Accumulation of Polyphosphates and Expression of High Molecular Weight Exopolyphosphatase in the Yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The effect of cultivation time and concentration of inorganic phosphate (P(i)) in the culture medium on the accumulation of polyphosphates (polyP) and the activity of two cytosolic exopolyphosphatases of the yeast Saccharomyces cerevisiae was studied: an exopolyphosphatase of 40 kD encoded by PPX1 and a high molecular weight exopolyphosphatase encoded by another gene. Depletion of polyP in the cells on P(i) starvation is a signal factor for the accumulation of polyP after the subsequent addition of 5-20 mM P(i) and glucose to the cells or spheroplasts. A high activity of both exopolyphosphatases does not prevent the accumulation of polyP. The expression of the high molecular weight exopolyphosphatase is due to the acceleration of metabolism in cells that have reached the stage of growth deceleration on the addition of P(i) and glucose or complete culture medium. This process may occur independently from the accumulation of polyP. The activity of exopolyphosphatase PPX1 depends less on the mentioned factors, decreasing 10-fold only under conditions of phosphate surplus at the stationary growth stage.     

Effect of a carbon source on polyphosphate accumulation in Saccharomyces cerevisiae

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

Effect of cycloheximide, iodoacetamide, and antimycin A on inorganic phosphate synthesis in Saccharomyces cerevisiae VKM Y-1173

The effect of inhibitors of protein synthesis (cycloheximide, CHI), glycolysis (iodoacetamide, IAA), and oxidative phosphorylation (antimycin A, ANM) on inorganic phosphate (polyP) synthesis during the first 0.5 h of their hypercompensation in Saccharomyces cerevisiae VKM Y-l173 grown on 2% glucose-containing media at low (hypoxia) or high aeration rates or in the presence of 1 vol % ethanol under high aeration conditions was studied. PolyP accumulation was highest in the medium with glucose under hypoxia; lower, with glucose at high aeration; and lowest, in the medium with ethanol. CHI had a small effect on the total polyP level but significantly stimulated ATP accumulation, irrespective of the culture growth conditions. The low-polymer acid-soluble polyP1 were synthesized most actively by the cells grown on glucose under hypoxia, alkali-soluble polyP3 were synthesized at en hanced aeration, and the most hig-molecular fraction, polyP5, was actively accumulated along with polyP3 at cultivation on ethanol. Regardless of the growth conditions, CHI inhibited accumulation of polyP4, the synthesis of which is associated with the synthesis of mannoproteins. IAA and ANM largely inhibited synthesis of all fractions at yeast growth under hypoxia and on ethanol, respectively. The results as a whole demonstrate the dependence of polyP formation on the main energy-generating cell processes and, at the same time, the absence of direct dependence of their synthesis on ATP concentration in Saccharomyces cerevisiae VKM Y-l 173.     

Accumulation of inorganic polyphosphate in phoU mutants of Escherichia coli and Synechocystis sp. strain PCC6803

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

Polyphosphate kinase of Acinetobacter sp. strain ADP1: purification and characterization of the enzyme and its role during changes in extracellular phosphate levels.

Polyphosphate (polyP) is a ubiquitous biopolymer whose function and metabolism are incompletely understood. The polyphosphate kinase (PPK) of Acinetobacter sp. strain ADP1, an organism that accumulates large amounts of polyP, was purified to homogeneity and characterized. This enzyme, which adds the terminal phosphate from ATP to a growing chain of polyP, is a 79-kDa monomer. PPK is sensitive to magnesium concentrations, and optimum activity occurs in the presence of 3 mM MgCl(2). The optimum pH was between pH 7 and 8, and significant reductions in activity occurred at lower pH values. The greatest activity occurred at 40 degrees C. The half-saturation ATP concentration for PPK was 1 mM, and the maximum PPK activity was 28 nmol of polyP monomers per microg of protein per min. PPK was the primary, although not the sole, enzyme responsible for the production of polyP in Acinetobacter sp. strain ADP1. Under low-phosphate (P(i)) conditions, despite strong induction of the ppk gene, there was a decline in net polyP synthesis activity and there were near-zero levels of polyP in Acinetobacter sp. strain ADP1. Once excess phosphate was added to the P(i)-starved culture, both the polyP synthesis activity and the levels of polyP rose sharply. Increases in polyP-degrading activity, which appeared to be mainly due to a polyphosphatase and not to PPK working in reverse, were detected in cultures grown under low-P(i) conditions. This activity declined when phosphate was added.     

