Publications by authors named "Tatiana Kulakovskaya"

30 Publications

  • Page 1 of 1

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

Biochemistry (Mosc) 2021 Jan;86(Suppl 1):S96-S108

Institute of Bioengineering, Federal Scientific Center for Biotechnology, Russian Academy of Sciences, Moscow, 117312, Russia.

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.
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http://dx.doi.org/10.1134/S0006297921140078DOI Listing
January 2021

Changes in cell wall structure and protein set in Candida maltosa grown on hexadecane.

Folia Microbiol (Praha) 2021 Apr 27;66(2):247-253. Epub 2020 Nov 27.

G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Pushchino Center for Biological Research of the Russian Academy of Sciences, prosp. Nauki 5, Pushchino, Moscow Region, 142290, Russia.

The yeast Candida maltosa is a model organism for studying adaptive changes in the structure and function of the cell wall when consuming water-insoluble nutrient sources. The cells of C. maltosa that utilize hydrocarbons contain supramolecular structures, so-called "canals" in the cell wall. Differences in protein profiles of culture liquids and cell wall extracts of C. maltosa grown on glucose and hexadecane were analyzed. Three proteins specific of cells grown on hexadecane were revealed using mass spectrometry: glycosyl hydrolase EPD2 in the culture liquid; a protein belonging to the cytochrome C family in the 0.5 mol/L NaCl extract; and PPIA_CANAL protein known as chaperone, in the 0.1% SDS extract. The possible role of these proteins in cell wall structures responsible for adaptation to hexadecane utilization is discussed.
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http://dx.doi.org/10.1007/s12223-020-00840-2DOI Listing
April 2021

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

Can J Microbiol 2021 Mar 10;67(3):226-230. Epub 2020 Sep 10.

FRC Pushchino Center for Biological Research, Russian Academy of Sciences, Skryabin Institute of Biochemistry and Physiology of Microorganisms, pr. Nauki 5, Pushchino, 142290 Russia.

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 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 cells treated with some heavy metal ions and detergents.
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http://dx.doi.org/10.1139/cjm-2020-0040DOI Listing
March 2021

Effect of Fe on inorganic polyphosphate level in autotrophic and heterotrophic cells of Rhodospirillum rubrum.

Arch Microbiol 2019 Nov 4;201(9):1307-1312. Epub 2019 Jul 4.

G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, FRC Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences, Prospect Nauki 5, Pushchino, 142290, Russia.

Inorganic polyphosphate is involved in metal homeostasis in microorganisms. The aim of the study was to reveal differences in polyphosphate metabolism of Rhodospirillum rubrum under autotrophic and heterotrophic cultivation in the presence of Fe (2.3 mg Fe L) and without Fe (traces). Heterotrophic conditions without Fe resulted in cell lysis and low biomass yield. High polyphosphate content and low exopolyphosphatase activity were observed in the cells cultivated autotrophically in the presence of Fe. The cells grown heterotrophically in the presence of Fe contained more phosphate and low-molecular polyphosphate; on the contrary, the content of the high molecular polyphosphate decreased in parallel with the increase in exopolyphosphatase activity. The possible involvement of Pi and polyphosphate to the formation of Fe-containing inclusions is discussed.
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http://dx.doi.org/10.1007/s00203-019-01697-xDOI Listing
November 2019

Ppn2 endopolyphosphatase overexpressed in Saccharomyces cerevisiae: Comparison with Ppn1, Ppx1, and Ddp1 polyphosphatases.

Biochimie 2019 Aug 6;163:101-107. Epub 2019 Jun 6.

Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky prosp. 33-2, Moscow, 119071, Russia.

