Inorganic polyphosphates and phosphohydrolases inHalobacterium salinarium

Halobacterium salinarium grown in a liquid medium consumed up to 75% of phosphates originally present in the growth medium and accumulated up to 100 μmol P_i/g wet biomass by the time it entered the growth retardation phase. The content of acid-soluble oligophosphates in the biomass was maximum at the early stage of active growth and drastically decreased when cells reached the growth-retardation phase. The total content of alkali-soluble and acid-insoluble polyphosphates changed very little throughout the cultivation period (five days). The polyphosphate content ofH. salinarium cells was close to that of yeasts and eubacteria. The pyrophosphatase, polyphosphatase, and nonspecific phosphatase activities ofH. salinarium cells were several times lower than those of the majority of eubacteria. The specific activity of pyrophosphatase, the most active hydrolase ofH. salinarium, gradually increased during cultivation, reaching 540 mU/mg protein by the end of the cultivation period. Half of the total pyrophosphatase activity of this halobacterium was localized in the cytosol. The molecular weight of pyrophosphatase, evaluated by gel filtration, was 86 kDa. The effective K_m of this enzyme with respect to pyrophosphate was 115 μM.     

Magnesium orthophosphate,a new form of reserve phosphates in the halophilic archaeon Halobacterium salinarium

The accumulation and utilization of reserve phosphates in the extremely halophilic archaeon Halobacterium salinarium were studied. The growth of H. salinarium was found to depend on the initial concentration of inorganic phosphate (Pi) in the culture medium and its content in the inoculum. Growing cells consumed 85-95% of Pi from the medium. Unlike the reserve phosphates of many other microorganisms, which are mainly polyphosphates, the reserve phosphates of H. salinarium cells contain no more than 15% polyphosphates, the rest being magnesium orthophosphate. The excessive consumption of Pi from the medium changed cell morphology and caused the death of part of the cell population. The cells that remained viable could grow in a Pi-deficient medium, utilizing about 70% of reserve magnesium phosphate as the phosphorus source.     

Untersuchungen über die Raten der Polyphosphatsynthese durch die Photophosphorylierung bei Ankistrodesmus braunii

Summary Short-time experiments with ³²P-labelled phosphate and chase experiments with equally labelled cells were carried out with synchronized algae under conditions of optimum phosphate uptake. In short-time experiments, in the presence as in the absence of CO₂, orthophosphate and organic phosphates are rapidly labelled, but their time curves show saturation behaviour after 10 to 20 min. Labelling of polyphosphates proceeds at a constant rate after a short lag period of about 5 min. In equally labelled algae ³²P-labelling correspondingly decreases in orthophosphate and in organic phosphates, but increases by about the same amount in the fraction of acid-insoluble polyphosphates. In the presence of external phosphate and in the light, polyphosphates show no visible decay within the 20 min of the chase experiments.A comparison of the two kinds of experiments suggests that polyphosphates are secondary products of photophosphorylation following only after orthophosphate and organic phosphates, probably after ATP. The rates of photophosphorylation are certainly much higher than the rates of labelling in organic phosphates because of the limiting phosphate uptake. Since the polyphosphates show no decay during the time of the experiments their turnover is low and the rates of polyphosphate labelling after a phosphate starvation period, and after the short lag period, can be regarded as approximate rates of polyphosphate synthesis. These rates are lower than the rates of phosphate uptake.In young cells of the synchronous culture phosphate replenishment after a 5-h starvation requires 2 to 3 h. After replenishment or in a culture undisturbed by phosphate starvation, the rates of polyphosphate accumulation, like the rates of phosphate uptake are much lower. In the presence of CO₂ they are constant for several hours, if related to culture volume with constant cell number. Polyphosphate accumulation is proportional to phosphate uptake under these conditions amounting to about one third. In the absence of CO₂, the rates decrease after 2 to 4 h of CO₂-starvation and, like in short-time experiments a large proportion of the phosphate taken up is used for polyphosphate accumulation. The low rates of long-time experiments may represent a steady state between formation and decay of polyphosphates. Since the cells kept in the absence of CO₂ are prevented from growing they actually accumulate more polyphosphates per cell volume, per chlorophyll, and per dry weight than the cells in the presence of CO₂.The rates of polyphosphate formation are discussed with respect to their turnover in the light observed by other investigators. They are regarded to be a result of competition for ATP together with the orthophosphate pool of the cells, and of the compartmentation. The rates of polyphosphate formation are rather low compared with the probable rates of ATP formation under various conditions of photophosphorylation. Therefore, the formation of polyphosphates is regarded as a process of secondary order of magnitude in the energy metabolism of algal cells.