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.     

Production and release of polyphosphate by a genetically engineered strain of Escherichia coli.

A recombinant strain of Escherichia coli MV1184, which contains plasmid-borne genes encoding the phosphate-specific transport (Pst) system and polyphosphate (polyP) kinase, accumulated high levels of Pi and released polyP into the medium. PolyP could be separated from the culture supernatant by DEAE-Toyopearl 650M chromatography and identified by high-resolution 31P nuclear magnetic resonance spectroscopy. Once E. coli recombinants accumulated high levels of polyP, they released polyP concomitantly with Pi uptake. PolyP release did not accompany the decrease in the cell density, indicating that it is not simply a result of cell lysis. PolyP release ceased when Pi became depleted in the medium and resumed upon addition of Pi to the medium. When Pi uptake was inhibited by 0.1 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP), no polyP release was observed. Furthermore, neither Pi uptake nor polyP release occurred when cells were incubated at 4 degrees C. These findings suggest that the occurrence of polyP release is a possible mechanism that limits a further increase in the cellular polyP concentration in E. coli recombinants. High-resolution 31P nuclear magnetic resonance spectroscopy also detected a surface pool of polyP in intact cells of the E. coli recombinant. The polyP resonance increased when cells were treated with EDTA and broadened upon the addition of a shift reagent, praseodymium. Although the mechanism of surface polyP accumulation is unclear, surface polyP seems to serve as the source for polyP release.     

Characterization of two phosphate transport systems in Acinetobacter johnsonii 210A.

The transport of P(i) was characterized in Acinetobacter johnsonii 210A, which is able to accumulate an excessive amount of phosphate as polyphosphate (polyP) under aerobic conditions. P(i) is taken up against a concentration gradient by energy-dependent, carrier-mediated processes. A. johnsonii 210A, grown under P(i) limitation, contains two uptake systems with Kt values of 0.7 +/- 0.2 microM and 9 +/- 1 microM. P(i) uptake via the high-affinity component is drastically reduced by N,N'-dicyclohexylcarbodiimide, an inhibitor of H(+)-ATPase, and by osmotic shock. Together with the presence of P(i)-binding activity in concentrated periplasmic protein fractions, these results suggest that the high-affinity transport system belongs to the group of ATP-driven, binding-protein-dependent transport systems. Induction of this transport system upon transfer of cells grown in the presence of excess P(i) to P(i)-free medium results in a 6- to 10-fold stimulation of the P(i) uptake rate. The constitutive low-affinity uptake system for P(i) is inhibited by uncouplers and can mediate counterflow of P(i), indicating its reversible, secondary nature. The presence of an inducible high-affinity uptake system for P(i) and the ability to decrease the free internal P(i) pool by forming polyP enable A. johnsonii 210A to reduce the P(i) concentration in the aerobic environment to micromolar levels. Under anaerobic conditions, polyP is degraded again and P(i) is released via the low-affinity secondary transport system.     

Regulation of bacterial phosphate taxis and polyphosphate accumulation in response to phosphate starvation stress

Phosphorus (P) is an essential constituent in all types of living organisms. Bacteria, which use inorganic phosphate (P_i), as the preferred P source, have evolved complex systems to survive during P_i starvation conditions. Recently, we found thatPseudomonas aeruginosa, a monoflagellated, obligately aerobic bacterium, is attracted to P_i. The evidence that the chemotactic response to P_i (P_i taxis) was observed only with cells grown in P_i-limiting medium suggests that P_i taxis plays an important role in scavenging P_i residues under conditions of P_i starvation. Many bacteria also exhibit rapid and extensive accumulation of polyphosphate (polyP), when P_i is added to cells previously subjected to P_i starvation stress. Since polyP can serve as a P source during P_i starvation conditions, it is likely that polyP accumulation is a protective mechanism for survival during P_i starvation. In the present review, we summarize our current knowledge on regulation of bacterial P_i taxis and polyP accumulation in response to P_i starvation stress.     

