Two internal pools of soluble polyphosphate in the cyanobacterium Synechocystis sp. strain PCC 6308: an in vivo 31P NMR spectroscopic study

Two intracellular pools of soluble polyphosphate were identified by in vivo ³¹P NMR spectroscopy in the cyanobacterium Synechocystis sp. strain PCC 6308. Polyphosphate was present in the cells after growth in sulfur-limited media containing excess phosphate. The presence of polyphosphate was confirmed by transmission electron microscopy and chemical analysis. ³¹P NMR spectroscopy of whole cells treated with EDTA revealed two pools of mobile polyphosphate. A downfield shift and narrowing of part of the broad polyphosphate resonance was observed after EDTA treatment, suggesting that EDTA binds metal ions normally associated with some of the polyphosphate. Phosphate, but not polyphosphate, leaked out of the cells after this treatment. Addition of magnesium ions caused the downfield shift in the polyphosphate resonance to move back toward its original value. These data show that only part of the cation-complexed polyphosphate is accessible to the added EDTA and suggest that there are two internal fractions of NMR-visible polyphosphate in the cells, only one of which loses its associated cations to EDTA. Spheroplast formation showed that polyphosphate was not present in the periplasm of the cells. Received: 3 July 1997 / Accepted: 26 September 1997     

Changes in polyphosphate composition and localization in Propionibacterium acnes after near-ultraviolet irradiation.

Electron microscopy showed that electron-dense granules accumulated in Propionibacterium acnes in larger amounts when the bacteria were grown on a phosphate-rich medium. X-ray microanalysis demonstrated that the granules contained mostly phosphorus and potassium, indicating that the cells contained polyphosphate granules. When cells were grown on a complex Bacto-agar medium, the amount and the size of the polyphosphate granules were reduced. Polyphosphate was also detected with 31P nuclear magnetic resonance (31P-NMR). Of the polyphosphates observed with 31P-NMR, 20% seemed to be located outside the cell membrane. Broad-band near-ultraviolet irradiation (emission maximum 366 nm) corresponding to doses that killed 37% of the cells increased the amount of polyphosphate in cells grown on the phosphate-rich medium. The fluorescent chromophore 4',6-diamidino-2-phenylindole (DAPI) shifted the fluorescence emission from 478 to 538 nm when bound to polyphosphate and excited at 340 nm. DAPI was used to detect polyphosphates generated after near-ultraviolet irradiation of the cells. Nonirradiated cells showed no increased fluorescence at 538 nm, indicating no polyphosphate is presented in the cells. We conclude that DAPI did not have "access" to the intracellular polyphosphate as long as the cells were not light damaged. This observation is important for the interpretation of near-UV damage to cells.     

X-ray Microanalysis of Phosphorus in Scenedesmus obtusiusculus

Results from energy dispersive x-ray microanalysis indicate considerable variation in phosphorus as well as in other elements found in polyphosphate granules in cells of Scenedesmus obtusiusculus starved of phosphorus for 24, 36 and 48 h. Comparison is made with cells at a 3-h stage of synchronization and after 48 h cultivation in a phosphorus-containing medium. After 48 h with a very limited external source of phosphorus, phosphorus was not detectable in‘polyphosphate granules’ in Epon-Araldite sections unless the resin and other organic molecules were removed by plasma microincineration. In spite of the low amounts of elements (Mg. Al, Si, P and Ca) detected in these granules they are still dense to electrons in unstained sections, like the granules in cells cultivated in the presence of phosphorus. That suggests that the localized differences in the structural packing of elements or radicals in the polyphosphale polymer is more important For visualization of unstained polyphosphate granules than the absolute concentration of phosphorus or other elements. In contrast to previous assumptions, the abundance of morphologically-observable‘polyphosphate’ granules can no longer be taken as an index of mobilizable phosphate reserves.     

