Environmental and physiological factors affecting the uptake of phosphate by Chlorobium limicola

The uptake of soluble phosphate by the green sulfur bacterium Chlorobium limicola UdG6040 was studied in batch culture and in continuous cultures operating at dilution rates of 0.042 or 0.064 h^–1. At higher dilution rates, washout occurred at phosphate concentrations below 7.1 μM. This concentration was reduced to 5.1 μM when lower dilution rates were used. The saturation constant for growth on phosphate (K _μ) was between 2.8 and 3.7 μM. The specific rates of phosphate uptake in continuous culture were fitted to a hyperbolic saturation model and yielded a maximum rate (Va _max) of 66 nmol P (mg protein)^–1 h^–1 and a saturation constant for transport (K _t) of 1.6 μM. In batch cultures specific rates of phosphate uptake up to 144 nmol P (mg protein)^–1 h^–1 were measured. This indicates a difference between the potential transport of cells and the utilization of soluble phosphate for growth, which results in a significant change in the specific phosphorus content. The phosphorus accumulated within the cells ranged from 0.4 to 1.1 μmol P (mg protein)^–1 depending on the growth conditions and the availability of external phosphate. Transport rates of phosphate increased in response to sudden increases in soluble phosphate, even in exponentially growing cultures. This is interpreted as an advantage that enables Chl. limicola to thrive in changing environments. Received: 9 February 1998 / Accepted: June 1998     

The effects of “Cell age” upon the lethal effects of physical and chemical mutagens in the yeast, Saccharomyces cerevisiae

Summary SummaryYeast cultures progressing from the exponential to the stationary phase of growth showed changes in cell sensitivity to physical agents such as UV light, heat shock at 52° C and the chemical mutagens ethyl methane sulphonate, nitrous acid and mitomycin C.Exponential phase cells showed maximum resistance to UV light and minimum resistance to heat shock and the three chemicals. The increased resistance of exponential phase cells to UV light was shown to be dependent upon the functional integrity of the RAD ₅₀ gene.Treatment of growing yeast cultures with radioactively labelled ethyl methane sulphonate indicated the preferential uptake of radioactivity during the sensitive exponential stage of growth. The results indicated that the differential uptake of the chemical mutagens was responsible for at least a fraction of the variations in cell sensitivity observed in yeast cultures at different phases of growth.     

Purification and kinetics of a protease-resistant,neutral, and thermostable phytase from Bacillus subtilis subsp. subtilis JJBS250 ameliorating food nutrition

Abstract

A novel protease-resistant and thermostable phytase from Bacillus subtilis subsp. subtilis JJBS250 was purified 36-fold to homogeneity with a combination of ammonium sulfate precipitation followed by Q-Sepharose and Sephadex G-50 chromatographic techniques. The estimated molecular mass of the purified phytase was 46?kDa by electrophoresis with optimal activity at pH 7.0 and 70?°C. About 19% of original activity was maintained at 80?°C for 10?min. Phytase activity was stimulated in presence of surfactants like Tween-20, Tween-80, and Triton X-100 and metal ions like Ca⁺², K⁺, and Co⁺² and it was inhibited by SDS and Mg⁺², Al⁺², and Fe⁺². Purified enzyme showed specificity to different salts of phytic acid and values of K_m and V_max were 0.293?mM and 11.49 nmoles s^?1, respectively for sodium phytate. The purified enzyme was resistant to proteases (trypsin and pepsin) that resulted in amelioration of food nutrition with simultaneous release of inorganic phosphate, reducing sugars, and soluble protein.     

Effect of foliar application of phosphorus on growth and mineral composition in peanut plants (Arachis hypogaea L.) under salt-stress

Summary Peanut (Arachis hypogaea L., Var. TMV-2) plants were raised in sand cultures under natural photoperiod. Salt treatment (0.4% NaCl on dry weight basis) was given 10 days after sowing. Nutrient solution was supplied 15 days after sowing to control and salinized plants. Phosphate (0.1% NaH₂PO₄ with 0.01% Tween-80) was sprayed to the plants to the drip point once daily from 20th to 25th day and from 30th to 35th day. The plants were harvested at 30th and 40th day for analysis. The plants grown under saline conditions showed a market reduction in growth. When phosphorus was supplied to saline plants by foliar application, there was an increase in dry weight, leaf area, stomatal frequency, and yield and the increase was more marked when the plants received phosphate for two periods. Phosphorus content decreased due to salinity which was restored by foliar spray of phosphate. With salinity, sodium accumulated while potassium and calcium were lowered. Phosphate spray decreased sodium and increased potassium and calcium in general.     