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Im Text verwendete Abkürzungen P₁ Trichloressigsäure lösliche Phosphate - davon P_i Orthophosphat - P_o organisches Phosphat - P_ul Hydrolyse-labiles TCE-unlösliches Phosphat - P_us Hydrolyse-stabiles TCE-unlösliches Phosphat - P_ges Gesamtphosphat, bei kurzzeitiger ³²P-Markierung Phosphataufnahme - Chl Chlorophyll     

In vivo P-31 NMR measurements of phosphate metabolism in Platymonas subcordiformis as related to external pH

The phosphate metabolism of Platymonas subcordiformis was investigated by 31P-NMR spectroscopy with special attention on the effect of external pH. Glycolyzing cells and cells energized by respiration or photosynthesis gave spectra dependent upon their metabolic state. The transition from deenergized to energized states is accompanied by a shift of cytoplasmic pH from 7.1–7.4, an increase of ATP level and-in well energized cells-the appearance of a new signal tentatively assigned to phosphoarginine.The spectra remain stable over a wide range of external pH. Cytoplasmic pH is well regulated in respiring cells for external pH in the range 5.3–12.3. The typical 0.4 units difference of internal pH in energized as compared to deenergized cells is not affected by external pH in the range 6–12. The intensity of a signal attributed to PEP is markedly increased at high external pH. pH regulation is less efficient below external pH of 6 in deenergized cells. Below pH 3.8 oxidative phosphorylation ceases. Upon raising cytoplasmic pH to 7.4 in deenergized cells polyphosphate chains start to disintegrate.Abbreviations PEP Phosphoenolpyruyate - P _i inorganic phosphate - PP _i inorganic pyrophosphate - poly P polyphosphates - PP-1, PP-2, PP-3 terminal, second, and third phosphate residue of polyphosphates - PP-4 core phosphate residues of polyphosphates - pH _i , pH _o internal (cytoplasmic) and external pH - NTP/NDP nucleotide triphosphate/-diphosphate - S/N signal to noise ratio     

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     

PHOSPHATE METABOLISM IN BLUE-GREEN ALGAE. II. CHANGES IN PHOSPHATE DISTRIBUTION DURING STARVATION AND THE “POLYPHOSPHATE OVERPLUS” PHENOMENON IN PLECTONEMA BORYANUM

Physiological aspects of phosphate utilization by the blue-green alga Plectonema boryanum were studied. It was found that the external phosphate concentration influenced the distribution of phosphorus-containing compounds in the cell. Culturing the alga in concentrations of 10, 100, and 1000 mg PO₄/l resulted in increases in the level of acid-soluble and acid-insoluble polyphosphates. The values reported for 100 and 1000 mg PO₄/l were the same, indicating that the cells were able to assimilate and utilize only fixed amounts of phosphates. The total phosphorus value for these cells was calculated to be 6.5 μg P per 10⁶ cells. Culturing the alga in 1 mg PO₄/l led to a decrease in phosphate concentration of all cell fractions. Cells grown in the absence of phosphate for 5 days had total cell phosphorus levels of 0.76 μg P per 10⁶ cells. Cells in culture for two months or longer were found to have total cell phosphorus levels of 0.73 μg P per 10⁶ cells. This was determined to be the minimum cell phosphorus level limiting growth. Transfer of cells from either culture condition to a medium containing phosphate led to an “overplus” phenomenon. This overplus phenomenon was characterized by increases in all cellular phosphorus fractions. The most dramatic increase was found in both the acid-soluble and acid-insoluble polyphosphates. These fractions often increased by more than an order of magnitude. The greatest phosphate uptake occurred within 1 hr of transfer of phosphate-starved cells into a medium containing a known amount of phosphate and is essentially complete at 4 hr. The total cell phosphorus levels for uptake never increased beyond 18.9 μg per 10⁶ cells.     

