External Orthophosphate Research Articles

Studies of the microbial P-cycle during a Lagrangian phosphate-addition experiment in the Eastern Mediterranean

Microbial uptake of orthophosphate was studied before and during a Lagrangian experiment where orthophosphate was added to the surface mixed layer in the Cyprus Gyre, Eastern Mediterranean, a region previously hypothesized to be characterized by P-limited growth of both phytoplankton and heterotrophic bacteria. The addition of ca. 110 nM orthophosphate to a ca. 16 km2 patch in situ led, within 1 day, to an increase in particulate-P from 8 to ca. 15 nM, a result in good agreement with a previous microcosm bioassay indicating this system to have a maximum capacity for orthophosphate consumption of between 10 and 25 nM phosphate. In samples of unperturbed water taken before the addition, outside, or below the experimental patch, orthophosphate turnover time (Tt) was <4 h, argued to be consistent with the assumption of diffusion-limited phytoplankton growth. Upon addition, Tt increased to 94 h. Estimates of maximum potential uptake rate (Vmax) for orthophosphate in unperturbed water exceeded by more than one order of magnitude the biological P-requirement (ν) as obtained from stoichiometric conversion of C-based primary and bacterial production values to estimated P-requirement. Upon addition of orthophosphate, Vmax decreased to a level comparable to ν. The observations are consistent with the assumption of P-starved cells before and P-replete cells with excess external orthophosphate after the addition. Orthophosphate uptake in unperturbed water was dominated by<1 μm organisms (mean ±SD between samples 0.56±0.03 μm). In samples with higher turnover time, orthophosphate uptake was shifted towards larger organisms, culminating after 5 days with a near doubling in mean size (1.08 μm). The size distribution of particulate-P standing stock had a mean size of 10 μm, indicating the presence of a substantial biomass of micro-organisms larger than those involved in P-uptake. Comparison of the measured particulate-P with microscope-based biomass estimates indicated a microbial food web dominated by heterotrophic organisms (70% of particulate-P), distributed with ca. 25% of total particulate-P in heterotrophic bacteria, ca. 40% in heterotrophic flagellates, and ca. 5% in ciliates. Concentration of bioavailable phosphate (Sn) estimated from the relationship Sn=νTi indicated Sn values<1 nM PO4 before the addition, increasing afterwards. Estimates of the sum Kt+Sn for the 0.6–0.2 μm size fraction were in the range 1–7 nM PO4 before and outside patch, suggesting this sum to be dominated by the half-saturation constant Kt. Kt+Sn increased to 69 nM after addition, then dropped over the following week back to background levels. As reported elsewhere in this volume, there was a decline in the observed chlorophyll concentrations, but a positive response in copepods. Less clear than the effects at the level of osmotroph physiology were the subsequent responses expected in the food web. Two possible mechanisms are discussed: (1) a positive response in bacterial production and the subsequent food chain of bacterial predators, and (2) a positive response in phytoplankton predators due to a shift in food quality rather than in food quantity.

Low Affinity Orthophosphate Carriers Regulate PHO Gene Expression Independently of Internal Orthophosphate Concentration in Saccharomyces cerevisiae

Phosphate is an essential nutrient that must be taken up from the growth medium through specific transporters. In Saccharomyces cerevisiae, both high and low affinity orthophosphate carriers allow this micro-organism to cope with environmental variations. Intriguingly, in this study we found a tight correlation between selenite resistance and expression of the high affinity orthophosphate carrier Pho84p. Our work further revealed that mutations in the low affinity orthophosphate carrier genes (PHO87, PHO90, and PHO91) cause deregulation of phosphate-repressed genes. Strikingly, the deregulation due to pho87Delta, pho90Delta, or pho91Delta mutations was neither correlated to impaired orthophosphate uptake capacity nor to a decrease of the intracellular orthophosphate or polyphosphate pools, as shown by (31)P NMR spectroscopy. Thus, our data clearly establish that the low affinity orthophosphate carriers affect phosphate regulation independently of intracellular orthophosphate concentration through a new signaling pathway that was found to strictly require the cyclin-dependent kinase inhibitor Pho81p. We propose that phosphate-regulated gene expression is under the control of two different regulatory signals as follows: the sensing of internal orthophosphate by a yet unidentified protein and the sensing of external orthophosphate by low affinity orthophosphate transporters; the former would be required to maintain phosphate homeostasis, and the latter would keep the cell informed on the medium phosphate richness.

