Direct Labeling of Polyphosphate at the Ultrastructural Level in Saccharomyces cerevisiae by Using the Affinity of the Polyphosphate Binding Domain of Escherichia coli Exopolyphosphatase

Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate and has many biological functions in prokaryotic and eukaryotic organisms. To investigate polyP localization, we developed a novel technique using the affinity of the recombinant polyphosphate binding domain (PPBD) of Escherichia coli exopolyphosphatase to polyP. An epitope-tagged PPBD was expressed and purified from E. coli. Equilibrium binding assay of PPBD revealed its high affinity for long-chain polyP and its weak affinity for short-chain polyP and nucleic acids. To directly demonstrate polyP localization in Saccharomyces cerevisiae on resin sections prepared by rapid freezing and freeze-substitution, specimens were labeled with PPBD containing an epitope tag and then the epitope tag was detected by an indirect immunocytochemical method. A goat anti-mouse immunoglobulin G antibody conjugated with Alexa 488 for laser confocal microscopy or with colloidal gold for transmission electron microscopy was used. When the S. cerevisiae was cultured in yeast extract-peptone-dextrose medium (10 mM phosphate) for 10 h, polyP was distributed in a dispersed fashion in vacuoles in successfully cryofixed cells. A few polyP signals of the labeling were sometimes observed in cytosol around vacuoles with electron microscopy. Under our experimental conditions, polyP granules were not observed. Therefore, it remains unclear whether the method can detect the granule form. The method directly demonstrated the localization of polyP at the electron microscopic level for the first time and enabled the visualization of polyP localization with much higher specificity and resolution than with other conventional methods.     

An acidocalcisomal exopolyphosphatase from Leishmania major with high affinity for short chain polyphosphate

We report the cloning, overexpression, purification, and characterization of the Leishmania major exopolyphosphatase (LmPPX). The product of this gene (LmPPX), the first related to polyphosphate (polyP) metabolism isolated from an eukaryotic organism different from yeast, has 388 amino acids and a molecular mass of 48 kDa. LmPPX differs from other exopolyphosphatases previously investigated. Heterologous expression of LmPPX in Escherichia coli produced a functional enzyme that was similar to the yeast exopolyphosphatase with respect to its Mg(2+) requirement, optimum pH, and sensitivity to cations, amino acids, and heparin but that, in contrast to the yeast enzyme and other exopolyphosphatases investigated before, acts on polyP of short chain lengths with higher rates and affinity. LmPPX is a processive enzyme, and it does not hydrolyze pyrophosphate, ATP, or p-nitrophenylphosphate. Confocal immunofluorescence microscopy using affinity-purified antibodies against the recombinant enzyme indicated an acidocalcisomal and cytosolic localization. High levels of short chain (21.4 +/- 3.0 mm) and long chain polyP (55.9 +/- 5.6 mm) were detected in L. major promastigotes. The unique characteristics of LmPPX and L. major polyP metabolism may facilitate the development of novel antileishmanial agents.     

Effects of inorganic polyphosphate on the proteolytic and DNA-binding activities of Lon in Escherichia coli

Lon belongs to a unique group of proteases that bind to DNA and is involved in the regulation of several important cellular functions, including adaptation to nutritional downshift. Previously, we revealed that inorganic polyphosphate (polyP) increases in Escherichia coli in response to amino acid starvation and that it stimulates the degradation of free ribosomal proteins by Lon. In this work, we examined the effects of polyP on the proteolytic and DNA-binding activities of Lon. An order-of-addition experiment suggested that polyP first binds to Lon, which stimulates Lon-mediated degradation of ribosomal proteins. A polyP-binding assay using Lon deletion mutants showed that the polyP-binding site of Lon is localized in the ATPase domain. Because the same ATPase domain also contains the DNA-binding site, polyP can compete with DNA for binding to Lon. In fact, an equimolar amount of polyP almost completely inhibited DNA-Lon complex formation, suggesting that Lon binds to polyP with a higher affinity than it binds to DNA. Collectively, our results showed that polyP may control the cellular activity of Lon not only as a protease but also as a DNA-binding protein.     