The Role of Polyphosphates in the Transport Mechanism of Glucose in Yeast Cells

Several cations inhibit anaerobic fermentation of glucose by intact yeast cells. Some ions (e.g. Hg⁺⁺) penetrate into the cytoplasm and cause an irreversible inhibition of fermentation. Other ions (e.g. UO₂⁺⁺, Ni⁺⁺, and Co⁺⁺) are reversibly bound to a substance at the outside of the yeast cell identified as polyphosphate. Although the cations are bound to exactly the same extent, their influences on fermentation differ greatly. Thorium ions are bound not only to the polyphosphates, but in addition, to phosphatides in the cell membrane. Under circumstances in which glucose is transported into the cell, the amount of polyphosphate in the outer face of the membrane decreases considerably. If yeast is poisoned with monoiodoacetate, the number of glucose molecules that can still be taken up equals the original number of cation-binding sites at the outer surface of the membrane. These data suggest that one molecule of glucose is taken up in connection with the disappearance of one polyphosphate monomer. The hypothesis is framed that the uptake of glucose into the yeast cell is associated with an enzymic phosphorylation (possibly of the carrier), with polyphosphate as phosphate donor. The inhibition of glucose uptake caused by certain metal ions may be the consequence of induced changes in the spatial arrangement of polyphosphate chains; the greater the change in configuration, the larger is the inhibition.     

Ultrastructure of the Acidophilic Aerobic Photosynthetic Bacterium Acidiphilium rubrum

The ultrastructure of cells of Acidiphilium rubrum, which is an acidophilic aerobic photosynthetic bacterium containing zinc-complexed bacteriochlorophyll a, was studied by electron microscopy with the rapid substitution technique. Thin-section electron microscopy indicated that any type of internal photosynthetic membranes was not present in this organism despite a relatively high content of the photopigment. The majority of cells had poly-β-hydroxybutyrate granules and electron-dense spherical bodies identified as being polyphosphate granules. When the organism was grown chemotrophically with 0.1% FeSO₄, it produced another group of electron-dense granules that were associated with the inner part of the cytoplasmic membrane. An energy-dispersive X-ray analysis showed that these membrane-bound, electron-dense granules contained iron. Received: 24 November 1999 / Accepted: 5 January 2000     

Genetic competence in Escherichia coli requires poly-beta-hydroxybutyrate/calcium polyphosphate membrane complexes and certain divalent cations.

In earlier studies of genetic competence in Escherichia coli induced with calcium-containing buffers, a strong correlation was found between transformation efficiency and the formation of poly-beta-hydroxybutyrate/calcium polyphosphate (PHB/Ca2+/PPi) complexes in the plasma membranes. In this study, we replaced Ca2+ with one of a number of other cations--monovalent, divalent, and trivalent--and found significant numbers of transformants (transformation efficiency, > 10(5)/micrograms of pBR322 DNA) only when the cells had high levels of PHB/Ca2+/PPi and the medium contained at least one of the divalent cations Ca2+, Mn2+, Sr2+, or Mg2+. Cells with high levels of the complexes were not competent when the medium did not contain these cations, but the cations were also ineffectual when the cells had few complexes. Surprisingly, Mn, Sr, and Mg were not incorporated into the complexes in place of Ca. These results indicate that PHB/Ca2+/PPi complexes and the above-mentioned divalent cations each have essential but disparate roles in genetic competence. Moreover, the strong selectivity of PHB/PPi for Ca2+ suggests the binding sites in the complexes are ionophoretic.     

