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.     

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.     

The effect of inactivation of the exo-and endopolyphosphatase genes PPX1 and PPN1 on the level of different polyphosphates in the yeast Saccharomyces cerevisiae

The inactivation of the PPX1 and PPN1 genes, which encode the major enzymes of polyphosphate degradation (exopolyphosphatase and endopolyphosphatase, respectively), was found to exert different effects on the content of different polyphosphates in the yeast Saccharomyces cerevisiae. The content of relatively low-molecular-weight acid-soluble polyphosphates in mutant yeast strains is inversely proportional to the exopolyphosphatase activity of the cytosol. At the same time, the mutation of these genes exerts no effect on salt-soluble polyphosphates. The content of high-molecular-weight alkali-soluble polyphosphates increases twofold in a mutant with inactivated genes of both exopolyphosphatase and endopolyphosphatase. The data obtained confirm the earlier suggestion that the metabolic pathways of particular polyphosphates in yeasts are different.     

The content and chain length of polyphosphates from vacuoles of Saccharomyces cerevisiae VKM Y-1173

The content of inorganic polyphosphates (polyP) in vacuoles of the yeast Saccharomyces cerevisiae is 15% of the total cellular polyP. Over 80% of the vacuole polyP are in an acid-soluble fraction. It was first established by ³¹P-NMR spectroscopy that a polymeric degree (n) of two subfractions obtained by precipitation with Ba²⁺ in succession at pH 4.5 and 8.2 was approximately 20 ± 5 and 5 ± 2 residues of ortho-phosphoric acid, respectively. Under a deficit of phosphate (P_i) in the cultivation medium, the polyP content in vacuoles decreased 7-fold with the same drastic reduction of their content in the cell. Unlike intact yeast cells, where polyP overcompensation is observed after their transfer from phosphate-free to phosphate-containing medium, the vacuoles do not show this effect. The data indicate the occurrence of special regulatory mechanisms of polyP synthesis in vacuoles differing from those in the whole cell.     

Inorganic polyphosphate in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> with a mutation disturbing the function of vacuolar ATPase

A mutation in the vma2 gene disturbing V-ATPase function in the yeast Saccharomyces cerevisiae results in a five- and threefold decrease in inorganic polyphosphate content in the stationary and active phases of growth on glucose, respectively. The average polyphosphate chain length in the mutant cells is decreased. The mutation does not prevent polyphosphate utilization during cultivation in a phosphate-deficient medium and recovery of its level on reinoculation in complete medium after phosphate deficiency. The content of short chain acid-soluble polyphosphates is recovered first. It is supposed that these polyphosphates are less dependent on the electrochemical gradient on the vacuolar membrane.     

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.     

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.     

Accumulation of Inorganic Polyphosphate in phoU Mutants of Escherichia coli and Synechocystis sp. Strain PCC6803

The biological process for phosphate (P_i) removal is based on the use of bacteria capable of accumulating inorganic polyphosphate (polyP). We obtained Escherichia coli mutants which accumulate a large amount of polyP. The polyP accumulation in these mutants was ascribed to a mutation of the phoU gene that encodes a negative regulator of the P_i regulon. Insertional inactivation of the phoU gene also elevated the intracellular level of polyP in Synechocystis sp. strain PCC6803. The mutant could remove fourfold more P_i from the medium than the wild-type strain removed.     

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.     

Phosphate Homeostasis in Conditions of Phosphate Proficiency and Limitation in the Wild Type and the phoP Mutant of Streptomyces lividans

Phosphate, as a constituent of the high energy molecules, ATP/GTP and polyphosphate, plays a crucial role in most of the metabolic processes of living organisms. Therefore, the adaptation to low Pi availability is a major challenge for bacteria. In Streptomyces, this adaptation is tightly controlled by the two component PhoR/PhoP system. In this study, the free intracellular Pi, ATP, ADP and polyP content of the wild type and the phoP mutant strain of S. lividans TK24 were analyzed at discrete time points throughout growth in Pi replete and limited media. PolyP length and content was shown to be directly related to the Pi content of the growth medium. In Pi repletion, ATP and high molecular weight (HMW) polyP contents were higher in the phoP mutant than in the WT strain. This supports the recently proposed repressive effect of PhoP on oxidative phosphorylation. High oxidative phosphorylation activity might also have a direct or indirect positive impact on HMW polyP synthesis. In Pi sufficiency as in Pi limitation, the degradation of these polymers was shown to be clearly delayed in the phoP mutant, indicating PhoP dependent expression of the enzymes involved in this degradation. The efficient storage of Pi as polyphosphate and/or its inefficient degradation in Pi in the phoP mutant resulted in low levels of free Pi and ATP that are likely to be, at least in part, responsible for the very poor growth of this mutant in Pi limitation. Furthermore, short polyP was shown to be present outside the cell, tightly bound to the mycelium via electrostatic interactions involving divalent cations. Less short polyP was found to be associated with the mycelium of the phoP mutant than with that of the WT strain, indicating that generation and externalization of these short polyP molecules was directly or indirectly dependent on PhoP.     