Polyphosphate accumulation by Rhodobacter sphaeroides grown under different environmental conditions with special emphasis on the effect of external phosphate concentrations.

Rhodobacter sphaeroides NR3 produced significant amounts of inorganic polyphosphates (polyPs) with visible volutin granules when grown phototrophically at external orthophosphate (Pi) concentrations of 10 mM and above with 20 mM malate as the sole carbon source. The maximum level of the accumulated polyPs, amounting to 6.2% of the cell dry weight, was found with 300 mM Pi. On the other hand, the stimulatory effect of excess Pi on polyP accumulation was not so pronounced in the cells grown under aerobic-dark conditions. The fractionation of the cellular phosphorous compounds revealed that the alkali-soluble and cold acid-soluble polyPs were the major polyPs in the high Pi-loaded phototrophic cultures. The phosphorus/carbon ratio in cell growth media was also found as an important factor affecting polyP accumulation in the bacterium. There was an antagonistic relationship between the polyP content and the RNA/DNA ratio during batch phototrophic growth. Ecophysiological implications of polyP accumulation in the phototrophic bacterium were also discussed.

Dependence of the Calvin cycle activity on kinetic parameters for the interaction of non-equilibrium cycle enzymes with their substrates.

Kinetic model studies and control analyses of the Calvin photosynthesis cycle have been performed to characterize the dependence of the cycle activity on maximum velocities and Km values for the interaction of the non-equilibrium cycle enzymes and ATP synthetase with their substrates under conditions of light and carbon dioxide saturation. The results show that Km values have no major influence on the cycle activity at optimal concentrations of external orthophosphate. The maximum cycle activity is controlled mainly by the catalytic capacities of ATP synthetase and sedoheptulose-bisphosphatase, and is close to the maximum cycle flux that can be supported by these two enzymes.

Phosphorus–Limited Growth Dynamics of Lotic Periphytic Diatom Communities: Areal Biomass and Cellular Growth Rate Responses

Three long-term phosphate enrichment experiments were conducted at the Experimental Troughs Apparatus (EXTRA), South Thompson River British Columbia to determine the relationship between external orthophosphate (PO43−) concentration and peak areal biomass (PB) of periphytic diatom communities. Levels of PO43−which saturated PB were two orders of magnitude greater than those required to saturate specific growth rates in thin film periphyton communities of similar taxonomic composition. With PO43−additions between 0.1 to 1.0 μg P∙L−1, PB responded in a hyperbolic fashion, initially increasing rapidly, then showing signs of saturation PB continued to increase in a slow, linear manner above 1.0 μg P∙L−1. Maximum PB (PBmax) was calculated to occur at ca. 28 μg P∙L−1. At higher PO43−concentrations (&gt; 30–50 μg P∙L−1) PB was no longer P limited. Below the saturation point, PB was approximated by a log–linear function of PO43−.

On the regulatory significance of inhibitors acting on non-equilibrium enzymes in the Calvin photosynthesis cycle

Control analyses and kinetic model studies have been performed in order to obtain quantitative information on the regulatory significance of 12 experimentally well-documented inhibitory interactions of Calvin cycle intermediates with the four non-equilibrium cycle enzymes. Evidence is presented to show that none of these interactions contributes significantly to the cycle flux control over the range of external orthophosphate concentrations where the reaction cycle shows close to optimal activity. Contrary to what has been generally supposed, the examined inhibitions appear to be of little interest for our understanding of the biological regulation of the Calvin photosynthesis cycle under conditions of light and carbon dioxide saturation.

Influence of external orthophosphate concentrations on some kinetic properties of activated sludge in an anaerobic-aerobic system

The addition of high levels of orthophosphate (P i) to the mixed liquor of activated sludge in a laboratory-scale batch anaerobic-aerobic system inhibited organic carbon uptake during the anaerobic phase. The degree of inhibition reached the maximum with P i at a concentration of 3 mM and above. When the external P i concentration was low, anaerobiosis induced the simultaneous occurrence of an increase of effluent P i and a decrease of the total phosphorus content of sludge, as usual. The decrease in the phosphorus level of sludge was due mainly to polyphosphate (polyP) degradation. The initial presence of 3 mM P i, however, suppressed both anaerobic P i release and polyP degradation. These results suggest that an excess of external P i inhibits polyP degradation and the resultant energy generation in sludge under anaerobic conditions, whereby anaerobic organic matter removal may be suppressed. The long-term operation of the anaerobic-aerobic system at high P i-loading rates resulted in producing sludge incapable of anaerobic P i release and aerobic enhanced P i removal.

A mathematical model of the Calvin photosynthesis cycle.

1. A mathematical model is presented for photosynthetic carbohydrate formation in C3 plants under conditions of light and carbon dioxide saturation. The model considers reactions of the Calvin cycle with triose phosphate export and starch production as main output processes, and treats concentrations of NADPH, NAD+, CO2, and H+ as fixed parameters of the system. Using equilibrium approximations for all reaction steps close to equilibrium steady-state and transient-state relationships are derived which may be used for calculation of reaction fluxes and concentrations of the 13 carbohydrate cycle intermediates, glucose 6-phosphate, glucose 1-phosphate, ATP, ADP, and inorganic (ortho)phosphate. 2. Predictions of the model were examined with the assumption that photosynthate export from the chloroplast occurs to a medium containing orthophosphate as the only exchangeable metabolite. The results indicate that the Calvin cycle may operate in a single dynamically stable steady state when the external concentration of orthophosphate does not exceed 1.9 mM. At higher concentrations of the external metabolite, the reaction system exhibits overload breakdown; the excessive rate of photosynthate export deprives the system of cycle intermediates such that the cycle activity progressively approaches zero. 3. Reactant concentrations calculated for the stable steady state that may obtain are in satisfactory agreement with those observed experimentally, and the model accounts with surprising accuracy for experimentally observed effects of external orthophosphate on the steady-state cycle activity and rate of starch production. 4. Control analyses are reported which show that most of the non-equilibrium enzymes in the system have a strong regulatory influence on the steady-state level of all of the cycle intermediates. Substrate concentration control coefficients for cycle enzymes may be positive, such that an increase in activity of an enzyme may raise the steady-state concentration of the substrate is consumes. 5. Under optimal external conditions (0.15-0.5 mM orthophosphate), reaction flux in the Calvin cycle is controlled mainly by ATP synthetase and sedoheptulose bisphosphatase; the cycle activity approaches the maximum velocity that can be supported by the latter enzyme. At lower concentrations of external orthophosphate the cycle activity is controlled almost exclusively by the phosphate translocator.(ABSTRACT TRUNCATED AT 400 WORDS)

A rapid-equilibrium model for the control of the Calvin photosynthesis cycle by cytosolic orthophosphate.

1. A simple model based on rapid-equilibrium assumptions is derived which relates the steady-state activity of the Calvin cycle for photosynthetic carbohydrate formation in C3 plants to the kinetic properties of a single cycle enzyme (fructose bisphosphatase) and of the phosphate translocator which accounts for the export of photosynthate from the chloroplast. Depending on the kinetic interplay of these two catalysts, the model system may exhibit a single or two distinct modes of steady-state operation, or may be unable to reach a steady state. 2. The predictions of the model are analysed with regard to the effect of external orthophosphate on the steady-state rate of photosynthesis in isolated chloroplasts under conditions of saturating light and CO2. Due to the possible existence of two distinct steady states, the model may account for the stimulatory as well as the inhibitory effects of external phosphate observed in experiments with intact chloroplasts. Stability arguments indicate, however, that only the steady-state case corresponding to phosphate inhibition of the rate of photosynthesis could be of physiological interest. 3. It is concluded that chloroplasts under physiological conditions most likely operate in a high-velocity steady state characterized by a negative Calvin cycle flux control coefficient for the phosphate translocator. This means that any factor enhancing the export capacity of the phosphate translocator can be anticipated to decrease the actual steady-state rate of photosynthate export due to a decreased steady-state rate of cyclic photosynthate production.

CHARACTERIZATION OF THE DIFFERENT FORMS OF PHOSPHORUS IN THE MYCELIUM OF THE ECTOMYCORRHIZAL FUNGUS, HEBELOMA CYLINDROSPORUM, CULTIVATED IN PURE CULTURE

SummaryThe ectomycorrhizal fungus, Hebeloma cylindrosporum, was grown in pure culture at different phosphorus concentrations, with or without shaking. Phosphorus fractions were separated by a modified Mudd, Yoshida &amp; Koite (1958) method. Acid‐soluble and acid‐insoluble phosphorus precipitated with barium acetate were identified by thin layer chromatography.In pure culture. Hebeloma cylindrosporum stores very little polyphosphate (4 to 9% of total P) and amounts are unrelated to the concentration of external phosphorus. Most of the phosphorus is stored as orthophosphate. Internal orthophosphate is well correlated with external orthophosphate and is readily utilized to form DNA and phospholipid. Internal soluble organic phosphorus can also be mobilized but to a lesser extent.

PHOSPHORUS UTILIZATION AND STORAGE IN BATCH CULTURES OF THE DINOFLAGELLATE PERIDINIUM CINCTUM F. WESTII1

ABSTRACT Peridinium cinctum f. westii (Lemm.) Lef., a dominant species in the phytoplankton of Lake Kinneret was studied in batch cultures. External orthophosphate concentrations were depleted by successive transfers to increasingly lower initial orthophosphate concentrations (P0) of 200, 50, 5, 0.6 and 0.2 μM (culture cycles of 45–64 days).Lower cell yields were obtained as P0 decreased, but growth rates were unaffected. The decrease in an intra‐cellular P pool unextractable by cold 6% trichloroacetic acid (TCA) was shown to be responsible for the effect on cell yields. A 31P nuclear magnetic resonance (NMR) study revealed that the major constituents of this internal phosphorus pool in phosphate‐rich cells were the polyphosphates, organized in rigid aggregates of low mobility. Electron‐dense bodies which were observed in phosphate‐enriched P. cinctum cells further supported the existence of an aggregate structure of the polyphosphates. It is concluded that P. cinctum stores excess orthophosphate as polyphosphates which accumulate in a rigid morphological structure.

Regulation of Phosphate Accumulation in the Unicellular CyanobacteriumSynechococcus

The phosphorus contents of acid-soluble pools, lipid, ribonucleic acid, and acid-insoluble polyphosphate were lowered in Synechococcus in proportion to the reduction in growth rate in phosphate-limited but not in nitrate-limited continuous culture. Phosphorus in these cell fractions was lost proportionately during progressive phosphate starvation of batch cultures. Acid-insoluble polyphosphate was always present in all cultural conditions to about 10% of total cell phosphorus and did not turn over during balanced exponential growth. Extensive polyphosphate formation occurred transiently when phosphate was given to cells which had been phosphate limited. This material was broken down after 8 h even in the presence of excess external orthophosphate, and its phosphorus was transferred into other cell fractions, notably ribonucleic acid. Phosphate uptake kinetics indicated an invariant apparent K(m) of about 0.5 muM, but V(max) was 40 to 50 times greater in cells from phosphate-limited cultures than in cells from nitrate-limited or balanced batch cultures. Over 90% of the phosphate taken up within the first 30 s at 15 degrees C was recovered as orthophosphate. The uptake process is highly specific, since neither phosphate entry nor growth was affected by a 100-fold excess of arsenate. The activity of polyphosphate synthetase in cell extracts increased at least 20-fold during phosphate starvation or in phosphate-restricted growth, but polyphosphatase activity was little changed by different growth conditions. The findings suggest that derepression of the phosphate transport and polyphosphate-synthesizing systems as well as alkaline phosphatase occurs in phosphate shortage, but that the breakdown of polyphosphate in this organism is regulated by modulation of existing enzyme activity.

Isolation of Protoplasts and Chloroplasts from Flag Leaves of Triticum aestivum L

Intact protoplasts and chloroplasts have been isolated from mature flag leaves of wheat (Triticum aestivum L.). Both showed high rates of photosynthesis, the best of which equaled those observed in the parent tissue (greater than 150 micromoles O(2) per milligram chlorophyll per hour). The presence of ethylenediaminetetraacetate and an alkaline medium (pH 8.4) were required in the isolation and assay for the achievement of maximum rates of photosynthesis by chloroplasts. Photosynthesis by isolated chloroplasts was inhibited at very low concentrations of external orthophosphate.

The influx of orthophosphate into squid giant axons.

1. The influx of (32)P, applied externally as orthophosphate, into the axoplasm of squid giant axons has been studied.2. An average orthophosphate influx of 20.9 f-mole/cm(2).sec is obtained if the (32)P found in the axoplasm is assumed to be indicative of orthophosphate which has crossed the axolemma.3. The influx does not show very much dependence on external orthophosphate concentration in the range 0.02-0.5 mM.4. The influx is reduced by cyanide, 2,4-dinitrophenol, ouabain and by the absence of external potassium.5. The (32)P appears to arrive in the axoplasm as orthophosphate.6. It is concluded that there is an inward movement of orthophosphate into the axons which is mediated by an active transport process and that this may have some connexion with the active transport of sodium and potassium.

The incorporation of radioactive phosphorus in the leucocyte during the extrusion of protein induced by staphylococcal leucocidin.

Incubation with leukocidin stimulates the incorporation of P/sup 32/ into the adenosine tri- and di-phosphate and guanosine tri- and di-phosphate of the leukocyte. Incubation with leukocidin does not increase the permeability of the leukocyte to orthophosphate and in the presence of P/sup 32/ does not increase the radioactivity of the intracellular orthophosphate. In the presence of fluoride, iodoacetate, or arsenate the incorporation of P/sup 32/ into the lipids and nucleotides of the leukocidin-treated leukocyte is partially inhibited. The extrusion of BETA -glucuronidase is not affected under these conditions. Omission of calcium from the medium stimulates the incorporation of P/sup 32/ into the lipids of the leukocidin-treated leukocyte and reduces the amount of incorporation into the nucleotides. Either component of leukocidin adsorbed on the leukocyte can be neutralized by antibody. Addition of antibody to the leukocidin-treated leukocyte does not inhibit the incorporation of P/sup 32/ into the nucleotides. Treatment with leukocidin decreases the rate of loss of radioactivity from the nucleotides of leukocytes labeled with P/sup 32/ and suspended in a medium of low specific radioactivity. Treatment with leukocidin reduces the amount of STAC/sup 14/!adenine incorporated into adenosine triphosphate and the amount of STAC/sup 14/!acetate incorporated into the lipids of themore » cell. It is suggested that the stimulation of incorporation of P/sup 32/ in the leukocidin-treated leukocyte does not result from- an increased rate of turnover of any phosphorus compound of the cell but can result from direct utilization at the cell surface of the external orthophosphate. (auth)« less