Effect of cycloheximide, iodoacetamide, and antimycin A on inorganic phosphate synthesis in Saccharomyces cerevisiae VKM Y-1173

The effect of inhibitors of protein synthesis (cycloheximide, CHI), glycolysis (iodoacetamide, IAA), and oxidative phosphorylation (antimycin A, ANM) on inorganic phosphate (polyP) synthesis during the first 0.5 h of their hypercompensation in Saccharomyces cerevisiae VKM Y-l173 grown on 2% glucose-containing media at low (hypoxia) or high aeration rates or in the presence of 1 vol % ethanol under high aeration conditions was studied. PolyP accumulation was highest in the medium with glucose under hypoxia; lower, with glucose at high aeration; and lowest, in the medium with ethanol. CHI had a small effect on the total polyP level but significantly stimulated ATP accumulation, irrespective of the culture growth conditions. The low-polymer acid-soluble polyP1 were synthesized most actively by the cells grown on glucose under hypoxia, alkali-soluble polyP3 were synthesized at en hanced aeration, and the most hig-molecular fraction, polyP5, was actively accumulated along with polyP3 at cultivation on ethanol. Regardless of the growth conditions, CHI inhibited accumulation of polyP4, the synthesis of which is associated with the synthesis of mannoproteins. IAA and ANM largely inhibited synthesis of all fractions at yeast growth under hypoxia and on ethanol, respectively. The results as a whole demonstrate the dependence of polyP formation on the main energy-generating cell processes and, at the same time, the absence of direct dependence of their synthesis on ATP concentration in Saccharomyces cerevisiae VKM Y-l 173.     

Study of the content of inorganic polyphosphates in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> grown on different carbon sources with different O<Subscript>2</Subscript> concentrations in the medium

The content of different fractions of inorganic polyphosphates (polyP) was studied in Saccharomyces cerevisiae VKM Y-1173 growing on a complete medium with glucose under hypoxia and active aeration as well as on ethanol. The highest growth rate was observed for aerobic fermentation, while the yield of biomass was maximal for cultivation on ethanol. In the mid-log growth phase, the amount of poly P was maximal in the cells grown on glucose under hypoxia and minimal on ethanol. In this latter case, the content of different poly P fractions changed unevenly: polyP3, polyP4, and polyP1 decreased by approximately 60%, 45%, and 30%, respectively; the salt-soluble polyP2 remained at almost the same level; while polyP5 abruptly increased 10-to 15-fold. These findings demonstrate that the metabolic pathways for poly P fractions are different. A significant drop in the amount of the main poly P fractions accompanied by a decrease of the poly P average chain length in the presence of carbon and P_i sources in the medium is evidence of active involvement of poly P as additional energy sources in the flows of energy in actively growing yeast cells.     

Polyphosphates and exopolyphosphatases in cytosol and mitochondria of Saccharomyces cerevisiae during growth on glucose or ethanol under phosphate surplus

Content and chain lengths of inorganic polyphosphates (polyP) as well as exopolyphosphatase activities were compared in cytosol and mitochondria of the yeast Saccharomyces cerevisiae during growth on glucose or ethanol under phosphate surplus. PolyP metabolism in cytosol and mitochondria was substantially dependent upon the carbon source. Acid-soluble polyP accumulated mainly in cytosol using either glucose or ethanol. The level of the accumulation was lower during growth on ethanol compared to that on glucose. Increase in polyP content in mitochondria was observed during growth on glucose, but not on ethanol. In cytosol the activity of exopolyphosphatase PPN1 was increased and the activity of exopolyphosphatase PPX1 was decreased independently of the carbon source under phosphate surplus conditions. Growth on ethanol caused exopolyphosphatase PPN1 to appear in the soluble mitochondrial fraction, while during growth on glucose only exopolyphosphatase PPX1 was present in this fraction.     

