Inorganic Polyphosphate and Physiological Properties of Saccharomyces cerevisiae Yeast Overexpressing Ppn2.

The effect of the yeast endopolyphosphatase Ppn2 overproduction on the metabolism of inorganic polyphosphates in Saccharomyces cerevisiae yeast was studied. Expression of the PPN2 gene under control of the strong constitutive promoter of glyceraldehyde 3-phosphate dehydrogenase gene (PKG1) led to a significant increase in the endopolyphosphatase activity stimulated by cobalt/zinc ions. This activity was present in both soluble and membrane subcellular fractions; it was higher toward long-chain polyphosphates and could be stimulated by ADP. The content of short-chain polyphosphates in the cells of the overexpressing strain was ~2.5 times higher compared to the parent strain. The cells overexpressing Ppn2 were more resistant to peroxide and alkali. The role of short-chain polyphosphates in the adaptation to these stress factors is discussed.

Inorganic Polyphosphate and Cancer.

This review presents data on the relationship between inorganic polyphosphate metabolism and carcinogenesis including participation of polyphosphates in the regulation of activity of mTOR and other proteins involved in carcinogenesis, the role of h-prune protein (human polyphosphatase) in cell migration and metastasis formation, the prospects for using polyphosphates and inhibitors of polyphosphate metabolism enzymes as agents for controlling cell proliferation and migration.

Transcriptome profile of yeast reveals the essential role of PMA2 and uncharacterized gene YBR056W-A (MNC1) in adaptation to toxic manganese concentration.

Adaptation of S. cerevisiae to toxic concentrations of manganese provides a physiological model of heavy metal homeostasis. Transcriptome analysis of adapted yeast cells reveals upregulation of cell wall and plasma membrane proteins including membrane transporters. The gene expression in adapted cells differs from that of cells under short-term toxic metal stress. Among the most significantly upregulated genes are PMA2, encoding an ortholog of Pma1 H+-ATPase of the plasma membrane, and YBR056W-A, encoding a putative membrane protein Mnc1 that belongs to the CYSTM family and presumably chelates manganese at the cell surface. We demonstrate that these genes are essential for the adaptation to toxic manganese concentration and propose an extended scheme of manganese detoxification in yeast.

Polyphosphates and Polyphosphatase Activity in the Yeast Saccharomyces cerevisiae during Overexpression of the DDP1 Gene.

The effects of overexpression of yeast diphosphoinositol polyphosphate phosphohydrolase (DDP1) having endopolyphosphatase activity on inorganic polyphosphate metabolism in Saccharomyces cerevisiae were studied. The endopolyphosphatase activity in the transformed strain significantly increased compared to the parent strain. This activity was observed with polyphosphates of different chain length, being suppressed by 2 mM tripolyphosphate or ATP. The content of acid-soluble and acid-insoluble polyphosphates under DDP1 overexpression decreased by 9 and 28%, respectively. The average chain length of salt-soluble and alkali-soluble fractions did not change in the overexpressing strain, and that of acid-soluble polyphosphate increased under phosphate excess. At the initial stage of polyphosphate recovery after phosphorus starvation, the chain length of the acid-soluble fraction in transformed cells was lower compared to the recipient strain. This observation suggests the complex nature of DDP1 involvement in the regulation of polyphosphate content and chain length in yeasts.

[Stress Resistance Mechanisms in the Indicator Fungi from Highly Radioactive Chernobyl Zone Sites].

Comparison of the levels of the protein carboxylic groups in response to peroxide stress revealed enhanced stress resistance in Purpureocillium lilacinum strains isolated from soils with high content of copper or radionuclides compared to the strains isolated from uncontaminated soils. While in background strains resistance to peroxide stress increased with glucose content in the medium increasing from 0.002 to 2%, the strains from radionuclides- or copper-contaminated soils did not exhibit this pattern. Respiratory activity and polyphosphate content were compared for radiation-resistant strain 1941 and strain SM from the area with zero radioactivity. For the protoplasts of strain 1941 isolated from the Chernobyl zone, elevated respiratory activity was revealed on the media with low glucose content. Under the control conditions, the content of inorganic polyphosphates (polyP) in strains 1941 and SM was the same. Under conditions of peroxide stress, only the background strain SM grown on the medium with low glucose concentration exhibited decreased levels of inorganic polyphosphates. Independent on glucose concentration in the medium, in both P. lilacinum strains, polyP content increased in the course of regeneration after peroxide stress.

[The role of mineral phosphorus compounds in naphthalene biodegradation by Pseudomonas putida].