Transferrin binding by mammalian cortical cells

Predominantly neuronal (neuronal) or non-neuronal (glial) cerebral cortical cell cultures were employed to study the kinetics and changes with maturation of¹²⁵I-diferric-transferrin uptake. The diferric-transferrin association curve of neuronal cultures at 37°C was nonphasic and indicated equilibrium at 90 minutes. Dissociation was completed by 70 minutes. Diferric-transferrin specific uptake (80% of total) in neuronal cells (evaluated at days 6, 9, 13, 16, and 23 in culture) increased with maturation. Scatchard transformation of the data revealed increasingB _max from day 6 to day 16 in culture (1626 to 2740 fmoles/mg protein). However, theK _uptake was statistically unchanged over time and equaled 48.7±13.9 nM (mean ±SD). In contrast, association studies of glial cultures documented equilibrium by 45 minutes and dissociation by 40 minutes. The concentration curves for differric-transferrin uptake in glial cells, evaluated at days 11, 15, and 18 in culture, revealed virtually identical uptake at the three ages studied, but the percent specific uptake (58%) was less than for neurons (88%). Scatchard transformation of the data revealed no statistical alteration ofB _max orK _uptake from days 11 to 18 in culture.B _max ranged from 595 to 751 fmol/mg protein; overallK _uptake was 48.3±13.2 nM (mean±SD).     

PHOSPHATE UPTAKE KINETICS IN EUGLENA GRACILIS (Z) (EUGLENOPHYCEAE) GROWN IN LIGHT/DARK CYCLES. II. PHASED PO4-LIMITED CULTURES1

The characteristics of phosphate uptake and photosynthetic capacity were studied in P-limited populations of Euglena gracilis Klebs (Z), using both P-limited batch cultures in stationary phase and cyclostat cultures grown on 14:10 LD. P uptake obeyed Michaelis-Menten kinetics between 0 and 150 μM PO₄ under both growth conditions. The value of V_max was 35% lower in the dark than in the light in the stationary phase cells. The value of K₈ was not affected by light conditions, and uptake was completely inhibited in the presence of 1 mm KCN. P uptake (at 2.0 μM PO₄) and photosynthetic capacity showed diel periodicity with peak rates occurring just before the beginning of the dark period for P uptake, and 8 h into the light period for photosynthetic capacity. V_max for P uptake increased by a factor of 1.5 over the light period, whereas K₈ remained constant at 1.4 μM PO₄. These patterns were displayed by both nondividing stationary phase cells and populations in which less than a third of the cells divided each day, indicating that the rhythmicity is not coupled to cell division.     

Inorganic phosphate transport inAcinetobacter lwoffi

The transport of inorganic phosphate has been studied inAcinetobacter lwoffi JW11. During growth on excess phosphate, only one transport system was present, with an apparent K_m of 1.4 M. When cells were starved for phosphate, a second uptake system with an apparent K_m of 110 nM was also synthesized. The two transport systems could be distinguished by differing sensitivities to the phosphate analogs arsenate and 2-aminoethylphosphonate. Both systems were inhibited by carbonylcyanidem-chlorophenylhydrazone, and to a lesser extent by Na azide. The high-affinity transport system was inactivated by osmotic shock treatment and by spheroplast formation. Preliminary evidence for a phosphate-binding protein in the osmotic shock fluid is presented. The isolation of a mutant constitutive for the high-affinity transport system is described.     