Dependence of inorganic polyphosphate chain length on the orthophosphate content in the culture medium of the yeast Saccharomyces cerevisiae

The content of inorganic linear polyphosphate (polyP) and the polymeric degree (n) of these compounds were determined in the process of growth of the yeast Saccharomyces cerevisiae VKM Y-1173 in a medium, which contained varying Pi amount with the constant level of all the necessary components. For this purpose, a combination of chemical methods of polyP extraction and 31P-NMR spectroscopy studies of their chain length were used. After 7 h of phosphate starvation, the yeast was shown to use almost completely the phosphate reserve in the form of polyP localized in various cell compartments to support their vitality. The polyP drop was followed by a considerable shortening of the polymer chain length of acid-soluble (polyP1) and two alkali-soluble (polyP3 and polyP4) fractions. Under the same conditions, the content of a salt-soluble fraction (polyP2) decreased almost 20-fold followed by a simultaneous increase of the chain length nearly 2-fold. As a result, fraction chain length ranged up to n = 40-45. Replacement of the yeast cells after phosphate starvation to a complete phosphate- and glucose-containing medium resulted in super-accumulation ("overcompensation") of polyP within 2 h mainly in polyP3 and, to a lesser degree, in polyP1, polyP2, and polyP5 fractions. In polyP4 fraction localized as polyP3 at the cell surface, the polyP super-accumulation was not detected. The increase of polyP amount in the fractions mentioned turned out not to be accompanied by simultaneous elongation of their chain length and occurred at the lowest level that is characteristic of a polymer level for each fraction. Further cultivation of the yeast on the complete medium during 2 h had little or no effect on polyP content in the cells but led to elongation of polyP chain length especially in the polyP3 and polyP4 fractions. A phenomenon of considerable elongation of polyP chain length against the background of their fixed content revealed in the yeast growing on the complete medium suggests that these organisms possess a previously unknown discrete way of polyP biosynthesis, which results first in the formation of comparatively low-molecular-mass chains followed by that of high-molecular-mass polymers.     

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.     

Effect of inhibitors on polyphosphate metabolism in the yeast Saccharomyces cerevisiae under hypercompensation conditions

After re-inoculation of the yeast Saccharomyces cerevisiae from phosphate-deficient to complete medium, the total content of polyphosphates increased tenfold during 2 h (hypercompensation), but the content of certain fractions increased differently. The content of acid-soluble polyphosphate increased to the maximal extent. The ratio of the activities of two exopolyphosphatases also changed in the cytosol. Activity of a low molecular weight exopolyphosphatase (40 kD) decreased almost twice, whereas activity of a high molecular weight exopolyphosphatase (830 kD) increased tenfold. Cycloheximide blocks the increase in activity of high molecular weight exopolyphosphatase and hence, under these conditions the latter is synthesized de novo. Inhibitors of energy metabolism and cycloheximide, an inhibitor of protein synthesis, differently influence accumulation of certain polyphosphate fractions under hypercompensation conditions. The effect of iodoacetamide, an inhibitor of glycolysis, on any fraction is negligible, while cycloheximide suppresses accumulation of only polyP4 fraction associated with the cell envelope and bafilomycin A1, an inhibitor of vacuolar H⁺-ATPase, suppresses accumulation of polyP3 fraction. The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) to variable extent inhibits accumulation of all the fractions. Analysis of the effect of inhibitors on accumulation of polyphosphates under hypercompensation conditions confirms various localization, heterogeneity, and multiplicity of the routes of biosynthesis of certain fractions of these macroergic phosphorus compounds and also suggests interrelation between their biosynthesis and the gradient of H⁺ electrochemical potential.     

Effect of <Emphasis Type="Italic">m</Emphasis>-carbonyl cyanide 3-chlorophenylhydrazone on inorganic polyphosphates synthesis in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> under different growth conditions

The effects of different concentrations of the protonophore uncoupler m-carbonyl cyanide 3-hchlorophenylhydrazone (CCCP) on the synthesis of inorganic polyphosphates (polyP) during the first 0.5 h of hypercompensation in the yeast Saccharomyces cerevisiae VKM Y-1173 growing on media with 2% glucose under low (hypoxia) or high aeration or with 1% (vol/vol) ethanol under high aeration were studied. It was shown that the yeast growth on ethanol was completely inhibited by 5 μM CCCP, while growth on glucose was inhibited by 25 μM CCCP, independently of aeration of the medium. The maximum rate of H2 absorption was shown at 2, 5, and 25 μM CCCP for the cells grown on ethanol, on glucose under high aeration, and on glucose under hypoxia, respectively. Against the decrease of total ATP level and total polyP, CCCP had a nonuniform effect on the synthesis of individual polyP fractions. CCCP maximally inhibited synthesis of the most actively formed fractions: polyPI during growth on glucose under hypoxia, polyPIII during growth on glucose under aeration, and polyPIII and polyPV during growth on ethanol. CCCP had no substantial effect on the synthesis of polyPII and polyPIV fractions, the formation of which seems to be less related to the electrochemical potential gradient of H⁺ ions.     

The concentration dynamics of inorganic polyphosphates during the cephalosporin C synthesis by Acremonium chrysogenum

The contents of five fractions of energy-rich inorganic polyphosphates (polyPs), ATP, and H⁺-ATPase activity in the plasma membrane were determined in a low-activity cephalosporin C (cephC) producer Acremonium chrysogenum ATCC 11550 and selected highly efficient producer strain 26/8 grown on glucose or a synthetic medium providing for active synthesis of this antibiotic. It was shown that strain 26/8 on the synthetic medium produced 26-fold higher amount of cephC as compared with strain ATCC 11550. This was accompanied by a drastic decrease in the cell contents of ATP and the high-molecular-weight fractions polyP2, polyP3, and polyP5 with a concurrent increase in the low-molecular-weight fraction polyP1. These data suggest that polyPs are involved in the cephC synthesis as a source of energy. H⁺-ATPase activity insignificantly changed at both low and high levels of cephC production. This confirms the assumption that A. chrysogenum has other alternative antibiotic transporters in addition to cefT. The obtained results can be used for optimizing commercial-scale cephC biosynthesis.     

Inorganic polyphosphate in mitochondria of Saccharomyces cerevisiae at phosphate limitation and phosphate excess

Isolated mitochondria of Saccharomyces cerevisiae cells grown on glucose possess acid-soluble inorganic polyphosphate (polyP). Its level strongly depends on phosphate (P(i)) concentration in the culture medium. The polyP level in mitochondria showed 11-fold decrease under 0.8 mM P(i) as compared with 19.3 mM P(i). When spheroplasts isolated from P(i)-starved cells were incubated in the P(i)-complete medium, they accumulated polyP and exhibited a phosphate overplus effect. Under phosphate overplus the polyP level in mitochondria was two times higher than in the complete medium without preliminary P(i) starvation. The average chain length of polyP in mitochondria was of <15 phosphate residues at 19.3 mM P(i) in the culture medium and increased at phosphate overplus. Deoxyglucose inhibited polyP accumulation in spheroplasts, but had no effect on polyP accumulation in mitochondria. Uncouplers (FCCP, dinitrophenol) and ionophores (monensin, nigericin) inhibited polyP accumulation in mitochondria more efficiently than in spheroplasts. Fast hydrolysis of polyP was observed after sonication of isolated mitochondria. Probably, the accumulation of polyP in mitochondria depended on the proton-motive force of their membranes.     

V-ATPase dysfunction suppresses polyphosphate synthesis in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae accumulates the high levels of inorganic polyphosphates (polyPs) performing in the cells numerous functions, including phosphate and energy storage. The effects of vacuolar membrane ATPase (V-ATPase) dysfunction were studied on polyP accumulation under short-term cultivation in the P_i–excess media after P_i starvation. The addition of bafilomycin A1, a specific inhibitor of V-ATPase, to the medium with glucose resulted in strong inhibition of the synthesis of long-chain polyP and in substantial suppression of short-chain polyP. The addition of bafilomycin to the medium with ethanol resulted in decreased accumulation of high-molecular polyP, while the accumulation of low-molecular polyP was not affected. The levels of polyP synthesis in the mutant strain with a deletion in the vma2 gene encoding a V-ATPase subunit were significantly lower than in the parent strain in the media with glucose and with ethanol. The synthesis of the longest chain polyP was not observed in the mutant cells. The synthesis of only the low-polymer acid-soluble polyP fraction occurred in the cells of the mutant strain. However, the level of polyP1 was nearly tenfold lower than compared to the cells of the parent strain. Both bafilomycin A1 and the mutation in vacuolar ATPase subunit vma2 lead to a considerable decrease of cellular polyP accumulation. Thus, the defects in ΔμH⁺ formation on the vacuolar membrane resulted in the decrease of polyP biosynthesis in S. cerevisiae.     

Inorganic polyphosphates in mitochondria

Current data concerning the crucial role of inorganic polyphosphates (polyP) in mitochondrial functions and dysfunctions in yeast and animal cells are reviewed. Biopolymers with short chain length (∼15 phosphate residues) were found in the mitochondria of Saccharomyces cerevisiae. They comprised 7–10% of the total polyP content of the cell. The polyP are located in the membranes and intermembrane space of mitochondria. The mitochondrial membranes possess polyP/Ca²⁺/polyhydroxybutyrate complexes. PolyP accumulation is typical of promitochondria but not of functionally active mitochondria. Yeast mitochondria possess two exopolyphosphatases splitting P_i from the end of the polyP chain. One of them, encoded by the PPX1 gene, is located in the matrix; the other one, encoded by the PPN1 gene, is membrane-bound. Formation of well-developed mitochondria in the cells of S. cerevisiae after glucose depletion is accompanied by decrease in the polyP level and the chain length. In PPN1 mutants, the polyP chain length increased under glucose consumption, and the formation of well-developed mitochondria was blocked. These mutants were defective in respiration functions and consumption of oxidizable carbon sources such as lactate and ethanol. Since polyP is a compound with high-energy bonds, its metabolism vitally depends on the cell bioenergetics. The maximal level of short-chain acid-soluble polyP was observed in S. cerevisiae under consumption of glucose, while the long-chain polyP prevailed under ethanol consumption. In insects, polyP in the mitochondria change drastically during ontogenetic development, indicating involvement of the polymers in the regulation of mitochondrial metabolism during ontogenesis. In human cell lines, specific reduction of mitochondrial polyP under expression of yeast exopolyphosphatase PPX1 significantly modulates mitochondrial bioenergetics and transport.     

Polyphosphate synthesis in yeast

Polyphosphate synthesis was studied in phosphate-starved cells of Saccharomyces cerevisiae and Kluyveromyces marxianus. Incubation of these yeasts for a short time with phosphate and either glucose or ethanol resulted in the formation of polyphosphate with a short chain length. With increasing incubation times, polyphosphates with longer chain lengths were formed. Polyphosphates were synthesized faster during incubation with glucose than with ethanol. Antimycin did not affect the glucose-induced polyphosphate synthesis in either yeast. Using ethanol as an energy source, antimycin A treatment blocked both polyphosphate synthesis and accumulation of orthophosphate in the yeast S. cerevisiae. However, in K. marxianus, polyphosphate synthesis and orthophosphate accumulation proceeded normally in antimycin-treated cells, suggesting that endogenous reserves were used as energy source. This was confirmed in experiments, conducted in the absence of an exogenous energy source.     

Molecular analysis of polyphosphate accumulation in bacteria

The dynamic behavior of inorganic polyphosphate (polyP), its accumulation and disappearance, is the most striking aspect of polyP metabolism in bacteria. Imbalance between polyP synthesis and degradation results in fluctuations of polyP by 100- to 1000-fold. We here review recent results with respect to this polyP metabolism in bacteria. PolyP accumulation in response to amino acid starvation, accompanied by increased levels of stringent factors, has been observed in Escherichia coli. Inhibition by stringent factors of polyphosphatase interrupts the dynamic balance between the synthesis and degradation of polyP, accounting for polyP accumulation. Polyphosphate kinase is required for activation of intracellular protein degradation, which is required for adaptation at the onset of amino acid starvation. The adaptation to amino acid starvation is mediated by the network of stringent response and polyP metabolism. PolyP accumulation independent of stringent response has also been observed. Novobiocin, an inhibitor for DNA gyrase, stimulated accumulation of polyP but not that of stringent factors. However, a temperature-sensitive DNA gyrase mutant did not exhibit polyP accumulation at the non-permissive temperature. Antagonistic relationship of polyP to nucleic acid synthesis, explored by Harold, appears to be more complicated. We discuss relationship of Pi regulation to polyP accumulation in E. coli and Klebsiella aerogenes. A function of polyP as an in vivo phosphagen affecting polyP accumulation is also discussed.     

