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.

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.

Mannan and phosphomannan from Kuraishia capsulata yeast.

Linear mannan and branched phosphomannan were identified as exopolysaccharides produced by Kuraishia capsulata yeast. Their structures were determined using nuclear magnetic resonance spectroscopy. The repeating unit of mannan was found to be a trisaccharide →6)-α-Manp-(1→2)-α-Manp-(1→2)-α-Manp-(1→, while the phosphomannan was shown to be built of ß-Manp-(1→2)-α-Manp-(1 disaccharide blocks linked by phosphodiester bonds via C-1 and C-6 of the reducing unit. The production of both polysaccharides was shown to depend on the phosphate concentration in the culture medium. In the absence of phosphate, only mannan was obtained, while an excess of KH2PO4 led to the exclusive production of phosphomannan. Chemical depolymerisation of phosphomannan led to the formation of disaccharide ß-Manp-(1→2)-(6-P)-Manp, representing the repeating unit of the hydrolysed polysaccharide. The treatment of the disaccharide with alkaline phosphatase resulted in the formation of disaccharide ß-Manp-(1→2)-Manp. The latest products can be transformed into glycosyl donors applicable further in the synthesis of oligosaccharides related to Candida cell wall polysaccharides.

Cell wall canals formed upon growth of Candida maltosa in the presence of hexadecane are associated with polyphosphates.

Canals are supramolecular complexes observed in the cell wall of Candida maltosa grown in the presence of hexadecane as a sole carbon source. Such structures were not observed in glucose-grown cells. Microscopic observations of cells stained with diaminobenzidine revealed the presence of oxidative enzymes in the canals. 4΄,6΄-diamino-2-phenylindole staining revealed that a substantial part of cellular polyphosphate was present in the cell wall of cells grown on hexadecane in condition of phosphate limitation. The content and chain length of polyphosphates were higher in hexadecane-grown cells than in glucose grown ones. The treatment of cells with yeast polyphosphatase PPX1 resulted in the decrease of the canal size. These data clearly indicated that polyphosphates are constituents of canals; they might play an important role in the canal structure and functioning.

Triterpenoid saponins from the roots of Acanthophyllum gypsophiloides Regel.

Two new triterpenoid saponins 1 and 2 were isolated from the methanol extract of the roots of Acanthophyllum gypsophiloides Regel. These saponins have quillaic acid or gypsogenin moieties as an aglycon, and both bear similar sets of two oligosaccharide chains, which are 3-O-linked to the triterpenoid part trisaccharide α-L-Arap-(1→3)-[α-D-Galp-(1→2)]-ß-D-GlcpA and pentasaccharide ß-D-Xylp-(1→3)-ß-D-Xylp-(1→3)-α-L-Rhap-(1→2)-[ß-D-Quip-(1→4)]-ß-D-Fucp connected through an ester linkage to C-28. The structures of the obtained saponins were elucidated by a combination of mass spectrometry and 2D NMR spectroscopy. A study of acute toxicity, hemolytic, anti-inflammatory, immunoadjuvant and antifungal activity was carried out. Both saponins 1 and 2 were shown to exhibit immunoadjuvant properties within the vaccine composition with keyhole limpet hemocyanin-based immunogen. The availability of saponins 1 and 2 as individual pure compounds from the extract of the roots of A. gypsophiloides makes it a prospective source of immunoactive agents.

Accumulation of phosphate and polyphosphate by Cryptococcus humicola and Saccharomyces cerevisiae in the absence of nitrogen.

The search for new phosphate-accumulating microorganisms is of interest in connection with the problem of excess phosphate in environment. The ability of some yeast species belonging to ascomycetes and basidiomycetes for phosphate (P (i) ) accumulation in nitrogen-deficient medium was studied. The ascomycetous Saccharomyces cerevisiae and Kuraishia capsulata and basidiomycetous Cryptococcus humicola, Cryptococcus curvatus, and Pseudozyma fusiformata were the best in P (i) removal. The cells of Cryptococcus humicola and S. cerevisiae took up 40% P (i) from the media containing P (i) and glucose (5 and 30 mM, respectively), and up to 80% upon addition of 5 mM MgSO(4) (.) The cells accumulated P (i) mostly in the form of polyphosphate (PolyP). In the presence of Mg(2+) , the content of PolyP with longer average chain length increased in both yeasts; they both had numerous inclusions fluorescing in the yellow region of the spectrum, typical of DAPI-PolyP complexes. Among the yeast species tested, Cryptococcus humicola is a new promising model organisms to study phosphorus removal from the media and biomineralization in microbial cells.

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.

Phosphate accumulation of Acetobacter xylinum.