Overproduction of YjbB reduces the level of polyphosphate in Escherichia coli: a hypothetical role of YjbB in phosphate export and polyphosphate accumulation

Intracellular phosphate (P(i) ) is normally maintained at a fairly constant concentration in Escherichia coli, mainly by P(i) transport systems and by the 'phosphate balance' between P(i) and polyphosphate (polyP). We have reported previously that excess uptake of P(i) in a phoU mutant results in elevated levels of polyP. Here, we found that the elevated levels of polyP in the mutant could be reduced by the overproduction of YjbB, whose N-terminal half contains Na(+) /P(i) cotransporter domains. The rate of P(i) export increased when the YjbB overproducer grew on a medium containing glycerol-3-phosphate. These results strongly suggested that YjbB reduced the elevated levels of polyP in the phoU mutant by exporting intracellular excess P(i) .     

Inorganic phosphate stimulates the toxic effects of Tl+ in rat liver mitochondria

We studied action of inorganic phosphate (P(i)) on toxic effects of Tl+ in isolated rat liver mitochondria. This is a convenient model to study the toxicity of heavy metals. P(i) markedly retarded contraction of energized mitochondria swollen in the TlNO3 medium and even stronger stimulated swelling and state 4 of succinate-energized mitochondria in the TlNO3 medium. A valinomycin-induced decrease of K+-diffusion potential was also accelerated by Tl+ in the presence of P(i). The mitochondrial permeability transition pore in the medium containing Ca2+, TlNO3, and nitrates of univalent cations was distinctly stimulated by P(i). However, P(i) did not affect both the Tl+-stimulated swelling of nonenergized mitochondria in the TlNO3 medium and swelling of energized mitochondria in the Tl acetate medium. Respiration stimulated by 2,4-dinitrophenol and monoamine oxidase activity of energized mitochondria were not affected by Tl+ regardless of the presence of P(i). We suggested that stimulation by P(i) of toxic action of Tl+ in mitochondria and cells could be due to even greater enhancement of uncoupling of mitochondria as shown by an additional increase of swelling and state 4, and in the greater probability of opening of MPTP in the presence of P(i) and Ca2+.     

Intracellular accumulation of polyphosphate by the yeast Candida humicola G-1 in response to acid pH

Cells of a newly isolated environmental strain of Candida humicola accumulated 10-fold more polyphosphate (polyP), during active growth, when grown in complete glucose-mineral salts medium at pH 5.5 than when grown at pH 7.5. Neither phosphate starvation, nutrient limitation, nor anaerobiosis was required to induce polyP formation. An increase in intracellular polyP was accompanied by a 4.5-fold increase in phosphate uptake from the medium and sixfold-higher levels of cellular polyphosphate kinase activity. This novel accumulation of polyP by C. humicola G-1 in response to acid pH provides further evidence as to the importance of polyP in the physiological adaptation of microbial cells during growth and development and in their response to environmental stresses.     

Effects of monovalent cations on phosphate accumulation and storage of the ectomycorrhizal fungus Suillus bovinus

Comparative in vivo ³¹P-NMR studies of the fungus Suillus bovinus (L.: Fr.) O. Kuntze in pure culture have produced interesting new data. To investigate the response of phosphate metabolism to a change in external monovalent cations, samples were exposed to a Hoagland solution containing different monovalent cations Li⁺, Na⁺, K⁺, or Rb⁺ at 10 mM concentration. A method of nutrient cycling during analysis where the cation was changed and the phosphate kept constant allowed us to determine the kinetics of phosphate accumulation, storage and incorporation into polyphosphate following exposure to the range of test cations. Different external monovalent cations had different effects upon changes in the content of both phosphate and polyphosphate. Treatment with Li⁺, Na⁺, or Rb⁺ resulted in a change in phosphate accumulation to 60, 73, and 107% and in content of the intracellular mobile polyphosphate (polyP) to 119, 112, and 94%, respectively, compared with the control taken as 100%. The effect of each cation is related to its position in the periodic table. Reversing this process, i.e., exchanging with K⁺, returned phosphate metabolism to normal. Although, the increase in depolarization of the cell membrane should affect the internal pH, fungal metabolism using energy requiring mechanisms appeared necessary to maintain the intracellular pH. Thus, increasing contents of mobile polyP were the consequence of an increasing energy demand. On the other hand, the increasing depolarization of the cell membrane following the sequence Rb⁺ < K⁺ < Na⁺ < Li⁺ inhibited the net P_i accumulation. Furthermore, it is postulated that the P_i accumulation was also regulated by the intracellular content in polyP.     