31P NUCLEAR MAGNETIC RESONANCE STUDY OF INTRACELLULAR PHOSPHATE POOLS AND POLYPHOSPHATE METABOLISM IN HETEROSIGMA AKASHIWO (HADA) HADA (RAPHIDOPHYCEAE)1

The effects of starvation and subsequent addition of phosphate-containing medium on the phosphate metabolic intermediates were studied by ³¹P-NMR spectroscope of perchloric acid extracts and intact cells of Heterosigma akashiwo (Hada) Hada. When orthophosphate in the medium was completely depleted the medium was enriched with orthophosphate (4.5 μM). In the phosphate starved condition, the P cell quota was 76 fmol·cell⁻¹ and the major components of phosphate intermediates were phosphodiester, sugar phosphate and orthophosphate (P_i). After addition of P_i, rapid uptake of P_i was observed and the P cell quota increased to 108 fmol·cell⁻¹ in 2 h, 134 fmol·cell⁻¹ in 5 h and 222 fmol·cell⁻¹ in 1 day after addition of phosphate. The ³¹P-NMR spectrum indicated that a major portion of P was stored as polyphosphate, in which the average chain length of polyphosphate increased from 10 to 20 phosphate residues in one day after addition of P_i.     

Accumulation of inorganic polyphosphates in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> under nitrogen deprivation: Stimulation by magnesium ions and peculiarities of localization

The yeast Saccharomyces cerevisiae was shown to have a high potential as a phosphate-accumulating organism under growth suppression by nitrogen limitation. The cells took up over 40% of phosphate from the medium containing 30 mM glucose and 5 mM potassium phosphate and over 80% of phosphate on addition of 5 mM magnesium sulfate. The major part of accumulated Pi was reserved as polyphosphates. The content of polyphosphates was ∼57 and ∼75% of the phosphate accumulated by the cells in the absence and presence of magnesium ions, respectively. The content of long-chain polyphosphates increased in the presence of magnesium ions, 5-fold for polymers with the average length of ∼45 phosphate residues, 3.7-fold for polymers with the average chain length of ∼75 residues, and more than 10-fold for polymers with the average chain length of ∼200 residues. On the contrary, the content of polyphosphates with the average chain length of ∼15 phosphate residues decreased threefold. According to the data of electron and confocal microscopy and X-ray microanalysis, the accumulated polyphosphates were localized in the cytoplasm and vacuoles. The cytoplasm of the cells accumulating polyphosphates in the presence of magnesium ions had numerous small phosphorus-containing inclusions; some of them were associated with large electron-transparent inclusions and the cytoplasmic membrane.     

Bioenergetic characterization of transient state phosphate uptake by the cyanobacterium Anacystis nidulans

A force flow relationship based on nonequilibrium thermodynamics was derived to analyze the variable transient state phosphate uptake phenomena of cyanobacteria seen under different growth conditions and external phosphate concentrations. This relationship postulates the following basic properties of the uptake system: First, a threshold value exists, below which incorporation is energetically impossible. Second, threshold values are influenced by the activity of the phosphate uptake system, such that a decrease of the activity increases the threshold level. Third, near the thermodynamic equilibrium the uptake rate is linearly dependent on the free energy of polyphosphate formation and the pH-gradient at the thylakoid membrane. Experiments performed with Anacystis nidulans showed that phosphate uptake characteristics conformed to the properties predicted by the linear force-flow relationship. Linearity extented into regions far form thermodynamic equilibrium, e.g. to high phosphate concentrations, when algae were preconditioned to high phosphate levels. Under phosphate limited growth linearity was confined to a small concentration range, threshold values decreased below 10 nM, and the external concentration approached threshold. The data suggest that the uptake system responds to changes in the external phosphate concentration in the same way as sensory systems to input stimuli by amplifying signals and adapting to them.Abbreviations chl chlorophyll - H _e ⁺ , H _C ⁺ , H _T ⁺ protons in the external medium, the cytoplasmic and thylakoid space respectively - P_c phosphate in the cytoplasmic space - P_e phosphate in the external medium - P_n, P_n+1 polyphosphates - pH_T pH-gradient across the thylakoid membrane     

The dependence of the cytoplasmic pH in aerobic and anaerobic cells of the green algae Chlorella fusca and Chlorella vulgaris on the pH of the medium as determined by 31P in vivo NMR spectroscopy