Long-chain polyphosphate in osteoblast matrix vesicles: Enrichment and inhibition of mineralization

Background

Inorganic polyphosphate (polyP) is a fundamental and ubiquitous molecule in prokaryotes and eukaryotes. PolyP has been found in mammalian tissues with particularly high levels of long-chain polyP in bone and cartilage where critical questions remain as to its localization and function. Here, we investigated polyP presence and function in osteoblast-like SaOS-2 cells and cell-derived matrix vesicles (MVs), the initial sites of bone mineral formation.

Molecular analysis of polyphosphate accumulation in bacteria

The dynamic behavior of inorganic polyphosphate (polyP), its accumulation and disappearance, is the most striking aspect of polyP metabolism in bacteria. Imbalance between polyP synthesis and degradation results in fluctuations of polyP by 100- to 1000-fold. We here review recent results with respect to this polyP metabolism in bacteria. PolyP accumulation in response to amino acid starvation, accompanied by increased levels of stringent factors, has been observed in Escherichia coli. Inhibition by stringent factors of polyphosphatase interrupts the dynamic balance between the synthesis and degradation of polyP, accounting for polyP accumulation. Polyphosphate kinase is required for activation of intracellular protein degradation, which is required for adaptation at the onset of amino acid starvation. The adaptation to amino acid starvation is mediated by the network of stringent response and polyP metabolism. PolyP accumulation independent of stringent response has also been observed. Novobiocin, an inhibitor for DNA gyrase, stimulated accumulation of polyP but not that of stringent factors. However, a temperature-sensitive DNA gyrase mutant did not exhibit polyP accumulation at the non-permissive temperature. Antagonistic relationship of polyP to nucleic acid synthesis, explored by Harold, appears to be more complicated. We discuss relationship of Pi regulation to polyP accumulation in E. coli and Klebsiella aerogenes. A function of polyP as an in vivo phosphagen affecting polyP accumulation is also discussed.     

Vacuolar localization of phosphorus in hyphae of Phialocephala fortinii, a dark septate fungal root endophyte

Phialocephala fortinii is a dark septate fungal endophyte that colonizes roots of many host species. Its effect on plant growth varies from being pathogenic to beneficial. The basic biology of this species has received little research, and thus the main objectives of this study were to determine cytological features of hyphae, including the nature of the vacuolar system, and whether polyphosphate was present in vacuoles. Both living hyphae and hyphae that had been rapidly frozen and freeze substituted before embedding were studied. A complex system of vacuoles, including a motile tubular vacuolar system, elongated vacuoles, and spherical vacuoles, was demonstrated in living hyphae by the fluorescent probe Oregon Green 488 carboxylic acid diacetate, using laser scanning confocal microscopy. The motile tubular vacuolar system was more prevalent at the hyphal tip than in more distal regions, whereas elongated vacuoles and spherical vacuoles were more abundant distal to the tip. All vacuoles contained polyphosphate as shown by labelling embedded samples with recombinant polyphosphate binding domain of Escherichia coli exopolyphosphatase, containing Xpress tag at the N-terminal end, followed by anti-Xpress antibody and a secondary antibody conjugated either to a fluorescent probe for laser scanning confocal microscopy or colloidal gold for transmission electron microscopy. The polyphosphate was dispersed in vacuoles. This was confirmed by staining embedded samples with 4',6-diamidino-2-phenylindole and viewing with UV light using epifluorescence microscopy. These cytological methods showed that the tubular vacuolar system had lower concentrations of polyphosphate than the spherical vacuoles. Lipid bodies were present around vacuoles.     

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.     