Saccharomyces cerevisiae has high level of inorganic polyphosphate and a multicomponent system of its metabolism, including polyphosphatases Ppx1, Ppn1, Ddp1, and Ppn2. The aim of the study was to construct the yeast strain overexpressing Ppn2 and to compare the properties of Ppn2, Ppx1, Ppn1, and Ddp1 purified from overexpressing strains of S. cerevisiae. We overexpressed Ppn2 in S. cerevisiae under a strong constitutive promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase-encoding gene and suggested biochemical criteria for distinguishing among yeast polyphosphatases, which is important for their identification and understanding of their functions. Ppn2, Ppn1, and Ddp1 had endopolyphosphatase activities, whereas Ppx1 did not. Ppx1 and Ppn1 exhibited high and Ddp1 and Ppn2 low exopolyphosphatase activity: 240, 500, 0.05 and 0.1 U/mg protein, respectively. The enzymes had distinct patterns of exopolyphosphatase activities stimulation by divalent metal ions. Ppn2, Ppn1 and Ddp1 displayed endopolyphosphatase activity in the presence of 1 mM Mg. The endopolyphosphatase activities of Ppn2 and Ppn1 were induced by 0.01 mM of Co or Zn, whereas that of Ddp1 required 0.1 mM of these cations. The endopolyphosphatase activity of Ppn1 was inhibited by 0.01 mg mL of heparin, while endopolphosphatase activity of Ppn2 was weakly sensitive to 0.25 mg mL of heparin. The Ppx1 and Ppn1 activity with guanosine tetraphosphate was nearly 80% of activity with long-chain polyphosphates. The Ppn1 hydrolyzed dATP, while Ppx1 did not. The differences in the mode of polyphosphate hydrolysis, substrate specificity, metal ion dependence and cell localization suggest distinct roles of these enzymes in yeast.
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http://dx.doi.org/10.1016/j.biochi.2019.06.001DOI Listing
August 2019

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

Cells 2019 05 15;8(5). Epub 2019 May 15.

Skryabin Institute of Biochemistry and Physiology of Microorganisms, FRC Pushchino Center for Biological Research of the Russian Academy of Sciences, pr. Nauki 5, Pushchino 142290, Russia.

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 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 encoding a low-affinity manganese transporter: Strong down-regulation in CRN/PPN1 increases manganese resistance by reduced manganese uptake. On the contrary, , 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 Δ, thus suggesting its essential function in manganese or phosphate transport.
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http://dx.doi.org/10.3390/cells8050461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6562782PMC
May 2019

The acid phosphatase Pho5 of Saccharomyces cerevisiae is not involved in polyphosphate breakdown.

Folia Microbiol (Praha) 2019 Nov 1;64(6):867-873. Epub 2019 Apr 1.

Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky prosp. 33-2, Moscow, 119071, Russia.

Inorganic polyphosphate is involved in architecture and functioning of yeast cell wall. The strain of Saccharomyces cerevisiae constitutively overexpressing acid phosphatase Pho5 was constructed for studying the Pho5 properties and its possible participation in polyphosphate metabolism. The parent strain was transformed by the vector carrying the PHO5 gene under a strong constitutive promoter of glyceraldehyde-3-phosphate dehydrogenase of S. cerevisiae. The culture liquid and biomass of transformant strain contained approximately equal total acid phosphatase activity. The levels of acid phosphatase activity associated with the cell wall and culture liquid increased in the transformant strain compared to the parent strain ~ 10- and 20-fold, respectively. The Pho5 preparation (specific activity of 46 U/mg protein and yield of 95 U/L) was obtained from culture liquid of overproducing strain. The overproducing strain had no changes in polyphosphate level. The activity of Pho5 with long-chained polyP was negligible. We concluded that Pho5 is not involved in polyphosphate metabolism. Purified Pho5 showed a similar activity with p-nitrophenylphosphate, ATP, ADP, glycerophosphate, and glucose-6-phosphate. The substrate specificity of Pho5 and its extracellular localization suggest its function: the hydrolysis of organic compounds with phosphoester bonds at phosphate limitation.
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http://dx.doi.org/10.1007/s12223-019-00702-6DOI Listing
November 2019

Inorganic polyphosphates and heavy metal resistance in microorganisms.

World J Microbiol Biotechnol 2018 Aug 27;34(9):139. Epub 2018 Aug 27.

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow, Russia, 142290.

The mechanisms of heavy metal resistance in microbial cells involve multiple pathways. They include the formation of complexes with specific proteins and other compounds, the excretion from the cells via plasma membrane transporters in case of procaryotes, and the compartmentalization of toxic ions in vacuoles, cell wall and other organelles in case of eukaryotes. The relationship between heavy metal tolerance and inorganic polyphosphate metabolism was demonstrated both in prokaryotic and eukaryotic microorganisms. Polyphosphates, being polyanions, are involved in detoxification of heavy metals through complex formation and compartmentalization. The bacteria and fungi cultivated in the presence of some heavy metal cations contain the enhanced levels of polyphosphate. In bacteria, polyphosphate sequesters heavy metals; some of metal cations stimulate an exopolyphosphatase activity, which releases phosphate from polyphosphates, and MeHPO ions are then transported out of the cells. In fungi, the overcoming of heavy metal stresses is associated with the accumulation of polyphosphates in cytoplasmic inclusions, vacuoles and cell wall and the formation of cation/polyphosphate complexes. The effects of knockout mutations and overexpression of the genes encoding polyphosphate-metabolizing enzymes on heavy metal resistance are discussed.
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http://dx.doi.org/10.1007/s11274-018-2523-7DOI Listing
August 2018

Inorganic polyphosphate in methylotrophic yeasts.