Intracellular Accumulation of Polyphosphate by the Yeast Candida humicola G-1 in Response to Acid pH

Cells of a newly isolated environmental strain of Candida humicola accumulated 10-fold more polyphosphate (polyP), during active growth, when grown in complete glucose-mineral salts medium at pH 5.5 than when grown at pH 7.5. Neither phosphate starvation, nutrient limitation, nor anaerobiosis was required to induce polyP formation. An increase in intracellular polyP was accompanied by a 4.5-fold increase in phosphate uptake from the medium and sixfold-higher levels of cellular polyphosphate kinase activity. This novel accumulation of polyP by C. humicola G-1 in response to acid pH provides further evidence as to the importance of polyP in the physiological adaptation of microbial cells during growth and development and in their response to environmental stresses.     

Regulation of calcium transport in bovine spermatozoa

Calcium uptake into bovine epididymal spermatozoa is enhanced by introducing phosphate in the suspending medium (Babcock et al. (1975) J. Biol. Chem. 250, 6488-6495). This effect of phosphate is found even at a low extracellular Ca2+ concentrations (i.e., 5 microM) suggesting that phosphate is involved in calcium transport via the plasma membrane. Bicarbonate (2 mM) cannot substitute for phosphate, and a relatively high bicarbonate concentration (20 mM) causes partial inhibition of calcium uptake in absence of Pi. In the presence of 1-2 mM phosphate, 20 mM bicarbonate enhances Ca2+ uptake. The data indicate that the plasma membrane of bovine spermatozoa contains two carriers for Ca2+ transport: a phosphate-independent Ca2+ carrier that is stimulated by bicarbonate and a phosphate-dependent Ca2+ carrier that is inhibited by bicarbonate. Higher phosphate concentrations (i.e., 10 mM) inhibit Ca2+ uptake into intact cells (compared to 1.0 mM phosphate) and this inhibition can be relieved partially by 20 mM bicarbonate. This effect of bicarbonate is inhibited by mersalyl. Calcium uptake into the cells is enhanced by adding exogenous substrates to the medium. There is no correlation between ATP levels in the cells and Ca2+ transport into the cell. ATP levels are high even without added exogenous substrate and this ATP level is almost completely reduced by oligomycin, suggesting that ATP can be synthesized in the mitochondria in the absence of exogenous substrate. Calcium transport into the sperm mitochondria (washed filipin-treated cells) is absolutely dependent upon the presence of phosphate and mitochondrial substrate. Bicarbonate cannot support Ca2+ transport into sperm mitochondria. There is good correlation between Ca2+ uptake into intact epididymal sperm and into sperm mitochondria with the various substrates used. This indicates that the rate of calcium transport into the cells is determined by the rate of mitochondrial Ca2+ uptake and respiration with the various substrates.     

The inhibition of mitochondrial dicarboxylate transport by inorganic phosphate, some phosphate esters and some phosphonate compounds

1. P(i) competitively inhibited succinate oxidation by intact uncoupled mitochondria in the presence of sufficient N-ethylmaleimide to block the phosphate carrier, with a K(i) of 2.5mm. 2. Of a large number of phosphate esters and phosphonate compounds, phenyl phosphate and phenylphosphonate were found to inhibit competitively uncoupled succinate oxidation by intact but not broken mitochondria. By comparison, benzoate was a relatively weak competitive inhibitor of succinate oxidation by intact mitochondria but a relatively potent inhibitor of succinate dehydrogenase. 3. Phenyl phosphate and phenylphosphonate were non-penetrant, and inhibited P(i)-dependent swelling of mitochondria suspended in isosmolar ammonium malate in a manner non-competitive with P(i). The inhibitors did not affect mitochondrial swelling when tested with P(i) alone. 4. It is concluded that: (i) phenyl phosphate and phenylphosphonate behaved as non-penetrant analogues of P(i), since their inhibitory properties were in strict contrast with those of benzoate; (ii) phenyl phosphate and phenylphosphonate interacted with the dicarboxylate carrier but not with the phosphate carrier; (iii) P(i) was effective as a competitive inhibitor of succinate oxidation because of its being either an alternative substrate for the dicarboxylate carrier or competitive with succinate for the intramitochondrial cations as proposed by Harris & Manger (1968).     