Monitoring of membrane-bound divalent cations in plant mitochondria using chlorotetracycline fluorescence

Chlorotetracycline (CTC) shows a strongly enhanced fluorescence upon addition of mitochondria isolated from Jerusalem artichoke ( Helianthus tuberosus L.) tubers in a low-cation medium. This indicates the presence of membrane-bound divalent cations. The chelation by CTC of the membrane-bound divalent cations does not affect the oxidation of exogenous NADH significantly. The removal of the bound divalent cations using ethyleneglycol-bis-(β-aminoethylether)-N,N'-tetraacetic acid (EGTA) and EDTA causes an 80% decrease in CTC fluorescence. Titration of CTC fluorescence (a direct measure of bound divalent cations) and 9-aminoacridine fluorescence (a measure of surface potential) with EGTA and EDTA gives similar curves, although CTC fluorescence responds more slowly to the addition of chelators. The same bound divalent cations appear to be monitored by CTC fluorescence or by 9-aminoacridine fluorescence.     

Distribution of polyphosphates in cell-compartments of Chlorella fusca as measured by 31P-NMR-spectroscopy

In suspensions of the green alga Chlorella fusca the influence of high pH and high ethylene-diamine-tetraacetic acid concentrations in the external medium, of French-press and perchloric acid extraction of the cells and of alkalization of the intracellular pH on the polyphosphate signal in ³¹P-nuclear magnetic resonance (³¹P NMR) spectra was investigated.The results show that part of the polyphosphates of asynchronous Chlorella cells are located outside the cytoplasmic membrane and complexed with divalent metal-ions. These polyphosphates are tightly bound to the cell wall and/or the cytoplasmic membrane and are not susceptible to hydrolyzation by strong acid at room temperature, in contrast to the intracytoplasmic polyphosphates.Upon alkalization of the internal pH of Chlorella cells, polyphosphates, previously not visible in the spectra become detectable by ³¹P-NMR-spectroscopy. ³¹P-NMR spectroscopic monitoring of polyphosphates during gradual alkalization of the extra-and intracellular space is proposed as a quick method for the estimation of the cellular polyphosphate content and distribution.Abbreviations CCCP Carbonylcyanide-m-chlorophenyl-hydrazone - NTP/NDP Nucleotide triphosphate/-diphosphate - PCA Perchloric acid - ³¹P-NMR ³¹P-nuclear magnetic resonance - PolyP polyphosphates - PP₁, PP₂, PP₃ terminal, second and third phosphate residue of polyphosphates, respectively - PP₄ core phosphate residues of polyphosphates     

The elemental composition dynamics of large polyphosphate granules in Acinetobacter strain 210A

Electron microscopy and energy dispersive X-ray micro-analysis were used to examine the elemental composition of large polyphosphate granules in unfixed and unstained intact cells of Acinetobacter strain 210A. When grown in medium with butyrate, Acinetobacter strain 210A possessed 1 or 2 large granules with a diameter of 0.4 m besides a relatively large number of small granules. The large granules were composed of phosphorus, magnesium and potassium. A decrease in the Mg/Ca-ratio of the medium from 5.95 to 0.0073 resulted in a decline in the intracellular Mg/Ca-ratio from 15 to 0.56. At a high intracellular Mg/Ca-ratio, magnesium was the dominant counterion in the polyphosphate granule. Calcium became the major cation in the polyphosphate bodies at a low intracellular Mg/Ca-ratio. Omission of Ca²⁺ or modification of the K/Mg ratio in the medium did not significantly affect the cation composition of the polyphosphate granules. The dissociation constants for Mg- and Ca-polyphosphate were 9.3×10^-2 mol/l and 1.5×10^-1 mol/l, respectively.     

EFFECT OF PHOSPHATE CONCENTRATION ON THE FINE STRUCTURE OF THE CYANOBACTERIUM,MICROCYSTIS AERUGINOSA KÜTZ. EMEND. ELENKIN

SUMMARY

Microcystis aeruginosa toxic strain UV-006 stored a fixed amount of polyphosphate in spherical granules located in the centroplasm. Twenty four hours of phosphate starvation induced use of stored polyphosphate, manifested by reduction in granule numbers. Reintroduction of 2, 4 or 8 mg l^?1 K₂HPO₄ resulted in redeposition of polyphosphate in a critical number of centroplasmic polyphosphate granules. Growth rate was unaffected by phosphate concentrations, although the final cell yield was slightly lower at 8 mg l ^?1