The effect of phosphate concentration in the culture medium on the growth and naphthalene degradation by Pseudomonas putida BS 3701 was studied. The limiting concentration of phosphate was 0.4 mM and 0.1 mM under cultivation in media with naphthalene and glucose, respectively The phosphate deficiency correlated with a decrease in the activities of naphthalene dioxygenase and salicylate hydroxylase and with salicylate accumulation in the culture medium. We suggest that this fact indicates the impaired regulation of gene expression of "upper" and "lower" pathways of naphthalene oxidation. Under naphthalene degradation, the cells accumulated three times more inorganic polyphosphates as compared with the consumption of glucose. The involvement of polyphosphates in the regulation of naphthalene metabolism has been considered.

PPX1 gene overexpression has no influence on polyphosphates in Saccharomyces cerevisiae.

The role of exopolyphosphatase PPX1 in polyphosphate metabolism in yeasts has been studied in strains of Saccharomyces cerevisiae with inactivated PPX1 and PPN1 genes transformed by the expression vector carrying the yeast PPX1 gene. Exopolyphosphatase activity in transformant strains increased 90- and 40-fold compared to the ΔPPX1 and ΔPPN1 strains, respectively. The purified recombinant exopolyphosphatase PPX1 was similar to the PPX1 of wild strains in its substrate specificity and requirement for divalent metal cations. It was more active with tripolyphosphate and low molecular mass polyphosphates than with high molecular mass polyphosphates and required Mg2+ for its activity. The high level of recombinant PPX1 expression caused no decrease in polyphosphate content in the cells of the transformant. This fact suggests the restricted role of PPX1 in polyphosphate metabolism in yeasts.

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.

Diversity of phosphorus reserves in microorganisms.

Phosphorus compounds are indispensable components of the Earth's biomass metabolized by all living organisms. Under excess of phosphorus compounds in the environment, microorganisms accumulate reserve phosphorus compounds that are used under phosphorus limitation. These compounds vary in their structure and also perform structural and regulatory functions in microbial cells. The most common phosphorus reserve in microorganism is inorganic polyphosphates, but in some archae and bacteria insoluble magnesium phosphate plays this role. Some yeasts produce phosphomannan as a phosphorus reserve. This review covers also other topics, i.e. accumulation of phosphorus reserves under nutrient limitation, phosphorus reserves in activated sludge, mycorrhiza, and the role of mineral phosphorus compounds in mammals.

Polyphosphates and exopolyphosphatase activities in the yeast Saccharomyces cerevisiae under overexpression of homologous and heterologous PPN1 genes.

The role of exopolyphosphatase PPN1 in polyphosphate metabolism in fungi has been studied in strains of Saccharomyces cerevisiae transformed by the yeast PPN1 gene and its ortholog of the fungus Acremonium chrysogenum producing cephalosporin C. The PPN1 genes were expressed under a strong constitutive promoter of the gene of glycerol aldehyde-triphosphate dehydrogenase of S. cerevisiae in the vector pMB1. The yeast strain with inactivated PPN1 gene was transformed by the above vectors containing the PPN1 genes of S. cerevisiae and A. chrysogenum. Exopolyphosphatase activity in the transformant with the yeast PPN1 increased 28- and 11-fold compared to the mutant and parent PPN1 strains. The amount of polyphosphate in this transformant decreased threefold. Neither the increase in exopolyphosphatase activity nor the decrease in polyphosphate content was observed in the transformant with the orthologous PPN1 gene of A. chrysogenum, suggesting the absence of the active form of PPN1 in this transformant.

Extracellular phosphomannan as a phosphate reserve in the yeast Kuraishia capsulata.

We have found that extracellular phosphomannan is the main phosphate reserve in the yeast Kuraishia capsulata, in contrast to other yeast species effectively absorbing Pi. Under nitrogen starvation, K. capsulata absorbed essentially all Pi from the medium containing 240 mM glucose, 2.5 mM MgSO4, and 11 mM KH2PO4. Inorganic polyphosphate level in the cells was about 14% of the Pi absorbed. Most of the Pi (~60%) was found in the fraction of extracellular phosphomannan that can be used as a carbon and phosphorus source by this yeast in deficient media.

Inorganic polyphosphate in the yeast Saccharomyces cerevisiae 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.

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 (approximately 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.

Properties of partially purified endopolyphosphatase of the yeast Saccharomyces cerevisiae.

Partially purified endopolyphosphatase from cytosol of the yeast Saccharomyces cerevisiae with inactivated genes PPX1 and PPN1 encoding exopolyphosphatases was obtained with ion-exchange and affinity chromatography. The enzyme activity was estimated by decrease of polyphosphate chain length determined by PAGE. The enzyme cleaved inorganic polyphosphate without the release of orthophosphate (P(i)) and was inhibited by heparin and insensitive to fluoride. Mg2+, Mn2+, and Co2+ (1.5 mM) stimulated the activity, and Ca2+ was ineffective. The molecular mass of the endopolyphosphatase determined by gel filtration was of ~20 kDa.

Production of antifungal cellobiose lipids by Trichosporon porosum.