Ribonucleic acid-permeable mutant of Escherichia coli

An RNA-permeable mutant was isolated from a tryptophan amber auxotrophic strain of Escherichia coli after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. The rationale of the isolation was based on the suppression of an amber mutation. A strain was selected, which could grow in minimal medium supplemented with transfer RNA prepared from an suI-carrying strain but not from an su⁻ strain. This mutant incorporated³H-labeled bulk RNA into the cells at a rate 40 times higher than did the parent strain. The level of tryptophan requirement, susceptibility to the lytic action of lysozyme and RNase activity in the culture medium of the mutant strain did not differ from those of the parent strain. The mutant strain incorporated ³H-labeled ribosomal RNA equally as well as it incorporated ³H-labeled transfer RNA and the incorporation was competitively inhibited by any species of cold RNA. However, the fate of ³H-labeled rRNA after incorporation resulted in degradation to yield acid-soluble fragments whereas tRNA after incorporation remained intact in the cell.     

The effect of inactivation of the exo- and endopolyphosphatase genes PPX1 and PPN1 on the level of different polyphosphates in the yeast Saccharomyces cerevisiae

The inactivation of the PPX1 and PPN1 genes, which encode the major enzymes of polyphosphate degradation (exopolyphosphatase and endopolyphosphatase, respectively), was found to exert different effects on the content of different polyphosphates in the yeast Saccharomyces cerevisiae. The content of relatively low-molecular-weight acid-soluble polyphosphates in mutant yeast strains is inversely proportional to the exopolyphosphatase activity of the cytosol. At the same time, the mutation of these genes exerts no effect on salt-soluble polyphosphates. The content of high-molecular-weight alkali-soluble polyphosphates increases twofold in a mutant with inactivated genes of both exopolyphosphatase and endopolyphosphatase. The data obtained confirm the earlier suggestion that the metabolic pathways of particular polyphosphates in yeasts are different.     

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.     

Effect of PPX1 inactivation on the exopolyphosphatase spectra in cytosol and mitochondria of the yeast Saccharomyces cerevisiae

Inactivation of PPX1 encoding exopolyphosphatase PPX1 in Saccharomyces cerevisiae results in a change in the exopolyphosphatase spectrum in the yeast cells. In the PPX1-deficient strain, elimination of an 45 kD exopolyphosphatase is observed in the cytosol, and activity of an exopolyphosphatase with molecular mass of 830 kD increases fivefold. The latter activity differs greatly in properties from the low-molecular-mass enzyme of the parent strain. In the soluble fraction of the mutant mitochondria, exopolyphosphatase of 45 kD characteristic of the soluble mitochondrial fraction in the parent strain is eliminated, and exopolyphosphatase with a molecular mass of 440 to 830 kD is found. On PPX1 inactivation, a membrane-bound form of mitochondrial exopolyphosphatase is unaffected in its activity level and properties. Therefore, the membrane-bound exopolyphosphatase of mitochondria and the high-molecular-mass enzyme of the cytosol of S. cerevisiae are not encoded by the PPX1 gene, unlike the soluble low-molecular-mass exopolyphosphatase of mitochondria, which is probably a product of this gene with a posttranslational modification. In the PPX1 mutant, exopolyphosphatase properties in the cell as a whole undergo modifications including the ability to hydrolyze polyphosphates (polyP) with different polymer degree.     

Alteration of the largest subunit of RNA polymerase II and its effect on chromosome stability in Schizosaccharomyces pombe

A mutation in the RNA polymerase II largest subunit (RpII LS) that is related to abnormal induction of sister chromatid exchange has previously been described the CHO-K1 cell mutant tsTM4. To elucidate the molecular basis of this effect we introduced the mutation into the homologous site in the Schizosaccharomyces pombe rpb1 gene, which encodes RpII LS. Since the tsTM4 mutant exhibited a decrease in the rate of DNA synthesis in cells arrested in S phase at the nonpermissive temperature, we focussed on the study of growth, the cell cycle, and chromosome stability at various temperatures. First, we examined the effects of the mutation on haploid yeast cells. The mutant showed slower growth than the wild type, but cell growth was not arrested at the nonpermissive temperature. When growing cells were shifted to the nonpermissive temperature, an accumulation of cells in G1 and/or G0 was observed. Tetrad analysis suggested that these phenotypes were associated with the mutation. In diploid cells, chromosome instability was detected by loss of intragenic complementation between two alleles of the ade6 gene. An abnormal fraction of cells containing an intermediate DNA content was also observed by FACS analysis. The accumulation of this fraction may reflect the fact that a large number of cells are in S phase or have an abnormal DNA content as a result of chromosome instability. These observations demonstrate that the S. pomberpb1 mutant exhibits a phenotype very similar to that of the CHO-K1 cell mutant tsTM4.     