Continued starvation decreased photosynthetic rate and growth ceased. Cells appeared senescent. Cyanophycin and polyglucoside reserves apparently increased in these cells, whilst thylakoids were reduced in number and reorientated away from. the cell wall and polyhedral bodies were lost. After the initial decrease, centroplasmic polyphosphate bodies increased to about half of the maximum numbers stored in cells grown in the presence of phosphate, suggesting that translocation of phosphorus from other areas in the phosphate-starved cell occurred.

Two further polyphosphate deposition areas were observed. DNA fibrils may have represented nucleation sites for developing polyphosphate granules. Intrathylakoidal deposits were rare.     

CHLAMYDOMONAS EUGAMETOS (CHLOROPHYTA) STORES PHOSPHATE IN POLYPHOSPHATE BODIES TOGETHER WITH CALCIUM1

When Chlamydomonas eugametos Moewus cells are starved of phosphate, they accumulate ³²P_i much faster than before starvation. Phosphate accumulation is stimulated by calcium. Less than 5% of the ³²P_i taken up by the cell is present in soluble molecules, suggesting that most is in a metabolically inactive, storage form. Nuclear magnetic resonance and X-ray microanalysis data are presented to show that it is stored as polyphosphate in electron-dense bodies hi the cytoplasm. The same bodies accumulate divalent cations, in particular calcium. The P/Ca ratio in the bodies was maintained between 5.4 (1-week-old cells) and 3.3 (5-week-old cells) during cultivation, suggesting that the calcium and phosphorus relations of the bodies are coupled. The possibility that these electron-dense bodies represent calcium stores that can be released to activate calcium signaling is discussed.     

ELECTRON MICROSCOPIC OBSERVATIONS ON THE ACCUMULATION OF DIVALENT CATIONS IN INTRAMITOCHONDRIAL GRANULES

Electron microscopic evidence is presented, from mitochondria in whole cells of toad urinary bladder and from isolated rat kidney mitochondria, indicating that the divalent cations calcium, strontium, and barium are accumulated in granules localized in the mitochondrial matrix. This accumulation occurs under conditions in which divalent ions are present in the medium bathing either whole cells or isolated mitochondria. The evidence indicates that the divalent ions are deposited on, or in a pre-existing granule, possibly in exchange for other ions. It suggests a possible role of the intramitochondrial granules in the regulation of the internal ionic environment of the mitochondrion. Certain biochemical and physiological implications of this phenomenon are discussed.     

Effects of ethidium bromide,tetramethylethidium bromide and betaine B on the ultrastructure of HeLa cell mitochondria in situ