Phosphate uptake in the yeast Candida tropicalis: purification of phosphate-binding protein and investigations about its role in phosphate uptake

The purification of a phosphate-binding protein (P_iBP₂) by immunoadsorption is described. The entire anti phosphate-binding protein 2 antibodies as well as the Fab fragments obtained from these antibodies inhibit P_i uptake by whole cells. The inhibition is a mixed type of inhibition (V _m and K _m are affected). These results should be regarded as a possible involvement of phosphate-binding protein 2 in P_i uptake. The binding of ¹²⁵I-labelled fragments prepared from anti phosphate-binding protein 2 antibodies to whole cells, to shocked cells and to protoplasts has been investigated. The results confirm the release of phosphate-binding protein by osmotic shock and during protoplast formation. From these findings, a cell-wall localisation, near the cell surface of the phosphate-binding protein should be proposed.Abbreviations P_i inorganic phosphate - P_iBP phosphate-binding protein - Tris Tris (hydroxymethyl)-aminoethane - MES (2(N-Morpholino) ethanesulfonic acid - BSA bovine serum albumin - EDTA ethylene diamine tetraacetic acid, disodium salt - PMSF phenylmethyl sulfonyl fluoride - SDS sodium dodecyl sulfate - Fab fragments, fragment antigen binding     

Identification of a sheath-associated protein involved in phosphate transport in Sphaerotilus natans

Summary Sphaerotilus natans was shown to have a fourfold lower K _mof phosphate transport when grown in medium containing 0.1 mm phosphate, compared to cells grown in 10.0 mm phosphate. Analysis of sheath proteins from cells grown at these two phosphate levels revealed a protein of 53 kDa present in the sheath of cells grown at a phosphate concentration of 0.1 mm. This sheath-associated, phosphate-regulated protein, designated SapP, was gel purified and used to raise a polyclonal antibody. Enzyme-linked immunosorbent assay was used to localize this protein to the surface of the sheathed cells. Phosphate uptake assays done in the presence of the antibody also showed a rise in the K _mof phosphate transport in cells grown in 0.1 mm phosphate, indicating that this protein is involved in high-affinity phosphate transport.Offprint requests to: C. F. Kulpa Jr     

Mechanisms of phosphate uptake into brush-border membrane vesicles from goat jejunum

This study concerns the uptake of inorganic phosphate into brush-border membrane vesicles prepared from jejunal tissues of either control or Ca-and/or P-depleted goats. The brush-border membrane vesicles showed a time-dependent accumulation of inorganic phosphate with a typical overshoot phenomenon in the presence of an inwardly directed Na⁺ gradient. The Na⁺-dependent inorganic phosphate uptake was completely inhibited by application of 5 mmol·l^-1 sodium arsenate. Half-maximal stimulation of inorganic phosphate uptake into brush-border membrane vesicles was found with Na⁺ concentrations in the order of 5 mmol·l^-1. Inorganic phosphate accumulation was not affected by a K⁺ diffusion potential (inside negative), suggesting an electroneutral transport process. Stoichiometry suggested an interaction of two or more Na ions with one inorganic phosphate ion at pH 7.4. Na⁺-dependent inorganic phosphate uptake into jejunal brush-border membrane vesicles from normal goats as a function of inorganic phosphate concentration showed typical Michaelis-Menten kinetic with V _max=0.42±0.08 nmol·mg^-1 protein per 15 s^-1 and K _m=0.03±0.01 mmol·l^-1 (n=4, x ±SEM). Long-term P depletion had no effect on these kinetic parameters. Increased plasma calcitriol concentrations in Ca-depleted goats, however, were associated with significant increases of V _max by 35–80%, irrespective of the level of P intake. In the presence of an inwardly directed Na⁺ gradient inorganic phosphate uptake was significantly stimulated by almost 60% when the external pH was decreased to 5.4 (pH_out/pH_in=5.4/7.4). The proton gradient had no effect on inorganic phosphate uptake in absence of Na⁺. In summary, in goats Na⁺ and calcitriol-dependent mechanisms are involved in inorganic phosphate transport into jejunal brush-border membrane vesicles which can be stimulated by protons.Abbreviations AP activity of alkaline phosphatase - BBMV brush-border membrane vesicles - EGTA ethyleneglycol-triacetic acid - n _app apparent Hill coefficient - P _i inorganic phosphate - PTH parathyroid hormone     