Polyphosphate Deficiency in Mycobacterium tuberculosis Is Associated with Enhanced Drug Susceptibility and Impaired Growth in Guinea Pigs

Inorganic polyphosphate (polyP), a linear polymer of hundreds of phosphate residues linked by ATP-like phosphoanhydride bonds, is found in all organisms and performs a wide variety of functions. This study shows that polyP accumulation occurs in Mycobacterium tuberculosis upon exposure to various stress conditions. M. tuberculosis possesses a single homolog of ppk-1, and we have disrupted ppk-1 in the M. tuberculosis genome by allelic replacement. The mutant strain exhibited negligible levels of intracellular polyP, decreased expression of sigF and phoP, and reduced growth in the stationary phase and displayed a survival defect in response to nitrosative stress and in THP-1 macrophages compared to the wild-type strain. We report that reduction in polyP levels is associated with increased susceptibility of M. tuberculosis to certain TB drugs and impairs its ability to cause disease in guinea pigs. These results suggest that polyP contributes to persistence of M. tuberculosis in vitro and plays an important role in the physiology of bacteria residing within guinea pigs.     

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.     

Effect of a carbon source on polyphosphate accumulation in Saccharomyces cerevisiae

The cells of Saccharomyces cerevisiae accumulate inorganic polyphosphate (polyP) when reinoculated on a phosphate-containing medium after phosphorus starvation. Total polyP accumulation was similar at cultivation on both glucose and ethanol. Five separate fractions of polyP: acid-soluble fraction polyP1, salt-soluble fraction polyP2, weakly alkali-soluble fraction polyP3, alkali-soluble fraction polyP4, and polyP5, have been obtained from the cells grown on glucose and ethanol under phosphate overplus. The dynamics of polyP fractions depend on a carbon source. The accumulation rates for fractions polyP2 and polyP4 were independent of the carbon source. The accumulation rates of polyP1 and polyP3 were higher on glucose, while fraction polyP5 accumulated faster on ethanol. As to the maximal polyP levels, they were independent of the carbon source for fractions polyP2, polyP3, and polyP4. The maximal level of fraction polyP1 was higher on glucose than on ethanol, but the level of fraction polyP5 was higher on ethanol. It was assumed that accumulation of separate polyP fractions has a metabolic interrelation with different energy-providing pathways. The polyphosphate nature of fraction polyP5 was demonstrated for the first time by ³¹P nuclear magnetic resonance spectroscopy, enzymatic assay, and electrophoresis.     

Contribution of dps to Acid Stress Tolerance and Oxidative Stress Tolerance in Escherichia coli O157:H7

An Escherichia coli O157:H7 dps::nptI mutant (FRIK 47991) was generated, and its survival was compared to that of the parent in HCl (synthetic gastric fluid, pH 1.8) and hydrogen peroxide (15 mM) challenges. The survival of the mutant in log phase (5-h culture) was significantly impaired (4-log₁₀-CFU/ml reduction) compared to that of the parent strain (ca. 1.0-log₁₀-CFU/ml reduction) after a standard 3-h acid challenge. Early-stationary-phase cells (12-h culture) of the mutant decreased by ca. 4 log₁₀ CFU/ml while the parent strain decreased by approximately 2 log₁₀ CFU/ml. No significant differences in the survival of late-stationary-phase cells (24-h culture) between the parent strain and the mutant were observed, although numbers of the parent strain declined less in the initial 1 h of acid challenge. FRIK 47991 was more sensitive to hydrogen peroxide challenge than was the parent strain, although survival improved in stationary phase. Complementation of the mutant with a functional dps gene restored acid and hydrogen peroxide tolerance to levels equal to or greater than those exhibited by the parent strain. These results demonstrate that decreases in survival were from the absence of Dps or a protein regulated by Dps. The results from this study establish that Dps contributes to acid tolerance in E. coli O157:H7 and confirm the importance of Dps in oxidative stress protection.