Inorganic polyphosphates in mitochondria

Current data concerning the crucial role of inorganic polyphosphates (polyP) in mitochondrial functions and dysfunctions in yeast and animal cells are reviewed. Biopolymers with short chain length (∼15 phosphate residues) were found in the mitochondria of Saccharomyces cerevisiae. They comprised 7–10% of the total polyP content of the cell. The polyP are located in the membranes and intermembrane space of mitochondria. The mitochondrial membranes possess polyP/Ca²⁺/polyhydroxybutyrate complexes. PolyP accumulation is typical of promitochondria but not of functionally active mitochondria. Yeast mitochondria possess two exopolyphosphatases splitting P_i from the end of the polyP chain. One of them, encoded by the PPX1 gene, is located in the matrix; the other one, encoded by the PPN1 gene, is membrane-bound. Formation of well-developed mitochondria in the cells of S. cerevisiae after glucose depletion is accompanied by decrease in the polyP level and the chain length. In PPN1 mutants, the polyP chain length increased under glucose consumption, and the formation of well-developed mitochondria was blocked. These mutants were defective in respiration functions and consumption of oxidizable carbon sources such as lactate and ethanol. Since polyP is a compound with high-energy bonds, its metabolism vitally depends on the cell bioenergetics. The maximal level of short-chain acid-soluble polyP was observed in S. cerevisiae under consumption of glucose, while the long-chain polyP prevailed under ethanol consumption. In insects, polyP in the mitochondria change drastically during ontogenetic development, indicating involvement of the polymers in the regulation of mitochondrial metabolism during ontogenesis. In human cell lines, specific reduction of mitochondrial polyP under expression of yeast exopolyphosphatase PPX1 significantly modulates mitochondrial bioenergetics and transport.     

V-ATPase dysfunction suppresses polyphosphate synthesis in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae accumulates the high levels of inorganic polyphosphates (polyPs) performing in the cells numerous functions, including phosphate and energy storage. The effects of vacuolar membrane ATPase (V-ATPase) dysfunction were studied on polyP accumulation under short-term cultivation in the P_i–excess media after P_i starvation. The addition of bafilomycin A1, a specific inhibitor of V-ATPase, to the medium with glucose resulted in strong inhibition of the synthesis of long-chain polyP and in substantial suppression of short-chain polyP. The addition of bafilomycin to the medium with ethanol resulted in decreased accumulation of high-molecular polyP, while the accumulation of low-molecular polyP was not affected. The levels of polyP synthesis in the mutant strain with a deletion in the vma2 gene encoding a V-ATPase subunit were significantly lower than in the parent strain in the media with glucose and with ethanol. The synthesis of the longest chain polyP was not observed in the mutant cells. The synthesis of only the low-polymer acid-soluble polyP fraction occurred in the cells of the mutant strain. However, the level of polyP1 was nearly tenfold lower than compared to the cells of the parent strain. Both bafilomycin A1 and the mutation in vacuolar ATPase subunit vma2 lead to a considerable decrease of cellular polyP accumulation. Thus, the defects in ΔμH⁺ formation on the vacuolar membrane resulted in the decrease of polyP biosynthesis in S. cerevisiae.     

Kinetics of growth and phosphate uptake in pure culture studies of Acinetobacter species

Acinetobacter has been found to be the major species responsible for mediating biological phosphate removal. The growth kinetics and phosphate uptake were investigated for an isolated Acinetobacter strain growing in a defined medium. The phosphate uptake is dependent on growth rate, temperature, and pH. Polyphosphate granules occurred in a balanced growth stage. The maximum phosphorus content in cells was 4.8% at the dilution rate of 12 day(-1). The specific phosphate uptake rate was found to be a quadratic polynomial function of the dilution rate. Increased calcium (up to 36 mg/L) and magnesium (up to 15 mg/L), and the addition of yeast extract (100 mg/L), primary effluent (20%), and fluoride (10 mg/L) did not affect phosphate uptake. Anaerobic conditioning (N(2) stripping), low pH (CO(2) stripping), and addition of sodium acetate under anaerobic conditions failed to stimulate immediate phosphate release. Nevertheless, After 21-24 h, the phosphate release was ca. 3, 5, and 15 mg P/g cell, respectively, for N(2) purging, the addition of acetate, and CO(2) purging. For two-stage completely stirred reactor operation, there was negligible phosphate overplus at the second reactor when phosphate was added, when the first reactor was subjected to phosphate limitation. When both phosphate and carbon limited the growth in the first reactor, there was slight phosphate accumulation under endogenous respiration conditions in the second reactor.     

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.