RESPONSES OF FRESHWATER ALGAE TO INHIBITORY VANADIUM CONCENTRATIONS: THE ROLE OF PHOSPHORUS1

We found that species-specific differences exist among a variety of freshwater algae and cyanobacteria in the extent to which growth and photosynthesis are inhibited by vanadium. A major factor controlling the degree of inhibition by vanadium was the phosphorus state (P-sufficient vs. P-deficient) of the organisms. In P-sufficient cultures, vanadium was inhibitory when the vanadium concentration exceeded the phosphate concentration. In P-deficient cultures, the depression of photosynthesis by vanadium increased with increasing phosphorus deficiency. Our conclusion that vanadium competed with phosphate for uptake sites was supported by the following three observations: 1) the decreased influx of ³²P-PO ₄ into P-deficient cells in the presence of vanadium, 2) the amelioration of vanadium inhibition of photosynthesis by the addition of phosphate, and 3) the accumulation of vanadium by cells. At vanadium concentrations that severely inhibited growth, the cells of Scenedesmus obliquus (Turp.) Kruger were larger than normal and contained more vacuoles, lipid, and starch bodies than normal cells. Four-celled coenobia were replaced by unicells. Scenedesmus acutusf: alternans Hortobagyi cells from vanadium-inhibited cultures had 7.5 times more vanadium per cell than control cultures and contained numerous granules that did not stain for polyphosphate and may be composed of condensed vanadate molecules. The cellular P quota and turnover time of PO₄in the medium are important regulators of the extent of inhibition by vanadium.     

The Influence of Phosphate Uptake on Cation Uptake in Fusarium oxysporum f. sp. vasinfectum

Fusarium oxysporum grown in a low phosphate medium was found to take up several times as much K from KH₂PO₄ as from KCI solutions. Large amounts of phosphate also were taken up from KH₂PO₄. Similar large uptakes of Na and phosphate took place from solutions of NaH₂PO₄. Substantial quanties of phosphate were taken up from solutions of Ca(H₂PO₄)₂ in the absence of any appreciable Ca uptake. When the fungus was grown in a medium containing high phosphate, little or no uptake of phosphate from KH₂PO₄ solutions occured and the K Uptake was at the same level as from KCI solutions. During large phosphate uptake sizable reductions in the organic acid content of the fungal cells were observed. Much, but not all, of the data could be explained on the basis of maintenance of charge balance within the cells. – The respiratory rate of fungus, grown in a low P medium, was markedly increased in KH₂PO₄ solution. Fungus, grown in a medium with high phosphate, had a higher respiratory rate which showed only a slight response to KH₂PO₄ solution. Fungus, grown in a medium with high phosphate, had a higher respiratory rate which showed only a slight response to KH₂PO₄.     

Effect of different non-ionic surfactants on the biodegradation of PAHs by diverse aerobic bacteria

The aim of this work was to evaluate the effect of several non-ionic surfactants (Tween-80, Triton X-100 and Tergitol NP-10) on the ability of different bacteria (Enterobacter sp., Pseudomonas sp. and Stenotrophomonas sp.) to degrade polycyclic aromatic hydrocarbons (PAHs). Bacterial cultures were performed at 25 °C in an orbital shaker under dark conditions in BHB medium containing 1% of surfactant and 500 mg l⁻¹ of each PAH. Experiments performed with Tween-80 showed the highest cell density values and maximum specific growth rate because this surfactant was used as a carbon source by all bacteria. High degree of PAHs degradation (>90%) was reached in 15 days in all experiments. Toxicity increased at early times using Tween-80 but decreased to low levels in a short time after the firsts 24 h. On the other hand, Triton X-100 and Tergitol NP-10 were not biodegraded and toxicity kept constant along time. However, PAHs-degradation rate was higher, especially by the action of Enterobacter sp. with Tween-80 or Triton X-100. Control experiments performed without surfactant showed a significant decrease in biomass growth rate with a subsequent loss of biodegradation activity likely due to a reduced solubility and bioavailability of PAHs in absence of surfactant.     