A nonradioactive method for the assay of polyphosphate kinase activity and its application in the study of polyphosphate metabolism in Burkholderia cepacia

Studies of polyphosphate (polyP) metabolism in microorganisms have been hampered by the lack of a convenient method for the assay in cell extracts of the activity of polyphosphate kinase (PPK), the enzyme principally responsible for microbial polyP biosynthesis. We report the development of such an assay, based on the well-established metachromatic reaction, with toluidine blue, of the polyP formed during the PPK-catalyzed reaction. The method was successfully used in the characterization of PPK activity in crude extracts of an environmental Burkholderia cepacia isolate. The development of a protocol for the physical recovery of polyP from solution is also reported.     

Streptomyces lividans potassium channel contains poly-(R)-3-hydroxybutyrate and inorganic polyphosphate

The Streptomyces lividans KcsA potassium channel, a homotetramer of 17.6 kDa subunits, was found to contain two nonproteinaceous polymers, namely, poly-(R)-3-hydroxybutyrate (PHB) and inorganic polyphosphate (polyP). PHB and polyP are ubiquitous cellular constituents with a demonstrated capacity for cation selection and transport. PHB was detected in both tetramer and monomer species of KcsA by reaction to anti-PHB IgG on Western blots, and estimated as 28 monomer units of PHB per KcsA tetramer by a chemical assay in which PHB is converted to its unique degradation product, crotonic acid. PolyP was detected in KcsA tetramers, but not in monomers, by metachromatic reaction to o-toluidine blue stain on SDS-PAGE gels. A band of free polyP was also visible, suggesting that polyP is released when tetramers dissociate. The exopolyphosphatase of Saccharomyces cerevisiae degraded the free polyP, but tetramer-associated polyP was not affected, indicating it was inaccessible to the enzyme. PolyP in KcsA was estimated as 15 monomer units per tetramer by an enzymatic assay in which polyphosphate kinase is used to transfer phosphates from polyP to [(14)C]ADP, yielding [(14)C]ATP. The experimentally determined isoelectric point of KcsA tetramer was 6.5-7.5, substantially more acidic than the theoretical pI of 10.3, and consistent with the inclusion of a polyanion. The results suggest that PHB is covalently bound to KcsA subunits while polyP is held within tetramers by ionic forces. It is posited that KcsA protein creates an environment in which PHB/polyP is selective for K(+). The basic amino acids attenuate the negative charge density of polyP, thereby transforming the cation binding preference from multivalent to monovalent, and discrimination between K(+) and Na(+) is accomplished by adjusting the ligand geometry in cation binding cavities formed by PHB and polyP.     

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.     

Ultrastructure of rapidly frozen and freeze-substituted germ tubes of an arbuscular mycorrhizal fungus and localization of polyphosphate

In arbuscular mycorrhizas (AM), the supply of phosphorus from the fungi is one of the most important benefits to the host plant. Here we describe for the first time the ultrastructure and polyphosphate (poly P) distribution in rapidly frozen and freeze-substituted germ tubes of the AM fungus Gigaspora margarita. At the ultrastructural level, phosphorus distribution was analysed using energy-filtering transmission electron microscopy, and poly P was detected using an enzyme-affinity method. Semithin sections and live cells were also stained with 4',6-diamidino-2-phenylindole, which is not specific but fluoresces yellow when viewed under UV irradiation by binding with poly P. The cryotechnique method showed that extensive elongate ellipsoid vacuoles containing a uniform electron-opaque material occupied most of the cell volume. Combining the results of multiple methods revealed that poly P was localized in a dispersed form in vacuoles and in the outer fungal cell wall. These results show the significant potential of AM fungi for phosphorus storage based on its localization in the extensive complement of vacuoles in thick hyphae. The mechanism of translocation of poly P in tubular vacuoles, and the role of poly P in the cell wall, need to be elucidated.     