The pH in the cytoplasm of aerobic and anaerobic cells of the green algae Chlorella fusca and Chlorella vulgaris was determined in dependence on the pH of the external medium, which was varied between pH 3 and pH 10. In aerobic cells of both species the cytoplasmic pH is maintained at a value above 7.2 even at an external pH of 3 and below 7.8 at an external pH of 10. In anaerobic cells the cytoplasmic pH shows linear dependence on external pH in the range of pH 6 to 9 (cytoplasmic pH 6.9 to 7.2), while below an external pH of 6 cytoplasmic pH is maintained at about 6.5.Abbreviations CCCP Carbonylcyanide-m-chlorophenyl-hydrazone - EDTA Ethylendiaminetetraacetic acid - MES 2-(N-Morpholino)-ethanesulfonic acid - MOPSO 3-(N-Morpholino)-2-hydroxy-propanesulfonic acid - NMR Nuclear Magnetic Resonance - pH cyt cytoplasmic pH - pH ex external pH - PIPES Piperazine-N,N-bis(2-ethanesulfonic acid) - PP_i Pyrophosphate - PP₁, PP₂, PP₃ 1st, 2nd, 3rd phosphate group of polyphosphates - PP₄ core phosphate groups of polyphosphates - TRIS Tris-hydroxymethyl-aminomethane     

Extracellular phosphomannan as a phosphate reserve in the yeast Kuraishia capsulata

We have found that extracellular phosphomannan is the main phosphate reserve in the yeast Kuraishia capsulata, in contrast to other yeast species effectively absorbing P_i. Under nitrogen starvation, K. capsulata absorbed essentially all P_i from the medium containing 240 mM glucose, 2.5 mM MgSO₄, and 11 mM KH₂PO₄. Inorganic polyphosphate level in the cells was about 14% of the P_i absorbed. Most of the P_i (～60%) was found in the fraction of extracellular phosphomannan that can be used as a carbon and phosphorus source by this yeast in deficient media.     

Cyclic condensed metaphosphates and linear polyphosphates in brown and red algae

The occurrence of linear condensed polyphosphates and cyclic condensed metaphosphates was studied by means of pulse-labeling with ³²P-orthophosphate (3–5 h) in a number of Phaeophyceae species: Pylaiella litoralis, Ilea fascia, Ectocarpus siliculosus and also Rhodophyceae species: Ceramium deslongchampsii, C. rubrum, Rhodomela confervoides, Porphyridium purpureum and P. aerugineum. Twodimensional cellulose thin layer chromatography revealed that in all species studied ³²P-radioactivity was generally present in all oligopolyphosphates containing 2 to 7 phosphate residues, in cyclic metaphosphates (tri-, tetra-, penta- and hexametaphosphates) and in high-molecular-weight condensed phosphates which remained at the starting point. Among the low-molecular-weight condensed inorganic phosphates the trimetaphosphate had a significantly higher specific activity than the other oligophosphates which were separated on the chromatography plates as measured by the direct scanning with a Geiger-Muller counter.The phosphate uptake strongly depends on the internal pool of reserve phosphates of the algae cells. The ³²P-orthophosphate incorporation of the cells is low and sluggish when growning in a synthetic medium or in sea water. Accordingly ³²P appeared preferentially in the low-molecular-weight fractions of condensed phosphates since the storage phosphates were not yet used. After previous incubation in a P-free culture medium of the algae the ³²P was rather rapidly incorporated and was found mostly in the highmolecular-weight condensed phosphates.During MAK-chromatography the high-molecular-weight fractions were eluted together with the nucleic acids (tRNA and DNA) while most of the low-molecular-weight fractions left the column immediately on elution.     