Culturing of stoneworts and submersed angiosperms with phosphate uptake exclusively from an artificial sediment

1. We aimed to demonstrate reproducible nutrition and growth of macrophytes in non‐axenic laboratory cultures preventing growth of phytoplankton and epiphytes. 2. Macrophyte shoot segments were planted in a mixture of commercial acid‐washed silica sand with crystalline tricalcium phosphate, and this artificial sediment was covered with a layer of pure silica sand. The liquid mineral media used did not contain phosphorus but were rich in all other nutrient elements. A CO₂ reservoir provided sustainable CO₂ supply to macrophyte cultures by gas diffusion through a polyethylene membrane. 3. Chara hispida, Chara tomentosa, Chara baltica, Elodea canadensis, Potamogeton pectinatus and Zanichellia palustris could be cultivated for long term without medium exchange and aeration. Microalgae growth was prevented by the absence of phosphate in the water column. Mobilisation of tricalcium phosphate and phosphate uptake by the rhizoids of C. hispida enabled sustainable rapid shoot growth and increased the concentration of inorganic phosphate in the shoot dry weight by five to six times in comparison with plants cultivated on pure silica sand. A significant growth support from tricalcium phosphate was also observed for E. canadensis, but the rate of phosphate uptake by the roots was not sufficient to maintain a storage pool of inorganic phosphate (P_i) in the growing shoots of this plant. 4. Membrane‐controlled CO₂ supply from a reservoir and artificial sediments like the one described provide attractive options for the laboratory culture of macrophytes.     

CALCIFICATION BY COCCOLITHOPHORIDS: EFFECTS OF pH AND Sr1

Cells of Coccolithus huxleyi which fail to deposit CaCO₃ and form coccoliths often occur as unwanted components in cultures used for studies of calcification. Non-calcified cells generally cannot be made to recalcify, but they can be removed from cultures by treatment at elevated pH or by a method based on faster sinking of calcified cells. Lowering the concentrations of nitrate, phosphate, or trace metals in the medium did not restore calcifying ability of non-calcified cells. However, addition of strontium did promote recalcification of decalcified Cricosphaera carterae grown under calcium limitation. Strontium seemed to promote coccolith attachment to cells rather than to affect calcium uptake or coccolith formation itself.     

Kinetic Behavior of Some Polyphosphate-Accumulating Bacteria Isolates in the Presence of Nitrate and Oxygen

This paper studies the phosphate uptake by pure cultures of Aeromonas hydrophila, Klebsiella oxytoca, Agrobacterium tumefaciens, and Aquaspirillum dispar in the presence of both nitrate and oxygen. It is shown that species were able to respire both electron acceptors for phosphate accumulation. A. tumefaciens and A. dispar accumulated overall phosphate both in oxic and anoxic culture conditions, whereas A. hydrophila and K. oxytoca eliminated overall phosphate only in oxic conditions. A. dispar was able to remove phosphate by reducing oxygen and nitrate simultaneously with the production of dinitrogen gas. The anoxic denitrification observed in the cultures of adapted and nonadapted cells to nitrate showed that only A. dispar have a denitrification rate superior when the cells were adapted to nitrate. Received: 15 December 1998 / Accepted: 21 January 1999     

The Influence of Photosynthetic Factors and Metaholic Inhibitors on the Uptake of Phosphate in P-Deficient Scenedesmus

The cells used in the present investigation had a phosphate content of about 20 per cent as compared with the status in normal cultures. The uptake of phosphate during a period of 4 hours was determined at a pH of 6,5, kept constant with the aid of a citrate buffer. In the absence of CO₂, light increased the uptake of phosphate with saturation around 14,000 erg/cm²s. With 5 per cent CO₂ in the air the relationship was more complicated, and the uptake of phosphate must he related to more than one process during active photosynthesis. The inhibiting effect of CO₂ in air was noticeable already at low concentrations both in light and in darkness. With the system used, this supports earlier indications for internal recycling of orthophosphate, CO₂ was inhibiting also in nitrogen in the light. Selenate in a concentration of 2 mM gave a slight and rather irregular inhibition.—Anaerobiosis had no effect in the light but gave a large decrease in the dark.—DNP (0.1 mM) was somewhat more active in the dark than in the light. The lower concentrations tested had no effect in either case.—Menadione (0.1 mM) inhibited strongly, and more in illuminated than in non-illuminated cells.     

