The Extracellular Vesicles Containing Inorganic Polyphosphate of Candida Yeast upon Growth on Hexadecane.

The cell wall of Candida yeast grown on presence of hexadecane as a sole carbon source undergoes structural and functional changes including the formation of specific supramolecular complexes-canals. The canals contain specific polysaccharides and enzymes that provide primary oxidization of alkanes. In addition, inorganic polyphosphate (polyP) was identified in Candida maltosa canals. The aim of the work was a comparative study of the features of cell walls and extracellular structures in yeast C. maltosa, C. albicans and C. tropicalis with special attention to inorganic polyphosphates as possible part of these structures when grown on the widely used xenobiotic hexadecane (diesel fuel). Fluorescence microscopy with DAPI has shown an unusual localization of polyP on the cell surface and in the exovesicles in the three yeast species, when growing on hexadecane. Electron-scanning microscopy showed that the exovesicles were associated with the cell wall and also presented in the external environment probably as biofilm components. Treatment of hexadecane-grown cells with purified Ppx1 polyphosphatase led to the release of phosphate into the incubation medium and the disappearance of polyP in vesicles and cell wall observed using microscopic methods. The results indicate the important role of polyP in the formation of extracellular structures in the Candida yeast when consuming hexadecane and are important for the design of xenobiotic destructors based on yeast or mixed cultures.

Effect of Deletions of the Genes Encoding Pho3p and Bgl2p on Polyphosphate Level, Stress Adaptation, and Attachments of These Proteins to Saccharomyces cerevisiae Cell Wall.

Inorganic polyphosphates (polyP), according to literature data, are involved in the regulatory processes of molecular complex of the Saccharomyces cerevisiae cell wall (CW). The aim of the work was to reveal relationship between polyP, acid phosphatase Pho3p, and the major CW protein, glucanosyltransglycosylase Bgl2p, which is the main glucan-remodelling enzyme with amyloid properties. It has been shown that the yeast cells with deletion of the PHO3 gene contain more high molecular alkali-soluble polyP and are also more resistant to exposure to alkali and manganese ions compared to the wild type strain. This suggests that Pho3p is responsible for hydrolysis of the high molecular polyP on the surface of yeast cells, and these polyP belong to the stress resistance factors. The S. cerevisiae strain with deletion of the BGL2 gene is similar to the Δpho3 strain both in the level of high molecular alkali-soluble polyP and in the increased resistance to alkali and manganese. Comparative analysis of the CW proteins demonstrated correlation between the extractability of the acid phosphatase and Bgl2p, and also revealed a change in the mode of Bgl2p attachment to the CW of the strain lacking Pho3p. It has been suggested that Bgl2p and Pho3p are able to form a metabolon or its parts that connects biogenesis of the main structural polymer of the CW, glucan, and catabolism of an important regulatory polymer, polyphosphates.

VTC4 Polyphosphate Polymerase Knockout Increases Stress Resistance of Saccharomyces cerevisiae Cells.

Inorganic polyphosphate (polyP) is an important factor of alkaline, heavy metal, and oxidative stress resistance in microbial cells. In yeast, polyP is synthesized by Vtc4, a subunit of the vacuole transporter chaperone complex. Here, we report reduced but reliably detectable amounts of acid-soluble and acid-insoluble polyPs in the Δvtc4 strain of Saccharomyces cerevisiae, reaching 10% and 20% of the respective levels of the wild-type strain. The Δvtc4 strain has decreased resistance to alkaline stress but, unexpectedly, increased resistance to oxidation and heavy metal excess. We suggest that increased resistance is achieved through elevated expression of DDR2, which is implicated in stress response, and reduced expression of PHO84 encoding a phosphate and divalent metal transporter. The decreased Mg2+-dependent phosphate accumulation in Δvtc4 cells is consistent with reduced expression of PHO84. We discuss a possible role that polyP level plays in cellular signaling of stress response mobilization in yeast.

Enzymes of Polyphosphate Metabolism in Yeast: Properties, Functions, Practical Significance.