Inorganic polyphosphate in mitochondria of Saccharomyces cerevisiae at phosphate limitation and phosphate excess

Isolated mitochondria of Saccharomyces cerevisiae cells grown on glucose possess acid-soluble inorganic polyphosphate (polyP). Its level strongly depends on phosphate (P(i)) concentration in the culture medium. The polyP level in mitochondria showed 11-fold decrease under 0.8 mM P(i) as compared with 19.3 mM P(i). When spheroplasts isolated from P(i)-starved cells were incubated in the P(i)-complete medium, they accumulated polyP and exhibited a phosphate overplus effect. Under phosphate overplus the polyP level in mitochondria was two times higher than in the complete medium without preliminary P(i) starvation. The average chain length of polyP in mitochondria was of <15 phosphate residues at 19.3 mM P(i) in the culture medium and increased at phosphate overplus. Deoxyglucose inhibited polyP accumulation in spheroplasts, but had no effect on polyP accumulation in mitochondria. Uncouplers (FCCP, dinitrophenol) and ionophores (monensin, nigericin) inhibited polyP accumulation in mitochondria more efficiently than in spheroplasts. Fast hydrolysis of polyP was observed after sonication of isolated mitochondria. Probably, the accumulation of polyP in mitochondria depended on the proton-motive force of their membranes.     

Haemophilus influenzae outer membrane protein P5 is associated with inorganic polyphosphate and polyhydroxybutyrate

Outer membrane protein P5 of nontypeable (acapsulate) Haemophilus influenzae (NTHi P5) forms large pores in planar lipid bilayers between symmetric solutions that unpredictably display a nonzero reversal potential. Moreover, NTHi P5 has a high theoretical isoelectric point, calculated as 9.58, which is not in agreement with the experimental isoelectric point, determined as 6.3-6.8, or with its preference for cations, disproportionately strong at one side. These anomalous results intimate that NTHi P5 is associated with a polyanion. Chemical and immunological analyses revealed the presence of inorganic polyphosphate (polyP), and the amphiphilic, solvating polyester, poly-(R)-3-hydroxybutyrate, frequently associated with polyP. A sharp reduction in cation selectivity was observed after addition of Saccharomyces cerevisiae exopolyphosphatase X to the bilayer, providing functional evidence for the involvement of polyP in selectivity. The results suggest that NTHi P5 associates with polyP and poly-(R)-3-hydroxybutyrate to create large, cation-selective pores in the outer membrane of H. influenzae.     

Inorganic polyphosphate is a potent activator of the mitochondrial permeability transition pore in cardiac myocytes

Mitochondrial dysfunction caused by excessive Ca2+ accumulation is a major contributor to cardiac cell and tissue damage during myocardial infarction and ischemia-reperfusion injury (IRI). At the molecular level, mitochondrial dysfunction is induced by Ca2+-dependent opening of the mitochondrial permeability transition pore (mPTP) in the inner mitochondrial membrane, which leads to the dissipation of mitochondrial membrane potential (ΔΨm), disruption of adenosine triphosphate production, and ultimately cell death. Although the role of Ca2+ for induction of mPTP opening is established, the exact molecular mechanism of this process is not understood. The aim of the present study was to test the hypothesis that the adverse effect of mitochondrial Ca2+ accumulation is mediated by its interaction with inorganic polyphosphate (polyP), a polymer of orthophosphates linked by phosphoanhydride bonds. We found that cardiac mitochondria contained significant amounts (280±60 pmol/mg of protein) of short-chain polyP with an average length of 25 orthophosphates. To test the role of polyP for mPTP activity, we investigated kinetics of Ca2+ uptake and release, ΔΨm and Ca2+-induced mPTP opening in polyP-depleted mitochondria. polyP depletion was achieved by mitochondria-targeted expression of a polyP-hydrolyzing enzyme. Depletion of polyP in mitochondria of rabbit ventricular myocytes led to significant inhibition of mPTP opening without affecting mitochondrial Ca2+ concentration by itself. This effect was observed when mitochondrial Ca2+ uptake was stimulated by increasing cytosolic [Ca2+] in permeabilized myocytes mimicking mitochondrial Ca2+ overload observed during IRI. Our findings suggest that inorganic polyP is a previously unrecognized major activator of mPTP. We propose that the adverse effect of polyphosphate might be caused by its ability to form stable complexes with Ca2+ and directly contribute to inner mitochondrial membrane permeabilization.     