Phosphate accumulation and the occurrence of polyphosphates and cyclic 2,3-diphosphoglycerate in Methanosarcina frisia

Methanosarcina frisia accumulates phosphate up to 14% of its dry weight. The phosphate is stored as long-chain polyphosphates as shown by ³¹P-NMR investigations. Further results show that the accumulation of phosphates is substrate-dependent. In the presence of H₂ and CO₂ as the only carbon and energy source 180 mg of PO _inf4 ^sup3- /g protein were accumulated, whereas 260 mg PO _inf4 ^sup3- /g protein were accumulated in the presence of methanol. This is far more than necessary for the maintenance of essential metabolic pathways. In addition, the ³¹P-NMR studies show the occurrence of cyclic 2,3-diphosphoglycerate in Methanosarcina frisia. The role of the phosphate metabolites in cell metabolism are discussed.Abbreviations M. Methanosarcina - CCP cyclic 2,3-diphosphoglycerate     

P-31 in-vivo NMR investigation on the function of polyphosphates as phosphate-and energysource during the regreening of the green alga Chlorella fusca

The green alga Chlorella fusca accumulates polyphosphates under conditions of nitrogen starvation while deassembling the photosynthetic apparatus. The polyphosphate content of cells regreening after resupply with nitrate under different culture conditions was investigated by P-31 in-vivo NMR spectroscopy. Neither phosphate deficiency nor anaerobiosis during the first hours of regreening inhibited the recovery of the cells. Polyphosphates were degraded during regeening. Differences in the amount of polyphosphates of phosphate supplied and deficient cells occurred only after more then 8 h. After 16 h phosphate deficient cells had still 75% of the polyphosphate content of phosphate suppled cells. In cells kept under anaerobic conditions polyphosphate degradation was much higher than in oxygen supplied cells. After 8 h they contained less than 50% of the polyphosphate content of oxygen supplied cells. These data suggest that polyphosphates serve as obligatory phosphate source during regreening and may be used as an energy source.Non standard abbreviations EDTA Ethylene diamine tetraacetic acid - FID Free induction decay - MOPSO 3-(N-morpholine)-2-hydroxy-propanesulfonic acid - NMR Nuclear magnetic resonance - PP Polyphosphates - PP₄ central phosphate groups of polyphosphates     

Phosphate transport across biomembranes and cytosolic phosphate homeostasis in barley leaves

Barley (Hordeum vulgare L.) plants were grown hydroponically with or without inorganic phosphate (P_i) in the medium. Leaves were analyzed for the intercellular and the intracellular distribution of P_i. Most of the leaf P_i was contained in mesophyll cells; P_i concentrations were low in the xylem sap, the apoplast and in the cells of the epidermis. The vacuolar concentration of P_i in mesophyll cells depended on P_i availability in the nutrient medium. After infiltrating the intercellular space of leaves with solutions containing P_i, P_i was taken up by the mesophyll at rates higher than 2.5 mol· (g fresh weight)^–1 · h^–1. Isolated mesophyll protoplasts did not possess a comparable capacity to take up P_i from the medium. Phosphate uptake by mesophyll protoplasts showed a biphasic dependence on P_i concentration. Uptake of P_i by P_i-deficient cells was faster than uptake by cells which had P_i stored in their vacuoles, although cytoplasmic P_i concentrations were comparable. Phosphate transport into isolated mesophyll vacuoles was dependent on their P_i content; it was stimulated by ATP. In contrast to the vacuolar P_i concentration, and despite different kinetic characteristics of the uptake systems for p_i of the plasmalemma and the tonoplast, the cytoplasmic p_i concentration was regulated in mesophyll cells within narrow limits under very different conditions of P_i availability in the nutrient medium, whereas vacuolar P_i concentrations varied within wide limits.Dedicated to Professor Wilhelm Simonis on the occasion of his 80th birthdayThis investigation was part of the research efforts of the Sonderforschungsbereich 176 of the Bayerische Julius-Maximilians-Universität Würzburg. We are grateful to Dr. Olaf Wolf for introducing us to the method for preparation of xylem sap of barley plants and to Mr. Yin Zuhua for fluorimetric experiments with the dye pyranine. T. Mimura is indebted to the Alexander-von-Humboldt-Stiftung for a postdoctoral research fellowship.     