Summary Several investigators have described the ultrastructural changes that occur in the mitochondria of cells in tissue cultures after treatment with the drug ethidium bromide (E). The mitochondria swell and the cristae become greatly altered and finally disappear; in the cristae-free region of the matrix electron-dense granules can be observed. It has been assumed that intercalation of E between the base pairs of the mitochondrial DNA induces the formation of the granular inclusions. To investigate whether intercalation is really the initial step in the generation of dense granules inside the matrix, we performed a comparative incubation study of HeLa-cell mitochondria in situ using three closely related dyes (D), i.e. E, tetramethylethidium bromide (TME) and betaine B (B). They strongly differ with regard to their affinity for DNA and their ability to cross membranes. E was used as a reference dye. TME does not intercalate, but is externally bound to DNA only weakly. The neutral B is not bound at all, but can cross membranes more easily than the cation E. Moreover, in aqueous solutions at pH7.0, B is in equilibrium with its protonated cation BH. BH and E have almost equal affinities for DNA. Therefore B may quickly pass the inner mitochondrial membranes and the cristae, and should then be bound inside the matrix, thus forming a BH-DNA complex. On the assumption that intercalation is necessary for the generation of intramitochondrial electron-dense bodies, we predicted that BH/B should be more efficient than E, while TME should be relatively ineffective. In experiments using HeLa cells, these predictions were found to be inaccurate. E, TME and BH/B produced almost the same mitochondrial alterations, but at different concentrations and after different incubation periods. In contrast to our expectations TME was much more effective than E and BH/B, with the last two behaving rather similarly.Therefore, it seems unlikely that the drugs penetrate the inner mitochondrial membrane system by simple physical diffusion or that intercalation is the preliminary step for the generation of dense granules inside the matrix. Instead, we assume that hydrophobic interaction between the dye cations E, BH and TME and the cristae is the main cause of the mitochondrial changes. The favoured binding partner of the dye cations may be the divalent anion, cardiolipin: this phospholipid is an essential part of the inner membrane system but is absent in other membranes of cells. By distributing the dyes between a lipophilic phase and water, it was shown that TME is more lipophilic than E and BH; this may explain the greater effectiveness of TME. The bound dye cations disturb the organization of the cristae, which become altered and finally disappear. We assume that the electron-dense granules in the matrix are mainly composed of the dyes and former membrane materials such as phospholipids and proteins, as well as perhaps some other hydrophobic matrix materials. This would also explain why it was impossible to digest the dense granules by DNase treatment. The drugs enter the mitochondrial matrix by disordering and finally destroying the cristae.     

ULTRASTRUCTURAL STUDIES ON THE CYANELLES OF GLAUCOCYSTIS NOSTOCHINEARUM ITZIGSOHN1

Ultrastructural studies conducted on the intracellular symbiont of Glaucocystis nostochinearum Itz., a unicellular alga with a debated taxonomic position, have shown that the endosymbiont, although somewhat aberrant, is a blue-green alga. Due possibly to its intracellular habitat, it lacks the characteristic cyanophycean double-layered cell wall and the cells appear to be completely naked, bound only by a single plasma membrane. The protoplasm of the cell is differentiated into the lamellated chromatoplasm, which contains the photosynthetic pigments and polyphosphate granules, and a nonlamellar ccntroplasm, in which the nucleic material is dispersed. The usual cyanophycean organelles, as well as the different vacuoles and granules, with the exception of the formed bodies, are missing. Approximately 10% of the cells sliows a peculiar homogeneous area at one tip, the nature of which is unknown. Binary fission of the organism is mentioned. Since this cyanelle has not yet been classified, we name it Skujapelta nuda nov. gen., nov. sp.; and because of its structural peculiarities we find it necessary to create a new family for it, Skujapeltaceae in the order Chroococcales.     

The binding of simian virus 40 large T antigen to the polyphosphate backbone of nucleic acids

Simian virus 40 (SV40) large tumor antigen (T antigen), a phosphoprotein found in nuclei of SV40-infected and -transformed cells, binds nonspecifically to DNA. To study this mechanism the binding properties of T antigen to double-stranded (ds) and single-stranded (ss) DNA-cellulose as well as to phosphocellulose were compared. After incubation of [35S] methionine or [3H] leucine/[32 P] phosphate radioactively-labeled cell extracts at different pH values (6.0, 7.3, 9.0) with DNA- or phosphocellulose, bound and unbound species of T antigen were purified and analyzed by SDS-polyacrylamide gel electrophoresis for both the yield and the possible correlation with protein phosphorylation. T antigens bound with comparable affinities to ds- and ss-DNA-cellulose and phosphocellulose. These results suggest the binding of T antigen to the polyphosphate backbone of DNA as a molecular mechanism for its nonspecific binding. The evidence for this observation was supported by blocking the binding of T antigen to DNA-cellulose by divalent cations (Ca2+, Mg2+). 3H/32P ratios of T antigen obtained by double-labeling cells for various times imply that higher phosphorylated forms of T antigen bound more strongly to ds- and ss-DNA as well as to phosphocellulose. Thus, in the presence of cellular proteins and other components the binding activity of T antigen to the polyphosphate backbone of DNA seems to be positively correlated with its phosphorylation. These observations are consistent with the hypothesis that the binding affinities of SV40 T antigen to host cell DNA may be regulated by its phosphorylation.     