A Highlights from MBoC Selection: Chromosome replication and segregation govern the biogenesis and inheritance of inorganic polyphosphate granules

Prokaryotes and eukaryotes synthesize long chains of orthophosphate, known as polyphosphate (polyP), which form dense granules within the cell. PolyP regulates myriad cellular functions and is often localized to specific subcellular addresses through mechanisms that remain undefined. In this study, we present a molecular-level analysis of polyP subcellular localization in the model bacterium Caulobacter crescentus. We demonstrate that biogenesis and localization of polyP is controlled as a function of the cell cycle, which ensures regular partitioning of granules between mother and daughter. The enzyme polyphosphate kinase 1 (Ppk1) is required for granule production, colocalizes with granules, and dynamically localizes to the sites of new granule synthesis in nascent daughter cells. Localization of Ppk1 within the cell requires an intact catalytic active site and a short, positively charged tail at the C-terminus of the protein. The processes of chromosome replication and segregation govern both the number and position of Ppk1/polyP complexes within the cell. We propose a multistep model in which the chromosome establishes sites of polyP coalescence, which recruit Ppk1 to promote the in situ synthesis of large granules. These findings underscore the importance of both chromosome dynamics and discrete protein localization as organizing factors in bacterial cell biology.     

Growth of polychlorinated-biphenyl-degrading bacteria in the presence of biphenyl and chlorobiphenyls generates oxidative stress and massive accumulation of inorganic polyphosphate

Inorganic polyphosphate (polyP) plays a significant role in increasing bacterial cell resistance to unfavorable environmental conditions and in regulating different biochemical processes. Using transmission electron microscopy of the polychlorinated biphenyl (PCB)-degrading bacterium Pseudomonas sp. strain B4 grown in defined medium with biphenyl as the sole carbon source, we observed large and abundant electron-dense granules at all stages of growth and following a shift from glucose to biphenyl or chlorobiphenyls. Using energy dispersive X-ray analysis and electron energy loss spectroscopy with an integrated energy-filtered transmission electron microscope, we demonstrated that these granules were mainly composed of phosphate. Using sensitive enzymatic methods to quantify cellular polyP, we confirmed that this polymer accumulates in PCB-degrading bacteria when they grow in the presence of biphenyl and chlorobiphenyls. Concomitant increases in the levels of the general stress protein GroEl and reactive oxygen species were also observed in chlorobiphenyl-grown cells, indicating that these bacteria adjust their physiology with a stress response when they are confronted with compounds that serve as carbon and energy sources and at the same time are chemical stressors.     

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) .     

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.     

Inorganic polyphosphate–an unusual suspect of the mitochondrial permeability transition mystery

Inorganic polyphosphate (polyP) is a naturally occurring polyanion made of ten to several hundred orthophosphates (P_i) linked together by phosphoanhydride bonds. PolyP is ubiquitously present in all organisms from bacteria to humans. Specific physiological roles of polyP vary dramatically depending on its size, concentration, tissue and subcellular localization. Recently we reported that mitochondria of ventricular myocytes contain significant amounts (280 ± 60 pmol/mg of protein) of polyP with an average length of 25 orthophosphates, and that polyP is involved in Ca²⁺-dependent activation of the mitochondrial permeability transition pore (mPTP). Here we extend our study to demonstrate the involvement of mitochondrial polyP in cardiac cell death. Furthermore, we show that polyP levels depend on the activity of the respiratory chain and are lower in myocytes from failing hearts. We conclude that polyP is a dynamically regulated macromolecule that plays an important role in mPTP-dependent cell death pathway.