The yeast Trichosporon porosum suppresses growth of ascomycetes and basidiomycetes belonging to 52 genera. It is due to secretion of a thermostable fungicidal agent. The suppression was maximal at pH 3.5-4.0. Fungicidal preparation obtained from the culture broth was shown to be a mixture of cellobiosides of dihydrodecane acid with different degree of acetylation of cellobiose residue. The preparation caused the death of Candida albicans and Filobasidiella neoformans cells in the concentrations of 0.2 and 0.03 mM, respectively.

Finding of endopolyphosphatase activity in the yeast Saccharomyces cerevisiae.

Endopolyphosphatase activity has been revealed in cytosol preparations of the yeast Saccharomyces cerevisiae with inactivated PPX1 and PPN1 genes encoding exopolyphosphatases. The enzyme cleaves inorganic polyphosphates with chain length of 15 to 208 phosphate residues to shorter chains without the release of orthophosphate (P(i)). The long chain polyphosphates are cleaved with preference over the short ones. Heparin, a known inhibitor of exopolyphosphatases, represses this activity. The endopolyphosphatase activity is not stimulated by Mg(2+) or Co(2+), in contrast to exopolyphosphatases. This activity along with a pyrophosphatase is supposed to be responsible for polyphosphate utilization as a phosphate reserve in a mutant devoid of exopolyphosphatases.

Inactivation of PPX1 and PPN1 genes encoding exopolyphosphatases of Saccharomyces cerevisiae does not prevent utilization of polyphosphates as phosphate reserve.

Cytosol polyphosphates (polyPs) are the main phosphate (P(i)) reserve in the yeast Saccharomyces cerevisiae. In this work, the participation of cytosol polyPs and exopolyphosphatases in maintenance of P(i) homeostasis under P(i) deficit in the cultivation medium has been studied in different strains of S. cerevisiae. The growth of yeast strains with inactivated genes PPX1 and PPN1 encoding the yeast exopolyphosphatases and a strain with double mutations in these genes in a P(i)-deficient medium is not disturbed. All the studied strains are able to maintain relatively constant P(i) levels in the cytosol. In P(i)-deficient medium, polyP hydrolysis in the cytosol of the parent and PPN1-deficient strains seems to be performed by exopolyphosphatase Ppx1 and proceeds without any change of the spectrum of polyP chain lengths. In the PPX1-deficient strain, long-chain polyPs are depleted first, and only then short-chain polyPs are hydrolyzed. In the double PPX1 and PPN1 mutant having low exopolyphosphatase activity, polyP hydrolysis in the cytosol starts with a notable delay, and about 20% of short-chain polyPs still remain after the polyP hydrolysis in other strains has almost been completed. This fact suggests that S. cerevisiae possesses a system, which makes it possible to compensate for inactivation of the PPX1 and PPN1 genes encoding exopolyphosphatases of the 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.

Efflux of potassium ions from cells and spheroplasts of Saccharomyces cerevisiae yeast treated with silver and copper ions.

Silver ions induce the efflux of potassium from cells of the yeast Saccharomyces cerevisiae but have no such effect on spheroplasts. Copper ions and the natural fungicide 2-O-3-hydroxyhexanoyl-beta-D-glucopyranosyl-(1-->4)-(6-O-acetyl-beta-D-glucopyranosyl-(1-->16)-2,15,16-trihydroxyhexadecanoic acid) induce the efflux of potassium ions from both cells and spheroplasts of S. cerevisiae. Silver and copper ions inhibit the activity of the plasma membrane H+-ATPase during the treatment of both cells and spheroplasts. It is supposed that the inability of silver ions to stimulate potassium efflux from spheroplasts results from damage to some components of K+ transport systems during preparation of spheroplasts.

Inorganic polyphosphates and exopolyphosphatases in different cell compartments of Saccharomyces cerevisiae.

The cytosol, nuclei, vacuoles, and mitochondria of the yeast Saccharomyces cerevisiae possess inorganic polyphosphates (polyPs). PolyP levels, spectra of polyP chain lengths, and their dependence on the growth phase are distinguished in the mentioned compartments. Inactivation of the PPX1 gene has no effect on the polyP metabolism under cultivation of the yeast in medium with glucose and 5-7 mM P(i). Inactivation of the PPN1 gene results in elimination of the high-molecular-mass exopolyphosphatases (approximately 120 to 830 kD) of the cytosol, nuclei, vacuoles, and mitochondria of S. cerevisiae suggesting that it is just PPN1 that encodes these enzymes. Expression of the low-molecular-mass exopolyphosphatase of approximately 45 kD encoded by the PPX1 gene decreases under PPN1 inactivation as well. While PPN1 inactivation has negligible effect on polyP levels, it results in increase in the long-chain polyPs in all the compartments under study.