Inorganic polyphosphates (polyP) are the linear polymers of orthophosphoric acid varying in the number of phosphate residues linked by the energy-rich phosphoanhydride bonds. PolyP is an essential component in living cells. Knowledge of polyP metabolizing enzymes in eukaryotes is necessary for understanding molecular mechanisms of polyP metabolism in humans and development of new approaches for treating bone and cardiovascular diseases associated with impaired mineral phosphorus metabolism. Yeast cells represent a rational experimental model for this research due to availability of the methods for studying phosphorus metabolism and construction of knockout mutants and strains overexpressing target proteins. Multicomponent system of polyP metabolism in Saccharomyces cerevisiae cells is presented in this review discussing properties, functioning, and practical significance of the enzymes involved in the synthesis and degradation of this important metabolite.

Phosphate efflux as a test of plasma membrane leakage in Saccharomyces cerevisiae cells.

Plasma membrane integrity is a key to cell viability. Currently, the main approach to assessing plasma membrane integrity is the detection of penetration of special dyes, such as trypan blue and propidium iodide, into the cells. However, this method needs expensive equipment: a fluorescent microscope or a flow cytometer. Besides, staining with propidium iodide occasionally gives false-positive results. Here, we suggest the phosphate (Pi) leakage assay as an approach to assess the increase in permeability of the plasma membrane of yeast cells. We studied the dependence of phosphate efflux and uptake into Saccharomyces cerevisiae cells on the composition of the incubation medium, time, and ambient pH. The difference in optimal conditions for these processes suggests that Pi efflux is not conducted by the Pi uptake system. The Pi efflux in water correlated with the proportion of cells stained with propidium iodide. This indicated that Pi efflux is associated with cytoplasmic membrane disruption in a portion of the yeast cell population. The assay of Pi efflux was used to evaluate membrane disruption in S. cerevisiae cells treated with some heavy metal ions and detergents.

The Reduced Level of Inorganic Polyphosphate Mobilizes Antioxidant and Manganese-Resistance Systems in Saccharomyces cerevisiae.

Inorganic polyphosphate (polyP) is crucial for adaptive reactions and stress response in microorganisms. A convenient model to study the role of polyP in yeast is the Saccharomyces cerevisiae strain CRN/PPN1 that overexpresses polyphosphatase Ppn1 with stably decreased polyphosphate level. In this study, we combined the whole-transcriptome sequencing, fluorescence microscopy, and polyP quantification to characterize the CRN/PPN1 response to manganese and oxidative stresses. CRN/PPN1 exhibits enhanced resistance to manganese and peroxide due to its pre-adaptive state observed in normal conditions. The pre-adaptive state is characterized by up-regulated genes involved in response to an external stimulus, plasma membrane organization, and oxidation/reduction. The transcriptome-wide data allowed the identification of particular genes crucial for overcoming the manganese excess. The key gene responsible for manganese resistance is PHO84 encoding a low-affinity manganese transporter: Strong PHO84 down-regulation in CRN/PPN1 increases manganese resistance by reduced manganese uptake. On the contrary, PHM7, the top up-regulated gene in CRN/PPN1, is also strongly up-regulated in the manganese-adapted parent strain. Phm7 is an unannotated protein, but manganese adaptation is significantly impaired in Δphm7, thus suggesting its essential function in manganese or phosphate transport.

Inorganic polyphosphate in methylotrophic yeasts.

Inorganic polyphosphate (polyP) is a significant regulatory and metabolic compound in yeast cells. We compared polyP content and localization, polyphosphatase activities, and transcriptional profile of polyP-related genes in industrially important methylotrophic yeasts, Hansenula polymorpha and Pichia pastoris. The increased need for phosphate, the decrease of long-chain polyP level, the accumulation of short-chain polyP, and enhanced endopolyphosphatase activity in the crude membrane fraction were observed in methanol-grown cells compared with glucose-grown cells of both species. Transcriptome analysis revealed notable differences in the expression patterns of key genes encoding proteins related to polyP metabolism. In methanol-grown cells, the genes encoding endopolyphosphatases and phosphate transporters were upregulated. The changes in polyP metabolism are probably related to the peculiarities of bioenergetics of methanol-grown cells.

The cadmium tolerance in Saccharomyces cerevisiae depends on inorganic polyphosphate.

The sensitivity to cadmium (Cd(II)), an important environmental pollutant, was studied in the cells of Saccharomyces cerevisiae strains with genetically altered polyphosphate metabolism. The strains overproducing polyphosphatases PPX1 or PPN1 were more sensitive to Cd(II) than the parent strain. The half maximal inhibitory concentrations were 0.02 and 0.05 mM for the transformants and the parent strain, respectively. Transformant strains cultivated in the presence of Cd(II) show a decrease in the content of short-chained cytosolic acid soluble polyphosphate. The role of this polyphosphate fraction in detoxification of heavy metal ions is discussed.

Polyphosphatase PPN1 of Saccharomyces cerevisiae: switching of exopolyphosphatase and endopolyphosphatase activities.

The polyphosphatase PPN1 of Saccharomyces cerevisiae shows an exopolyphosphatase activity splitting phosphate from chain end and an endopolyphosphatase activity fragmenting high molecular inorganic polyphosphates into shorter polymers. We revealed the compounds switching these activities of PPN1. Phosphate release and fragmentation of high molecular polyphosphate prevailed in the presence of Co2+ and Mg2+, respectively. Phosphate release and polyphosphate chain shortening in the presence of Co2+ were inhibited by ADP but not affected by ATP and argininе. The polyphosphate chain shortening in the presence of Mg2+ was activated by ADP and arginine but inhibited by ATP.

Purification and properties of recombinant exopolyphosphatase PPN1 and effects of its overexpression on polyphosphate in Saccharomyces cerevisiae.

Inorganic polyphosphate performs many regulatory functions in living cells. The yeast exopolyphosphatase PPN1 is an enzyme with multiple cellular localization and probably variable functions. The Saccharomyces cerevisiae strain with overexpressed PPN1 was constructed for large-scale production of the enzyme and for studying the effect of overproduction on polyphosphate metabolism. The ΔPPN1 strain was transformed by the vector containing this gene under a strong constitutive promoter of glycerol aldehyde-triphosphate dehydrogenase of S. cerevisiae. Exopolyphosphatase activity in the transformant increased 28- and 11-fold compared to the ΔPPN1 and parent strains, respectively. The content of acid-soluble polyphosphate decreased ∼6-fold and the content of acid-insoluble polyphosphate decreased ∼2.5-fold in the cells of the transformant compared to the ΔPPN1 strain. The recombinant enzyme was purified. The substrate specificity, cation requirement, and inhibition by heparin were found to be similar to native PPN1. The molecular mass of a subunit (∼33 kD) and the amino acid sequence of the recombinant enzyme were the same as in mature PPN1. The recombinant enzyme was localized mainly in the cytoplasm (40%) and vacuoles (20%). The overproducer strain had no growths defects under phosphate deficiency or phosphate excess. In contrast to the parent strains accumulating polyphosphate, the transformant accumulated orthophosphate under phosphate surplus.

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 Pi-excess media after Pi 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.

The antifungal effect of cellobiose lipid on the cells of Saccharomyces cerevisiae depends on carbon source.

The cellobiose lipid of Cryptococcus humicola, 16-(tetra-O-acetyl-ß-cellobiosyloxy)-2-hydroxyhexadecanoic acid, is a natural fungicide. Sensitivity of the cells of Saccharomyces cerevisiae to the fungicide depends on a carbon source. Cellobiose lipid concentrations inducing the leakage of potassium ions and ATP were similar for the cells grown in the medium with glucose and ethanol. However, the cells grown on glucose and ethanol died at 0.05 mg ml(-1) and 0.2 mg ml(-1) cellobiose lipid, respectively. Inorganic polyphosphate (PolyP) synthesis was 65% of the control with 0.05 mg ml(-1) cellobiose lipid during cultivation on ethanol. PolyP synthesis was not observed during the cultivation on glucose at the same cellobiose lipid concentration. The content of longer-chain polyP was higher during cultivation on ethanol. We speculate the long-chained polyP participate in the viability restoring of ethanol-grown cells after treatment with the cellobiose lipid.

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 (31)P nuclear magnetic resonance spectroscopy, enzymatic assay, and electrophoresis.