Appl Microbiol Biotechnol 2018 Jun 21;102(12):5235-5244. Epub 2018 Apr 21.

FRC Biotechnology, Institute of Bioengineering, Russian Academy of Sciences, pr. Shestidesyatiletiya Oktyabrya 7-1, Moscow, 117312, Russia.

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.
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http://dx.doi.org/10.1007/s00253-018-9008-3DOI Listing
June 2018

The biosorption of cadmium and cobalt and iron ions by yeast Cryptococcus humicola at nitrogen starvation.

Folia Microbiol (Praha) 2018 Jul 19;63(4):507-510. Epub 2018 Jan 19.

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Prospect Nauki 5, Pushchino, Moscow Region, Russia, 142290.

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.
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http://dx.doi.org/10.1007/s12223-018-0583-6DOI Listing
July 2018

Mannan and phosphomannan from Kuraishia capsulata yeast.

Carbohydr Polym 2018 Feb 28;181:624-632. Epub 2017 Nov 28.

Laboratory of Glycoconjugate Chemistry, N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospect 47, 119991 Moscow, Russia. Electronic address:

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 β-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 KHPO led to the exclusive production of phosphomannan. Chemical depolymerisation of phosphomannan led to the formation of disaccharide β-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 β-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.
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http://dx.doi.org/10.1016/j.carbpol.2017.11.103DOI Listing
February 2018

The cadmium tolerance in Saccharomyces cerevisiae depends on inorganic polyphosphate.

J Basic Microbiol 2017 Nov 15;57(11):982-986. Epub 2017 Aug 15.

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Russia.

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.
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http://dx.doi.org/10.1002/jobm.201700257DOI Listing
November 2017

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

FEMS Yeast Res 2017 05;17(3)

Laboratory of 3-D structures of microorganisms, G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Prospect Nauki, 5 Pushchino, Moscow Region, 142290 Russian Federation.

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.
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http://dx.doi.org/10.1093/femsyr/fox026DOI Listing
May 2017

Manganese tolerance in yeasts involves polyphosphate, magnesium, and vacuolar alterations.

Folia Microbiol (Praha) 2016 Jul 8;61(4):311-7. Epub 2015 Dec 8.

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Prospect Nauki 5, Pushchino, Moscow region, 142290, Russia.

Basidiomycetous and ascomycetous yeast species were tested for manganese tolerance. Basidiomycetous Cryptococcus humicola, Cryptococcus terricola, Cryptococcus curvatus and ascomycetous Candida maltosa, Kluyveromyces marxianus, Kuraishia capsulata, Lindnera fabianii and Sacharomyces cerevisiae were able to grow at manganese excess (2.5 mmol/L), while the growth of basidiomycetous Rhodotorula bogoriensis was completely suppressed. The lag phase duration increased and the exponential growth rate decreased at manganese excess. The increase of cell size and enlargement of vacuoles were characteristics for the cells grown at manganese excess. The alterations in inorganic polyphosphate content and cellular localization were studied. L. fabianii, K. capsulata, C. maltosa, and Cr. humicola accumulated the higher amounts of inorganic polyphosphates, while Cr. terricola and Cr. curvatus demonstrated no such accumulation. The polyphosphate content in the cell wall tested by DAPI staining increased in all species under the study; however, this effect was more pronounced in Cr. terricola and Cr. curvatus. The accumulation of Mg(2+) in the cell wall under Mn(2+) excess was observed in Cr. humicola, Cr. curvatus and Cr. terricola. The accumulation of polyphosphate and magnesium in the cell wall was supposed to be a factor of manganese tolerance in yeasts.
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http://dx.doi.org/10.1007/s12223-015-0440-9DOI Listing
July 2016

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

PLoS One 2015 5;10(3):e0119594. Epub 2015 Mar 5.

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, pr. Nauki 5, Pushchino, 142290, Russia.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0119594PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4350845PMC
January 2016

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

J Biosci Bioeng 2015 Jan 15;119(1):52-6. Epub 2014 Jul 15.

Bioengineering Centre, Russian Academy of Sciences, pr. Shestidesyatiletiya Oktyabrya 7-1, Moscow 117312, Russia.

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.
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http://dx.doi.org/10.1016/j.jbiosc.2014.06.006DOI Listing
January 2015

Synthesis of magneto-sensitive iron-containing nanoparticles by yeasts.

J Ind Microbiol Biotechnol 2014 Apr 28;41(4):657-63. Epub 2014 Feb 28.

Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 5 Prospekt Nauki, Pushchino, Moscow Region, 142290, Russia,

Industrial production of magneto-sensitive nanoparticles, which can be used in the production of target drug delivery carriers, is a subject of interest for biotechnology and microbiology. Synthesis of these nanoparticles by microorganisms has been described only for bacterial species. At the same time, it is well known that yeasts can form various metal-containing nanoparticles used, for instance, in semiconductors, etc. This paper describes the first results of the biosynthesis of magneto-sensitive nanoparticles by yeasts. The organisms we used-Saccharomyces cerevisiae and Cryptococcus humicola-represented two different genera. Magneto-sensitive nanoparticles were synthesized at room temperature in bench-scale experiments. The study included transmission electron microscopy of the yeast cells and their energy dispersive spectrum analyses and revealed the presence of iron-containing nanoparticles. Both yeast cultures synthesized nanoparticles at high concentrations of dissolved iron. Electron microscopy showed that nanoparticles were associated mainly with the yeast cell wall. Formation of magneto-sensitive nanoparticles was studied under conditions of applied magnetic fields; a possible stimulating role of magnetic field is suggested. On the whole, the paper reports a novel approach to green biosynthesis of magneto-sensitive nanoparticles.
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http://dx.doi.org/10.1007/s10295-014-1417-4DOI Listing
April 2014

Cytoplasmic inorganic polyphosphate participates in the heavy metal tolerance of Cryptococcus humicola.

Folia Microbiol (Praha) 2014 Sep 16;59(5):381-9. Epub 2014 Feb 16.

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Prospect Nauki 5, Pushchino, Moscow Region, 142290, Russia.

The basidiomycetous yeast Cryptococcus humicola was shown to be tolerant to manganese, cobalt, nickel, zinc, lanthanum, and cadmium cations at a concentration of 2.5 mmol/L, which is toxic for many yeasts. The basidiomycetous yeast Cryptococcus terreus was sensitive to all these ions and did not grow at the above concentration. In the presence of heavy metal cations, С. humicola, as opposed to C. terreus, was characterized by the higher content of acid-soluble inorganic polyphosphates. In vivo 4',6'-diamino-2-phenylindole dihydrochloride staining revealed polyphosphate accumulation in the cell wall and cytoplasmic inclusions of С. humicola in the presence of heavy metals. In C. terreus, polyphosphates in the presence of heavy metals accumulate mainly in vacuoles, which results in morphological changes in these organelles and, probably, disturbance of their function. The role of polyphosphate accumulation and cellular localization as factors of heavy metal tolerance of Cryptococcus humicola is discussed.
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http://dx.doi.org/10.1007/s12223-014-0310-xDOI Listing
September 2014

Adaptation of Saccharomyces cerevisiae to toxic manganese concentration triggers changes in inorganic polyphosphates.

FEMS Yeast Res 2013 Aug 3;13(5):463-70. Epub 2013 Jun 3.

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow region, Russia.

The ability of Saccharomyces cerevisiae to adapt to toxic Mn(2+) concentration (4 mM) after an unusually long lag phase has been demonstrated for the first time. The mutants lacking exopolyphosphatase PPX1 did not change the adaptation time, whereas the mutants lacking exopolyphosphatase PPN1 reduced the lag period compared with the wild-type strains. The cell populations of WT and ΔPPN1 in the stationary phase at cultivation with Mn(2+) contained a substantial number of enlarged cells with a giant vacuole. The adaptation correlated with the triggering of polyphosphate metabolism: the drastic increase in the rate and chain length of acid-soluble polyphosphate. The share of this fraction, which is believed to be localized in the cytoplasm, increased to 76%. Its average chain length increased to 200 phosphate residues compared with 15 at the cultivation in the absence of manganese. DAPI-stained inclusions in the cytoplasm were accumulated in the lag phase during the cultivation with Mn(2+).
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http://dx.doi.org/10.1111/1567-1364.12049DOI Listing
August 2013

V-ATPase dysfunction suppresses polyphosphate synthesis in Saccharomyces cerevisiae.