Modification of corn cob meal with quarternary ammonium groups

Corn cob meal was modified with quarternary ammonium groups and subsequently extracted with 80% ethanol, water, and 5% NaOH. The fractions obtained had lower polydispersities, values, and yields than unmodified material. The yields are lower than those obtained on bagasse under the same conditions. The modification caused the drastic degradation of the ethanol-lignin (EL) fraction. The one-step extraction with NaOH/H₂O₂ gave 28·8% yield of material (calculated on the starting material) which contained 12·0% Klason lignin, and had the highest polydispersity (4·3, ). The water-soluble fractions consisted of arabinoglucuronoxylan and alkali-soluble fractions of xylan without other sugar moieties. The water-soluble fraction from NaOH/H₂O₂ extraction contained arabinoglucuronoxylan with modified arabinose and acid units. By this method higher yields could be obtained than on bagasse treated by the sequential extraction.     

The biosorption of cadmium and cobalt and iron ions by yeast <Emphasis Type="Italic">Cryptococcus humicola</Emphasis> at nitrogen starvation

Yeasts Cryptococcus humicola accumulated cadmium, cobalt, and iron (~?50, 17, and 4% of the content in the medium, respectively) from the medium containing glucose, phosphate, and 2 mmol/L of metal salts. The effects of metal absorption on the levels of orthophosphate (Pi) and inorganic polyphosphate (polyP) varied for the metals under study. The levels of Pi and polyP increased in the case of cadmium and cobalt, respectively. In the case of iron, no changes in the levels of Pi and polyP were observed. Multiple DAPI-stained polyP inclusions were observed in the cytoplasm of cadmium-containing cells. The intensity of DAPI staining of the cell wall especially increased in case of cobalt and iron accumulation.     

Biotechnological synthesis of water-soluble food-grade polyphosphate with Saccharomyces cerevisiae

Inorganic polyphosphate (polyP) is the polymer of phosphate. Water-soluble polyPs with average chain lengths of 2–40 P-subunits are widely used as food additives and are currently synthesized chemically. An environmentally friendly highly scalable process to biosynthesize water-soluble food-grade polyP in powder form (termed bio-polyP) is presented in this study. After incubation in a phosphate-free medium, generally regarded as safe wild-type baker's yeast (Saccharomyces cerevisiae) took up phosphate and intracellularly polymerized it into 26.5% polyP (as KPO₃, in cell dry weight). The cells were lyzed by freeze-thawing and gentle heat treatment (10 min, 70°C). Protein and nucleic acid were removed from the soluble cell components by precipitation with 50 mM HCl. Two chain length fractions (42 and 11P-subunits average polyP chain length, purity on a par with chemically produced polyP) were obtained by fractional polyP precipitation (Fraction 1 was precipitated with 100 mM NaCl and 0.15 vol ethanol, and Fraction 2 with 1 final vol ethanol), drying, and milling. The physicochemical properties of bio-polyP were analyzed with an enzyme assay, ³¹P nuclear magnetic resonance spectroscopy, and polyacrylamide gel electrophoresis, among others. An envisaged application of the process is phosphate recycling from waste streams into high-value bio-polyP.     

