultivation conditions the total pool of adenosine phosphates was found to increase, mainly due to accumulation of ATP, during the exponential phase of cell growth and to decrease during transition of the culture into the stationary phase. Changes in the intracellular content of adenosine phosphates were parallel with changes in the respiratory activity of yeast cells cultivated under batch conditions. Yeast cells harvested at the exponential growth phase were sensitive to dehydration, losing a notable amount of adenosine phosphates as well as respiratory capacity during drying, leading to a massive dying-off of the cells. Yeast at the stationary phase was resistant to drying, and, during this process, accumulated ATP by mitochondrial oxidation of endogenous carbohydrates. The accumulated ATP was used by the dried yeast cells as an energy source in the first minutes of reactivation. On the basis of our results we recommend that the ATP content of dried yeast cells should be used as an indicator of their capacity to recover their viability by reactivation.     

Metabolic studies with NMR spectroscopy of the alga Dunaliella salina trapped within agarose beads

A technique for the entrapment of the unicellular algae Dunaliella salina in agarose beads and their perfusion during NMR measurements is presented. The trapped cells maintained their ability to proliferate under normal growth conditions, and remained viable and stable under steady-state conditions for long periods during NMR measurements. Following osmotic shock in the dark, prominent changes were observed in the intracellular level of ATP and polyphosphates, but little to no changes in the intracellular pH or orthoposphate content. When cells were subjected to hyperosmotic shock, the ATP level decreased. The content of NMR-visible polyphosphates decreased as well, presumably due to the production of longer, NMR-invisible structures. Following hypoosmotic shock, the ATP content increased and longer polyphosphates were broken down to shorter, more mobile polymers.     

Polyphosphates as an energy source for growth of Saccharomyces cerevisiae

Cells of the yeast Saccharomyces cerevisiae with a low content of polyphosphates (polyP) are characterized by disturbance of growth in medium with 0.5% glucose. The parent strain with polyP level reduced by phosphate starvation had a longer lag phase. The growth rate of strains with genetically determined low content of polyP due to their enhanced hydrolysis (CRN/pMB1_PPN1 Sc is a superproducer of exopolyphosphatase PPN1) or reduced synthesis (the BY4741 vma2Δ mutant with impaired vacuolar membrane energization) was lower in the exponential phase. The growth of cells with high content of polyP was accompanied by polyP consumption. In cells of strains with low content of polyP, CRN/pMB1_PPN1 Sc and BY4741 vma2Δ, their consumption was insignificant. These findings provide more evidence indicating the use of polyP as an extra energy source for maintaining high growth rate.     

Adenosine triphosphate pool levels and endogenous metabolism in Arthrobacter crystallopoietes during growth and starvation

The adenosine triphosphate (ATP) content of Arthrobactery crystallopoietes was measured during growth, starvation and recovery from starvation. During exponential growth of the cells as spheres in a glucose salts medium, the level of ATP per cell remained constant at 8.0×10^-10 g/cell. Morphogenesis to rodshaped cells and an increased growth rate following addition of casein hydrolysate was accompanied by an almost two-fold increase in the ATP level. As division of the rod-shaped cells proceeded, the level of ATP declined. After growing as rods for 12–14 h the cells underwent fragmentation to spheres during which time the ATP level again increased to the original value of 8.0×10^-10 g/cell. As the spherical cells resumed growth on the residual glucose, their ATP content declined for a short period and then remained relatively constant. During starvation of sphere or rod-shaped cells for one week, the ATP level declined by approximately 70% during the first 40–50 h and then remained constant. The endogenous metabolism rate of spherical cells declined during the first 10–20 h of starvation and then remained constant at approximately 0.02% of the cell carbon being utilized per h. Addition of glucose to spherical cells which had been starved for one week increased both the ATP content per cell and their rate of endogenous metabolism. The ATP content fluctuated and then remained at a level higher than maintained during starvation while endogenous metabolism quickly declined.Non-Standard Abbreviations ATP adenosine triphosphate - GS glucose mineral salts - HC casein hydrolysate - PVP polyvinylpyrrolidone - DMSO dimethylsulfoxide - MOPS morpholinopropane sulfonic acid - EDTA ethylene diaminetetraacetic acid     

Protein degradation in extracts of exponential and stationary phase Vibrio cells

The degradation of the foreign protein [14C]methyl apohaemoglobin ([14C-me]globin) was stimulated by ATP in cell-free extracts from exponential phase and shaken and standing stationary phase Vibrio cells. A marked stimulation by ATP of the degradation of [14C-me]globin was observed with exponential phase cell extracts which were preincubated for 30 min at 30 degrees C. Maximum stimulation was obtained with 3 mM-ATP and optimum degradation was at pH 8.0-8.5. Preincubation of extracts from both types of stationary phase cells did not affect the degree of ATP stimulation. The amount of ATP stimulation of [14C-me]globin degradation by exponential phase extracts decreased markedly when the cells were starved in a growth limiting minimal medium before preparation of the cell extracts. In the exponential and both types of stationary phase extracts most of the activity was located in the cytoplasmic fractions. Although the periplasmic preparations contained a minor portion of the total activity, this activity showed a greater percentage stimulation by ATP. In the absence of ATP the specific proteolytic activities of the extracts from exponential and both types of stationary phase cells were similar. The proteolytic activities in all the cell extracts were inhibited to the same extent by phenylmethylsulphonyl fluoride, but the exponential and both types of stationary phase cell extracts were inhibited to different extents by EDTA and p-hydroxymercuribenzoate. The results suggest that the proteolytic systems responsible for the degradation of abnormal proteins are different in exponential and stationary phase Vibrio cells.