Folia Microbiol (Praha) 2013 Nov 2;58(6):437-41. Epub 2013 Feb 2.

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142290, Pushchino, Moscow Region, Russia.

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.
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http://dx.doi.org/10.1007/s12223-013-0226-xDOI Listing
November 2013

Triterpenoid saponins from the roots of Acanthophyllum gypsophiloides Regel.

Beilstein J Org Chem 2012 23;8:763-75. Epub 2012 May 23.

Laboratory of Glycoconjugate Chemistry, N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky prospect 47, 119991 Moscow, Russian Federation.

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)]-β-D-GlcpA and pentasaccharide β-D-Xylp-(1→3)-β-D-Xylp-(1→3)-α-L-Rhap-(1→2)-[β-D-Quip-(1→4)]-β-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.
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http://dx.doi.org/10.3762/bjoc.8.87DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3388865PMC
September 2012

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

FEMS Yeast Res 2012 Sep 28;12(6):617-24. Epub 2012 May 28.

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Russia.

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.
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http://dx.doi.org/10.1111/j.1567-1364.2012.00812.xDOI Listing
September 2012

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

Springerplus 2012 25;1:18. Epub 2012 Sep 25.

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region 142290 Russia.

The cellobiose lipid of Cryptococcus humicola, 16-(tetra-O-acetyl-β-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.
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http://dx.doi.org/10.1186/2193-1801-1-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3725880PMC
August 2013

Phosphate accumulation of Acetobacter xylinum.

Arch Microbiol 2009 May 24;191(5):467-71. Epub 2009 Mar 24.

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow, Russia.

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.
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http://dx.doi.org/10.1007/s00203-009-0470-2DOI Listing
May 2009

Effect of a carbon source on polyphosphate accumulation in Saccharomyces cerevisiae.

FEMS Yeast Res 2008 Sep 17;8(6):877-82. Epub 2008 Jul 17.

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow, Russia.

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.
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http://dx.doi.org/10.1111/j.1567-1364.2008.00420.xDOI Listing
September 2008

Formation of insoluble magnesium phosphates during growth of the archaea Halorubrum distributum and Halobacterium salinarium and the bacterium Brevibacterium antiquum.

FEMS Microbiol Ecol 2005 Mar 25;52(1):129-37. Epub 2004 Nov 25.

Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia.

Stationary phase cells of the halophilic archaea Halobacterium salinarium and Halorubrum distributum, growing at 3-4 M NaCl, and of the halotolerant bacterium Brevibacterium antiquum, growing with and without 2.6 NaCl, took up approximately 90% of the phosphate from the culture media containing 2.3 and 11.5 mM phosphate. The uptake was blocked by the uncoupler FCCP. In B. antiquum, EDTA inhibited the phosphate uptake. The content of polyphosphates in the cells was significantly lower than the content of orthophosphate. At a high phosphate concentration, up to 80% of the phosphate taken up from the culture medium was accumulated as Mg(2)PO(4)OH x 4H(2)O in H. salinarium and H. distributum and as NH(4)MgPO(4) x 6H(2)O in B. antiquum. Consolidation of the cytoplasm and enlargement of the nucleoid zone were observed in the cells during phosphate accumulation. At phosphate surplus, part of the H. salinarium and H. distributum cell population was lysed. The cells of B. antiquum were not lysed and phosphate crystals were observed in the cytoplasm.
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http://dx.doi.org/10.1016/j.femsec.2004.10.012DOI Listing
March 2005

Ustilagic acid secretion by Pseudozyma fusiformata strains.

FEMS Yeast Res 2005 Jul;5(10):919-23

G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142290 Pushchino, Moscow region, Russia.

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.
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http://dx.doi.org/10.1016/j.femsyr.2005.04.006DOI Listing
July 2005

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

FEMS Yeast Res 2004 Dec;5(3):247-52

G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142290 Pushchino, Moscow region, Russia.

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.
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http://dx.doi.org/10.1016/j.femsyr.2004.07.008DOI Listing
December 2004

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

FEMS Yeast Res 2003 Jun;3(4):401-4

G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142290, Moscow region, Pushchino, Russia.

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).
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http://dx.doi.org/10.1016/S1567-1356(02)00202-7DOI Listing
June 2003

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

Biochim Biophys Acta 2002 Feb;1558(2):161-70

Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Moscow Region, Russia.

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.
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http://dx.doi.org/10.1016/s0005-2736(01)00428-xDOI Listing
February 2002