The cells of Acetobacter xylinum decreased phosphate concentration in the medium from 5 to 2.5 or 0.3 mM during incubation in the presence of Mg(2+) and glucose, or Mg(2+) and casamino acids, respectively. The prevalence of orthophosphate or polyphosphate in the biomass of A. xylinum depends on the medium composition. Under phosphate uptake in the presence of glucose, the content of orthophosphate in the biomass changed little, while that of polyphosphate increased fourfold. At incubation with casamino acids, the content of orthophosphate increased 15 times, while that of polyphosphate increased only 2.5 times. Some part of orthophosphate in this case seems to be bound with the cell surface. The polyphosphate chain length in the cells of A. xylinim increases under phosphate uptake. This increase is more noticeable in the presence of glucose. Casamino acids can be replaced by alpha-ketoglutaric acid in combination with (NH(4))(2)SO(4), or arginine, or glutamine, the catabolism of which results in formation of NH(4) (+) and alpha-ketoglutarate.

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.

Ustilagic acid secretion by Pseudozyma fusiformata strains.

Eight strains of Pseudozyma fusiformata were examined for antifungal activity. All of them had the same spectrum of action and were active against many species of yeasts, yeast-like and filamentous fungi. They secreted glycolipids, which were purified from the culture liquid by column and thin-layer chromatography. According to nuclear magnetic resonance and mass-spectroscopy experiments all strains produced ustilagic acid, a cellobioside-containing 2,15,16-trihydroxypalmitic acid as aglycon, 3-hydroxycaproic acid and acetic acid as O-acylic substituents.

Characterization of an antifungal glycolipid secreted by the yeast Sympodiomycopsis paphiopedili.

An antifungal glycolipid was purified from the culture liquid of the ustilaginomycetous yeast Sympodiomycopsis paphiopedili by column and thin-layer chromatography. According to nuclear magnetic resonance and mass-spectroscopy experiments it was a cellobioside containing 2,15,16-trihydroxypalmitic acid as an aglycon. The minimal effective concentrations leading to ATP leakage and growth inhibition were 45 and 160 microg ml(-1) for Cryptococcus terreus and Candida albicans, respectively.

ATP leakage from yeast cells treated by extracellular glycolipids of Pseudozyma fusiformata.

The ustilaginaceous yeast Pseudozyma fusiformata secreted glycolipids which were lethal to many yeasts and fungi more active at pH of about 4.0, and in the temperature range of 20-30 degrees C. Purified glycolipids enhanced non-specific permeability of the cytoplasmic membrane in sensitive cells, which resulted in ATP leakage and susceptibility of the cells to staining with bromocresol purple. Cells of Saccharomyces cerevisiae lost the ability to acidify the medium. Basidiomycetous yeasts were more sensitive to the glycolipids than ascomycetous ones. The minimal effective glycolipid concentration was 0.13 and 0.26 mg ml(-1) for Cryptococcus terreus and Filobasidiella neoformans, while for Candida albicans and Saccharomyces cerevisiae it was 1.0 and 1.6 mg ml(-1).

The mycocidal, membrane-active complex of Cryptococcus humicola is a new type of cellobiose lipid with detergent features.

The chemical composition of the mycocidal complex (formerly known as microcin) secreted by Cryptococcus humicola was investigated by chemical, mass spectrometric and nuclear magnetic resonance methods. The results indicate that the mycocidal complex is composed of glycolipids with a highly acetylated (up to five acetyl groups) cellobiose backbone [beta-D-Glcp-(1'-->4)-beta-D-Glcp] linked to the omega-hydroxyl group of alpha,omega-dihydroxy palmitate [16:0-alpha,omega-di-OH] with an unsubstituted carboxyl group. The acyl chain forming aglycon can be replaced by [18:0-(alpha,omega-di-OH)], [18:0-(alpha,omega-1,omega-tri-OH)], and [18:0-(alpha,omega-2,omega-tri-OH)]. The complex has a comparatively high surface activity; 0.5 mg/ml of it reduced the surface tension of 0.1 M NaHCO(3) from 71 mN/m to 37 mN/m and interfacial tension against n-hexadecane from 39 mN/m to 10 mN/m. The critical micelle concentration of the complex at pH 4.0, determined by the fluorometric method with N-phenyl-1-naphthylamine as fluorescent probe and by the De Nouy ring method, was 2 x 10(-5) M (taking the average molecular mass of the complex to be 750); it did not depend on the presence of 100 mM KCl and was an order of magnitude higher at pH 7.0. By fluorescence resonance energy transfer spectroscopy with N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-phosphatidylethanolamine as energy donor and N-(rhodamine B sulfonyl)-phosphatidylethanolamine as energy acceptor the complex was shown to intercalate into the liposomal lipid matrix. Primary lesions caused by the complex in planar lipid bilayers were revealed as short-living current fluctuations of a broad spectrum of amplitudes. The mycocidal effect of the complex is suggested to be associated with its detergent-like properties.