Effect of reduced pH on inorganic polyphosphate accumulation by Burkholderia cepacia complex isolates

AIMS: Burkholderia cepacia complex (Bcc) isolates causing pulmonary infection in cystic fibrosis (CF) patients grow within an acidic environment in the lung. As exposure to acid pH has been shown to increase intracellular inorganic polyphosphate (polyP) formation in some bacteria, we investigated the inter-relationship between acidic pH and polyP accumulation in Bcc isolates. METHODS AND RESULTS: The formation of polyP by one Burkholderia cenocepacia clinical isolate was initially examined at a range of pH values by measuring total intracellular polyP accumulation and phosphate uptake. The pattern of polyP accumulation corresponded with the pattern of phosphate uptake with the maximum for both occurring at pH 5.5. Phosphate uptake and formation of polyP by this isolate was further determined over 48 h at pH 5.5, 6.5 and 7.5; formation of polyP was maximal at pH 5.5 at all time points studied. Sixteen of 17 additional clinical and environmental Bcc isolates examined also exhibited maximum phosphate uptake at pH 5.5. CONCLUSIONS: Both clinical and environmental Bcc isolates, of five genomovars, show enhanced formation of polyP in an acidic environment. Given both the speculated role of polyP in pathogenesis, cell signalling and biofilm formation and the acidic nature of the CF lung, this may be of considerable clinical importance. SIGNIFICANCE AND IMPACT OF THE STUDY: Growth of Bcc in an acidic environment, such as that found in the lungs of CF patients may be influenced in part by polyP accumulation.     

Polyphosphate accumulation by Pseudomonas putida CA-3 and other medium-chain-length polyhydroxyalkanoate-accumulating bacteria under aerobic growth conditions

Pseudomonas putida CA-3 accumulates polyphosphate (polyP) and medium-chain-length polyhydroxyalkanoate (mclPHA) concurrently under nitrogen limitation. Five other mclPHA-accumulating Pseudomonas strains are capable of simultaneous polyP and mclPHA biosynthesis. It appears that polyP is not the rate-limiting step for mclPHA accumulation in these Pseudomonas strains.     

Strictly polyphosphate-dependent glucokinase in a polyphosphate-accumulating bacterium,Microlunatus phosphovorus

ATP-dependent glucokinase is suggested to have evolved from a hypothetical polyphosphate (polyP)-dependent glucokinase (polyP-GK) via a bifunctional polyP/ATP glucokinase (polyP/ATP-GK). Here we showed that polyP-GK is present in a polyP-accumulating bacterium, Microlunatus phosphovorus. The polyP-GK produced glucose-6-P(i) from glucose and polyP, but it could not phosphorylate glucose with ATP. The polyP-GK was most closely related to the polyP/ATP-GK of Mycobacterium tuberculosis.     

Formation of Polyphosphate by Polyphosphate Kinases and Its Relationship to Poly(3-Hydroxybutyrate) Accumulation in Ralstonia eutropha Strain H16

A protein (PhaX) that interacted with poly(3-hydroxybutyrate) (PHB) depolymerase PhaZa1 and with PHB granule-associated phasin protein PhaP2 was identified by two-hybrid analysis. Deletion of phaX resulted in an increase in the level of polyphosphate (polyP) granule formation and in impairment of PHB utilization in nutrient broth-gluconate cultures. A procedure for enrichment of polyP granules from cell extracts was developed. Twenty-seven proteins that were absent in other cell fractions were identified in the polyP granule fraction by proteome analysis. One protein (A2437) harbored motifs characteristic of type 1 polyphosphate kinases (PPK1s), and two proteins (A1212, A1271) had PPK2 motifs. In vivo colocalization with polyP granules was confirmed by expression of C- and N-terminal fusions of enhanced yellow fluorescent protein (eYFP) with the three polyphosphate kinases (PPKs). Screening of the genome DNA sequence for additional proteins with PPK motifs revealed one protein with PPK1 motifs and three proteins with PPK2 motifs. Construction and subsequent expression of C- and N-terminal fusions of the four new PPK candidates with eYFP showed that only A1979 (PPK2 motif) colocalized with polyP granules. The other three proteins formed fluorescent foci near the cell pole (apart from polyP) (A0997, B1019) or were soluble (A0226). Expression of the Ralstonia eutropha ppk (ppk_Reu) genes in an Escherichia coli Δppk background and construction of a set of single and multiple chromosomal deletions revealed that both A2437 (PPK1a) and A1212 (PPK2c) contributed to polyP granule formation. Mutants with deletion of both genes were unable to produce polyP granules. The formation and utilization of PHB and polyP granules were investigated in different chromosomal backgrounds.