Conformation of carboxymethyl amylose in aqueous solutions

Intrinsic viscosity and specific optical rotation measurements have been carried out for Carboxymethyl amylose (CMA) solutions in 0.15M sodium chloride at various pH values. Potentiometric titrations of CMA have been performed at various polymer concentrations as well as in presence of different divalent cations. The amount of divalent cation (Ni⁺²) bound to CMA has been determined by using a new method af analysis based on polyelectrolyte theory. Finally from the results it is shown that CMA may exist as a random coil in solution.     

Inorganic polyphosphates and sensitivity of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> cells to membrane-damaging agents

The leakage of ATP and potassium ions from the cells of Saccharomyces cerevisiae with different levels of inorganic polyphosphate was studied under the action of two detergents (natural cellobiose lipid 16-[6-O-acetyl-2′-O-(3-hydroxyhexanoyl)-β-cellobiosyloxy)-2,15-dihydroxyhexadecanoic acid and sodium dodecyl sulfate) and silver cations. Cellobiose lipid had practically the same membrane-damaging activity against the cells grown in phosphate-containing medium, under phosphate starvation, and under polyphosphate hypercompensation. The cells grown under the latter conditions were less sensitive to sodium dodecyl sulfate and silver cations. The possible protective action of polyphosphates against the membrane-damaging agents under study is discussed.     

Detection of a yeast polyphosphate fraction localized outside the plasma membrane by the method of phosphorus-31 nuclear magnetic resonance

Non-penetrating cations, like UO2+(2) and Eu3+, are bound to the outside of yeast cells in a reversible fashion. Binding of these ions was attended with a decrease of the 31P NMR polyphosphate signal. Subsequent addition of EDTA to the suspension restored the original spectrum. These experiments confirm the localization of a polyphosphate fraction outside the plasma membrane of yeast.     

Ultrastructural changes in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> cells under stress conditions

Ultrastructural organization of the aerobic yeast Yarrowia lipolytica was studied under conditions of oxidative, heat, and ethanol stresses. It was shown that the following uniform changes in cell ultrastructure did not depend on the type of stress: enlargement of mitochondria, enhanced number and enlargement of peroxisomes, and formation of lipid granules. Similar ultrastructural changes also occurred during the transition of cells to the stationary growth phase. It was shown for the first time that accumulation of polyphosphate granules occurred as a stress response in yeasts. Moreover, numerous globular structures of unknown nature appeared on the cell wall surface under oxidative or heat stress. Under ethanol stress, the cells developed clearly marked deep invaginations of the cytoplasmic membrane. (The same changes in the cytoplasmic membrane were observed in the cells grown on ethanol.) Variations of the cell envelope structure along with the formation of polyphosphate granules were not observed in the stationary growth phase. Ultrastructural changes in the cells under stress conditions are in agreement with the previous data on survival, respiratory activity, and variations of the antioxidant systems.     

Differentiation of polyphosphate and poly-β-hydroxybutyrate granules in an Acinetobacter sp. isolated from activated sludge

Cells containing polyphosphate 71 micrograms P (mg protein)-1 and no poly-beta-hydroxybutyrate showed metachromatic granules but no lipid granules; cells containing poly-beta-hydroxybutyrate (15% of dry weight) showed fluorescence lipid granules but no metachromatic granules; whereas cells containing both polyphosphate and poly-beta-hydroxybutyrate showed both types of granules. These observations, together with a critical review of the literature, show a clear distinction between metachromatic (or volutin) granules and lipid granules.