Phosphorus-31 nuclear magnetic resonance studies of intracellular pH,phosphate compartmentation and phosphate transport in yeasts

³¹P NMR spectra were obtained from suspensions of Candida utilis, Saccharomyces cerevisiae and Zygosaccharomyces bailii grown aerobically on glucose. Direct introduction of substrate into the cell suspension, without interruption of the measurements, revealed rapid changes in pH upon addition of the energy source. All ³¹P NMR spectra of the yeasts studied indicated the presence of two major intracellular inorganic phosphate pools at different pH environments. The pool at the higher pH was assigned to cytoplasmic phosphate from its response to glucose addition and iodoacetate inhibition of glycolysis. After addition of substrate the pH in the compartment containing the second phosphate pool decreased. A parallel response was observed for a significant fraction of the terminal and penultimate phosphates of the polyphosphate observed by ³¹P NMR. This suggested that the inorganic phosphate fraction at the lower pH and the polyphosphates originated from the same intracellular compartment, most probably the vacuole. In this vacuolar compartment, pH is sensitive to metabolic conditions. In the presence of energy source a pH gradient as large as 0.8 to 1.5 units could be generated across the vacuolar membrane. Under certain conditions net transport of inorganic phosphate across the vacuolar membrane was observed during glycolysis: to the cytoplasm when the cytoplasmic phosphate concentration had become very low due to sugar phosphorylation, and into the vacuole when the former concentration had become high again after glucose exhaustion.Non-Standard Abbreviations NMR nuclear magnetic resonance - ppm parts per million - PP polyphosphate - P_i,c cytoplasmic inorganic phosphate - P_i,v vacuolar inorganic phosphate - pH_in,c cytoplasmic pH - pH_in,v vacuolar pH - FCCP carbonyl p-trifluoromethoxyphenylhydrazone     

Properties of polyphosphatase ofAcinetobacter johnsonii 210A

Polyphosphatase, an enzyme which hydrolyses highly polymeric polyphosphates to P_i, was purified 77-fold fromAcinetobacter johnsonii 210A by Q-Sepharose, hydroxylapatite and Mono-Q column chromatography. The native molecular mass estimated by gel filtration and native gel electrophoresis was 55 kDa. SDS-polyacrylamide gel electrophoresis indicated that polyphosphatase ofAcinetobacter johnsonii 210A is a monomer. The enzyme was specific for highly polymeric polyphosphates and showed no activity towards pyrophosphate and organic phosphate esters. The enzyme was inhibited by iodoacetamide and in the presence of 10 mM Mg²⁺ by pyro- and triphosphate. The apparent K_m-value for polyphosphate with an average chain length of 64 residues was 5.9 µM and for tetraphosphate 1.2 mM. Polyphosphate chains were degraded to short chain polymers by a processive mechanism. Polyphosphatase activity was maximal in the presence of Mg²⁺ and K⁺.     

Intracellular Accumulation of the Monomeric Precursors of Polyphosphates and Polyhydroxyalkanoates in Acinetobacter calcoaceticusand Escherichia coliCells

The formation of polyhydroxyalkanoates granules in anaerobically grown Escherichia coliM-17 cells was found to be preceded by the intracellular accumulation of carbonic acids (predominantly, acetic acid), amounting to 9% of the cytosol. The intracellular concentration of acidic metabolites increased after the lyophilization of the bacterial biomass and decreased after its long-term storage (3.5–13.5 years). The decrease in the concentration of acidic metabolites is likely due to the dehydration of dimeric carbonic acids in the viscoelastic cytosol of resting bacterial cells. The hydrophobic obligately aerobic cells of Acinetobacter calcoaceticusIEGM 549 are able to utilize a wide range of growth substrates (from acetate and citrate to hydrophobic hydrocarbons), which is considerably wider than the range of the growth substrates of E. coli(predominantly, carbohydrates). The minimal essential and optimal concentrations of orthophosphates in the growth medium of A. calcoaceticuswere found to be tens of times lower than in the case of E. coli.The intracellular content of orthophosphates in A. calcoaceticuscells reached 35–77% of the total phosphorus content (P_total), providing for the intense synthesis of polyphosphates. The P_totalof the A. calcoaceticuscells grown in media with different proportions between the concentrations of acetate and phosphorus varied from 0.7 to 3.3%, averaging 2%. This value of P_totalis about two times higher than that observed for fermenting E. colicells. Lowering the cultivation temperature of A. calcoaceticusfrom 37–32 to 4°C augmented the accumulation of orthophosphates in the cytoplasm, presumably owing to a decreased requirement of growth processes for orthophosphate. In this case, if the concentration of phosphates in the cultivation medium was low, they were completely depleted.