Nitrate uptake by bean (Phaseolus vulgaris L.) roots under phosphate deficiency

Bean (Phaseolus vulgaris L.) plants were cultured for 19 d on complete or on phosphate deficient culture media. Low inorganic phosphate concentration in the roots decreased ATP level and nitrate uptake rate. The mechanisms which may control nitrate uptake rate during phosphate deficiency were examined. Plasma membrane enriched fractions from phosphate sufficient and phosphate deficient plants were isolated and compared. The decrease in total phospholipid content was observed in plasma membranes from phosphate deficient roots, but phospholipid composition was similar. No changes in ATPase and proton pumping activities measured in isolated plasma membrane of phosphate sufficient and phosphate deficient bean roots were noted. The electron microscope observations carried out on cortical meristematic cells of the roots showed that active ATPases were found in plasma membrane of both phosphate sufficient and phosphate deficient plants. The decrease in inorganic phosphate concentration in roots led to increased nitrate accumulation in roots, accompanied by a corresponding alterations in NO₃ distribution between shoots and roots. Nitrate reductase activity in roots of phosphate deficient plants estimated in vivo and in vitro was reduced to 50–60% of the control. The increased NO₃ concentration in root tissue may be explained by decreased NR activity and lower transport of nitrate from roots to shoots. Therefore, the reduction of nitrate uptake during phosphate starvation is mainly a consequence of nitrate accumulation in the roots.     

Monoclonal antibodies directed against protoplasts of soybean cells

Splenocytes, derived from mice that had been immunized with protoplasts prepared from suspension cultures of root cells of Glycine max (L.) Merr. (SB-1 cell line), were fused with a murine myeloma cell line. The resulting hybridoma cultures were screened for the production of antibodies directed against the soybean protoplasts and were then cloned. One monoclonal antibody, designated MVS-1, was found to bind to the outer surface of the plasma membrane on the basis of several criteria: (a) agglutination of the protoplasts; (b) binding of fluorescence-labeled immunoglobulin on protoplasts yielding a ring staining pattern with prominent intensity at the edges; and (c) saturable binding by protoplasts of ¹²⁵I-labeled Antibody MVS-1. The antigenic target of Antibody MVS-1, identified by immunoblotting techniques, contained a polypeptide of relative molecular mass (M_r) approx. 400000 under both reducing and non-reducing conditions. When the antigenic target of Antibody MVS-1 was chromatographed in potassium phosphate buffer, the position of elution corresponded to that of a high-molecular-weight species (M_r 400000). These results provide the protein characterization required for the analysis of the mobility of Antibody MVS-1 bound to the plasma membrane of SB-1 cells.Abbreviations D diffusion coefficient - M_r relative molecular mass - PBS phosphate-buffered saline (8.00 g NaCl, 1.15 g Na₂HPO₄, 0.20 g NaH₂PO₄ per 1 L, pH 7.2) - TPBS phosphate-buffered saline containing 0.5% Tween-20 - TX-100, TX-114 Triton X-100, X-114 - SDS sodium dodecyl sulfate     

Immunological evidence for the involvement of cell wall proteins in phosphate uptake in the yeast Saccharomyces cerevisiae

Immunological cross-reactivity between cell wall proteins obtained from two yeast genera (Candida tropicalis and Saccharomyces cerevisiae) is reported. Specific retention of two cell wall proteins from Saccharomyces cerevisiae by an immunoabsorbent column coupled with antibodies against phosphate binding protein 2 (PiBP2) from Candida tropicalis allowed to generate antibodies against the proteins from S. cerevisiae. These antibodies were effective in inhibiting phosphate uptake by S. cerevisiae cells. The proteins from S. cerevisiae displayed a phosphate binding activity which was inhibited in the presence of the forementioned antibodies. These results and the observation that the amount of these proteins in the shock fluid was dependent of the growth conditions (i.e., in the presence or in the absence of phosphate) support the idea that these proteins are involved in the high affinity phosphate transport system.Abbreviations Pi inorganic phosphate - PiBP2 phosphate binding protein 2 obtained from Candida tropicalis - Tris Tris(hydroxymethyl)-aminoethane - MES [2-(N-Morpholino)] ethanesulfonic acid - EDTA ethylene diamine tetraacetic acid, disoldium salt - PMSF phenylmethyl sulfonyl fluoride - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis