Fractionation of sediment phosphate with chelating compounds: a simplification, and comparison with other methods

The NTA/EDTA fractionation of sediment-bound, inorganic phosphate was improved and made more effective. The iron-bound phosphate is now extracted with Ca-EDTA plus dithionite and followed by an extraction of calcium-bound phosphate by Nat-EDTA at pH = 4.5. A comparison is made with two other sequential extraction procedures: the SEDEX and the Hieltjes & Lijklema extractions. The EDTA extractions have an advantage over the other two methods.     

Interactions between respiration and inorganic phosphate uptake in phosphate-limited cells of Chlamydomonas reinhardtii

Phosphate addition to P-limited cells of Chlamydomonas reinhardtii resulted in an immediate increase in the rate of respiratory O₂ consumption. The respiration rate continued to increase for several minutes after the addition of P₁. Similar patterns of P₁ stimulation of respiratory O₂ consumption were observed in the presence of cyanide (cytochrome oxidase inhibitor) and propyl gallate (alternative oxidase inhibitor). Stimulation of O₂ consumption was accompanied by rapid changes in levels of glycolytic intermediates. These changes were consistent with activation of ATP-dependent phosphofructokinase and pyruvate kinase. The adenylate pool exhibited only minor perturbations, P₁, uptake resulted in extracellular acidification, which continued for several minutes after the exhaustion of added P₁, whereas exhaustion of extracellular P₁ resulted in a rapid decline in the O₂ consumption rate. These results are consistent with control of respiration in P-limited cells occurring largely at the level of glycolysis.     

Methods and applications to control the uptake rate of carbon

Methods to control carbon and nutrient uptake at different availability of carbon were tested on plants of birch (Betula pendula Roth.) and tomato (Lycopersicon esculentum Mill. cv. Solentos). The present paper accounts for the methods and the possibility to maintain steady-state, i.e., a long-term and stable physiological state of acclimated plants. Steady-state comprises, by definition, equality between constant relative growth rates, and relative uptake rates of carbon and nutrients. Two methods were tested. The first, not previously applied, method (a), was based on a constant relative addition rate of carbon, R_AC. In the second method (b), a constant concentration of CO₂ in the air, c_a, was used to attain non-limiting conditions. The methods are analogous to those used by us to control plant nutrition, and the generality of fluxes to quantify supply as well as uptake and growth was verified. Thus, different R_AC resulted in clear-cut responses, from strong reduction to non-limitation of uptake and growth, whereas different c_a levels in the range 100 to 700 ppm had comparatively small effects, with an unclear causality. Non-limiting conditions were achieved at c_a≥ 200 ppm. Effects reported in the literature have been based upon the control of c_a, similarly to method (b), whereas results comparable to those obtained with method (a) are lacking. Transpiration rate increased rapidly at c_a < 200 ppm CO₂, and at low R_AC levels, ≤ 0.1 day^?1, wilting tendencies were observed. Elevated c_a, 500 or 700 ppm, did not increase the relative growth rate (R_G) but reduced transpiration and increased both nitrogen productivity (growth rate per unit of nitrogen in the plant) and transpiration productivity (growth rate per unit of water transpired by the plant). Obviously, effects of c_a may be due to changed transpiration rate rather than to changed quantitative availability of CO₂. Relative uptake (R_UC) and growth (R_G) rates were closely equal to the R_AC applied (R_AC? R_UC? R_G); i.e., the purely mathematical conditions defining steady-state were fulfilled. This unambiguous and straightforward test of reliability confirms that experimental artefacts did not produce uncontrolled or unintended effects, so that the new technique allows an accurate control of CO₂ uptake and plant growth. The results add to previous databases and reference systems, where limiting conditions grade and classify plant performance as deviations from maximum growth. Evidently, methodology in experimentation and in evaluation of plant responses, can be based upon unifying concepts and general theories.     

Genotypic variation of rice in phosphorus acquisition from iron phosphate: Contributions of root morphology and phosphorus uptake kinetics

To elucidate the contributions of rice root morphology and phosphorus uptake kinetics to P uptake by rice from iron phosphate, a sand culture experiment with either sufficient P supply (control treatment, 10 mg P/l as NaH₂PO₄) or Fe-P as the only source of P (40 mg P/pot as FePO₄ × 4H₂O) and a solution culture experiment supplied with either sufficient P (10 mg P/l) or deficient P (0.5 mg P/l) were conducted. Eight rice cultivars, which differed in P uptake from Fe-P, were investigated. Plant P uptake, root morphology, and P uptake kinetics were determined. There were significant (P < 0.05) genotypic variations in both plant dry weight and P uptake per plant among eight rice (Oryza sativa L.) cultivars when supplied with Fe-P as the P source. The Fe-P treatment significantly (P < 0.05) decreased plant dry weight, P uptake per plant, and P concentration in plant dry matter of all cultivars in comparison with the control plants. In Fe-P treated plants, significant (P < 0.05) genotypic variation was shown in root morphology, including root length, surface area, volume, and number of lateral roots. The P uptake per plant from Fe-P by rice was significantly (P < 0.05) correlated with root surface area and root volume as well as with the number of lateral roots, suggesting that the ability of rice to absorb P from Fe-P was closely related to root morphology. Low P supply in solution significantly increased the I _max (P < 0.05), but significantly decreased the K _M (P < 0.05) for P absorption by all rice cultivars. We supposed that kinetic characteristics of root P uptake could not account for the ability of rice to absorb P from Fe-P. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 2, pp. 260–266. The text was submitted by the authors in English.     

Enzymatic and structural characterization of an archaeal thiamin phosphate synthase

Studies on thiamin biosynthesis have so far been achieved in eubacteria, yeast and plants, in which the thiamin structure is formed as thiamin phosphate from a thiazole and a pyrimidine moiety. This condensation reaction is catalyzed by thiamin phosphate synthase, which is encoded by the thiE gene or its orthologs. On the other hand, most archaea do not seem to have the thiE gene, but instead their thiD gene, coding for a 2-methyl-4-amino-5-hydroxymethylpyrimidine (HMP) kinase/HMP phosphate kinase, possesses an additional C-terminal domain designated thiN. These two proteins, ThiE and ThiN, do not share sequence similarity. In this study, using recombinant protein from the hyperthermophile archaea Pyrobaculum calidifontis, we demonstrated that the ThiN protein is an analog of the ThiE protein, catalyzing the formation of thiamin phosphate with the release of inorganic pyrophosphate from HMP pyrophosphate and 4-methyl-5-β-hydroxyethylthiazole phosphate (HET-P). In addition, we found that the ThiN protein can liberate an inorganic pyrophosphate from HMP pyrophosphate in the absence of HET-P. A structure model of the enzyme–product complex of P. calidifontis ThiN domain was proposed on the basis of the known three-dimensional structure of the ortholog of Pyrococcus furiosus. The significance of Arg320 and His341 residues for thiN-coded thiamin phosphate synthase activity was confirmed by site-directed mutagenesis. This is the first report of the experimental analysis of an archaeal thiamin synthesis enzyme.     

Growth and uptake kinetics of phosphate by benthic microalga Nitzschia sp. isolated from Hiroshima Bay,Japan

In the present study, we experimentally investigated the phosphate uptake kinetics of benthic microalga Nitzschia sp. isolated from Hiroshima Bay, Japan. The maximum uptake rate (ρ_max) obtained by short‐term experiments was 6.84 pmol cell^?1 h^?1 for phosphate. The half‐saturation constant for uptake (K_S) was 61.2 µmol cell^?1 h^?1. Both the ρ_max and Ks of this species were extremely high, suggesting that Nitzschia sp. is adapted to benthic environments, where nutrient concentrations are much higher than in the water column. The specific maximum growth rate (µ'_max) and minimum cell quota (Q₀) for the P‐limited condition, obtained by a semi‐continuous growth experiment, were 0.48 day^?1 and 0.045 pmol cell^?1, respectively. It is concluded that Nitzschia sp. could be a ‘storage strategist’ species, meaning it adapts so as to minimize the influence of fluctuations in phosphate conditions resulting from the change in redox conditions of sediment due to bioturbation.     

Growth and phosphate uptake kinetics of the toxic dinoflagellate Alexandrium tamarense from Hiroshima Bay in the Seto Inland Sea, Japan

Shellfish poisoning by the toxic dinoflagellate Alexandrium tamarense (Lebour) Balech occurred for the first time in Hiroshima Bay, Japan, in 1992. Oyster culture in the bay produces as much as 60% of the total production in Japan, and it suffered severe damage. In the present study, we experimentally investigated the growth rate and phosphate uptake kinetics of A. tamarense, Hiroshima Bay strain. A short-term phosphate uptake experiment revealed that the maximum uptake rate was 1.4 pmol P cell^-1 per h and the half-saturation constant was 2.6 umol L^-1. In semicontin-uous culture, the maximum specific growth rate and the minimum phosphorus cell quota were 0.54 day^-1 and 0.56 pmol P cell^-1, respectively. These uptake rates suggest that A. tamarense is a poor phosphorus competitor compared with other species. However, the large phosphorus storage capacity (Q_pmax/q_o= 36), the surge phosphorus uptake ability (V_s/V_i= 4.1) and the low growth rate would be advantageous for surviving brief periods of phosphorus limitation which frequently occur in Hiroshima Bay.     

Inorganic phosphate uptake in Trypanosoma cruzi is coupled to K cycling and to active Na extrusion

Background

Orthophosphate (P_i) is a central compound in the metabolism of all organisms, including parasites. There are no reports regarding the mechanisms of P_i acquisition by Trypanosoma cruzi.

Methods

³²P_i influx was measured in T. cruzi epimastigotes. The expression of P_i transporter genes and the coupling of the uptake to Na⁺, H⁺ and K⁺ fluxes were also investigated. The transport capacities of different evolutive forms were compared.

Results

Epimastigotes grew significantly more slowly in 2 mM than in 50 mM P_i. Influx of P_i into parasites grown under low P_i conditions took place in the absence and presence of Na⁺. We found that the parasites express TcPho84, a H⁺:P_i-symporter, and TcPho89, a Na⁺:P_i-symporter. Both P_i influx mechanisms showed Michaelis–Menten kinetics, with a one-order of magnitude higher affinity for the Na⁺-dependent system. Collapsing the membrane potential with carbonylcyanide-p-trifluoromethoxyphenylhydrazone strongly impaired the influx of P_i. Valinomycin (K⁺ ionophore) or SCH28028 (inhibitor of (H⁺ + K⁺)ATPase) significantly inhibited P_i uptake, indicating that an inwardly-directed H⁺ gradient energizes uphill P_i entry and that K⁺ recycling plays a key role in P_i influx. Furosemide, an inhibitor of the ouabain-insensitive Na⁺-ATPase, decreased only the Na⁺-dependent P_i uptake, indicating that this Na⁺ pump generates the Na⁺ gradient utilized by the symporter. Trypomastigote forms take up P_i inefficiently.

Conclusions

P_i starvation stimulates membrane potential-sensitive P_i uptake through different pathways coupled to Na⁺ or H⁺/K⁺ fluxes.

General significance

This study unravels the mechanisms of P_i acquisition by T. cruzi, a key process in epimastigote development and differentiation to trypomastigote forms.     

Present and potential uses of the natural abundance of stable isotopes in plant science, with illustrations from the marine environment

The use of stable isotope natural abundance measurements in plant ecophysiological research is discussed in the context of studies of ¹³C/¹²C ratios in marine plants, with emphasis on the uniqueness of the information given by natural abundance measurements and of the importance of complementary data obtained by other techniques in making full use of the natural abundance data. (1) Inorganic C acquisition and assimilation in marine plants can involve diffusive entry of CO₂, or the occurrence of a CO₂-concentrating mechanism frequently involving active HCO₃^? influx. For diffusive CO₂ entry, the δ¹³C measurements can give unique information on the fractional limitation of photosynthesis by CO₂ transport which, with photosynthetic rate measurements, can be used to compute transport conductances. For active HCO₃^?, influx, the δ¹³C values uniquely permit computation of the ratio of the bidirection fluxes (influx/efflux) which, with photon yield data, can be used to given information on the mechanism of the efflux. The analyses are absolutely dependent on external (non-δ¹³C) data distinguishing between diffusive CO₂ entry and the occurrence of a CO₂ concentrating mechanism. (2) δ¹³C measurements on marine photolithotrophs and on members of other trophic levels collected from the sea can give unique data on food webs, with measurements of δ values for other isotopes and compositional data adding precision to the interpretations. (3) Measurements of in situδ¹³C values for extant marine photolithotrophs, compared with δ¹³C values for ancient atmospheric CO₂, can give unique information on the mechanism of atmospheric CO₂ draw-down at the start of glacials; other information permits more concrete conclusions to be drawn.     

Photophosphorylation in Scenedesmus in vivo: O2 evolution, ATP pools and transients, and phosphate binding during photoreduction of NO3−, NO2− and CO2

The relation between light-induced electron transport with NO₃^?, NO₂^? or CO₂ as acceptors, ATP pools and transients in dark-light-dark transitions, and phosphate uptake was examined in phosphorus-starved cells of Scenedesmus obtusiusculus Chod. Net O₂ evolution at saturating light was around 6 μmol × (mg chlorophyll × h)^?1 in the absence of any acceptor, but reached average rates of 21, 65 and 145 μmol × (mg chlorophyll × h)^?1 upon additions of 5 mM KNO₃, KNO₂ and KHCO₃, respectively. The apparent rate of photophosphorylation in transition experiments was only a few percent of the rate calculated from CO₂-dependent O₂ evolution. Blocking non-cyclic electron transport with DCMU inhibited phosphate assimilation, but acceleration of non-cyclic electron flow by addition of NO₃^? or NO₂^? did not stimulate phosphate assimilation as compared to the situation without an acceptor. A functional non-cyclic system might primarily be needed for an efficient shuttle transfer of ATP from the chloroplast to the cytoplasm. An inhibition of the non-cyclic system due to lack of reducible substrates accelerates the cyclic system and thus indicates a regulation mechanism between the two systems.     

Cloning and functional characterization of an enzyme from Helicobacter pylori that catalyzes two steps of the methylerythritol phosphate pathway for isoprenoid biosynthesis

Background

The methylerythritol phosphate pathway for isoprenoid biosynthesis is an attractive target for the design of new specific antibiotics for the treatment of gastrointestinal diseases associated with the presence of the bacterium Helicobacter pylori since this pathway which is essential to the bacterium is absent in humans.

Results

This work reports the molecular cloning of one of the genes of the methylerythritol phosphate pathway form H. pylori (ispDF; HP_1440) its expression in Escherichia coli and the functional characterization of the recombinant enzyme. As shown by genetic complementation and in vitro functional assays the product of the ispDF gene form H. pylori is a bifunctional enzyme which can replace both CDP-methylerythritol synthase and methylerythritol cyclodiphosphate synthase from E. coli.

General significance

Designing inhibitors that affect at the same time both enzyme activities of the H. pylori bifunctional enzyme (i.e. by disrupting protein oligomerization) would result in more effective antibiotics which would be able to continue their action even if the bacterium acquired a resistance to another antibiotic directed against one of the individual activities.

Conclusion

The bifunctional enzyme would be an excellent target for the design of new, selective antibiotics for the treatment of H. pylori associated diseases.     

A study of the growth condition and solubilization of phosphate from hydroxyapatite byPantoea agglomerans

The growth conditions ofPantoea agglomerans, a phosphate solubilizing organism, were studied in our laboratory to determine the optimal conditions.Pantoea agglomerans showed the highest growth rate at 30°C, pH 7.0 and 2 vvm, after 50 h cultivation. A certain relationship between pH and phosphate concentration, was evident when the glucose concentration in the medium was changed. Increasing glucose concentration increased the pH buffer action of the broth. At glucose concentrations higher than the optimum concentration of 0.2 M, the cell growth was retarded.P. agglomerans consumed glucose as a substrate to produce organic acids which caused the pH decrease in the culture medium. The phosphate concentration in the medium was increased by the presence of the organic acids, which solubilized insoluble phosphates such as hydroxyapatite.     

Phosphate-solubilizing microorganisms isolated from rhizospheric and bulk soils of colonizer plants at an abandoned rock phosphate mine

The abandoned “Monte-Fresco” rock phosphate mine in Táchira, Venezuela, was sampled to study the biodiversity of phosphate-solubilizing microorganisms (PSM). Rhizosphere and bulk soils were sampled from colonizer plant species growing at a mined site where pH and soluble P were higher than the values found at a near by unmined and shrubby soil. Counting and isolating of PSM choosing strains showing high solubilization halos in a solid minimal medium with hydroxyapatite as phosphate source were evaluated using ammonia or nitrate as nitrogen sources and dextrose, sucrose, and mannitol as carbohydrate sources. A larger number of PSM were found in the rhizospheric than in the bulk soil. Six fungal strains belonging to the genus Penicillium and with high hydroxyapatite dissolution capacities were isolated from bulk soil of colonizer plants. Five of these strains had similar phenotypes to Penicillium rugulosum IR-94MF1 but they solubilized hydroxyapatite at different degrees with both nitrogen sources. From 15 strains of Gram-negative bacteria isolated from the rhizosphere of colonizer plants, 5 were identified as diazotrophic free-living encapsulated Azotobacter species able to use ammonium and/or nitrate to dissolve hydroxyapatite with glucose, sucrose and/or mannitol. Different nitrogen and carbohydrate sources are parameters to be considered to further characterize the diversity of PSM.     

Synthesis of 11-phenoxyundecyl phosphate and its use as a substrate-acceptor in the reaction with UDP-GlcNAc: polyprenyl phosphate GlcNAc-phosphotransferase from <Emphasis Type="Italic">Salmonella arizona</Emphasis> O:59

A new scheme of synthesis of 11-phenoxyundecyl phosphate from 11-bromoundecanoic acid was suggested; its ability to serve as an acceptor of 2-acetamido-2-deoxy-α-D-glucopyranosyl phosphate in a reaction catalyzed by UDP-N-acetylglucosamine: polyprenyl phosphate N-acetylglucosamine phosphotransferase from Salmonella arizona O:59 was demonstrated.     

Interaction of superoxide dismutase with phospholipid liposomes an uptake,spin label and calorimetric study

The effect of superoxide dismutases from five species upon phospholipid bilayers has been investigated. The uptake by egg phosphatidylcholine bilayers of the holo and apo forms of bovine superoxide dismutase increases with enzyme concentration and only a fraction of each is removed by treatment with trypsin. These uptake data indicate that both forms of the enzyme associate with and are embedded within lipid bilayers. From the spectrum of the spin label 2-(3-carboxypropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl, the binding of superoxide dismutase to egg phosphatidylcholine bilayers can be shown to disorder the lipid packing. The disordering by the bovine holoenzyme is small but increases with increasing enzyme concentration and period of incubation. The disordering effects of the apoenzyme are much larger and are reversible by Cu²⁺, Zn²⁺ reconstitution of the apoenzyme. The disordering effect of the apoenzyme is further confirmed by differential scanning calorimetry. The gel to liquid crystalline phase transition of egg phosphatidylcholine is lowered 7°C by 25% by weight apo-superoxide dismutase to lipid. Human, dog, swordfish and yeast superoxide dismutases also disorder, and to a greater extent than the bovine enzyme. The greatest perturbation is produced by yeast superoxide dismutase; a 20% decrease in the order parameter by 50% by weight enzyme to lipid.     

Detection and characterization of a sialoglycosylated bacterial ABC-type phosphate transporter protein from patients with visceral leishmaniasis

We report the discovery and characterization of a glycosylated bacterial ABC-type phosphate transporter isolated from the peripheral blood mononuclear cell (PBMC) fraction of patients with visceral leishmaniasis (VL). Three disease-associated 9-O-acetylated sialoglycoproteins (9-O-AcSGPs) of 19, 56 and 65 kDa, respectively, had been identified and their purity, apparent mass and pI established by SDS-PAGE and isoelectric focusing. Western blot analyses showed that the 9-O-acetylated sialic acid is linked via α2→6 linkage to a subterminal N-acetylgalactosamine. For the 56 kDa protein, N- as well as O-glycosylations were demonstrated by specific glycosidase treatment and found to account for more than 9 kDa of the protein mass. The presence of sialic acids was further confirmed through thin layer chromatography, fluorimetric HPLC and electrospray ionization-mass spectrometry. The protein was identified by mass spectrometry and de novo sequencing of five tryptic fragments as a periplasmic ABC-type phosphate transporter of Pseudomonas aeruginosa. The amino acid sequences of the assigned peptides had 83–100% identity with the NCBI entry for a Pseudomonas transporter protein. Based on the recently reported X-ray structure of a human phosphate-binding protein, we predicted a 3D structural model for the 56 kDa protein using homology and threading methods. The most probable N- and O-glycosylation sites were identified by combinations of sequence motif-searching bioinformatics tools, solvent accessibility calculations, structural environment analyses and mass spectrometric data. This is the first reported glycosylation as well as sialylation of the periplasmic component of an ABC-type phosphate transporter protein and of one of few identified bacterial glycoproteins. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Environmental factors controlling temporal and spatial variability in the soil‐atmosphere exchange of CO2, CH4 and N2O from an Australian subtropical rainforest

The temporal variations in CO₂, CH₄ and N₂O fluxes were measured over two consecutive years from February 2007 to March 2009 from a subtropical rainforest in south‐eastern Queensland, Australia, using an automated sampling system. A concurrent study using an additional 30 manual chambers examined the spatial variability of emissions distributed across three nearby remnant rainforest sites with similar vegetation and climatic conditions. Interannual variation in fluxes of all gases over the 2 years was minimal, despite large discrepancies in rainfall, whereas a pronounced seasonal variation could only be observed for CO₂ fluxes. High infiltration, drainage and subsequent high soil aeration under the rainforest limited N₂O loss while promoting substantial CH₄ uptake. The average annual N₂O loss of 0.5 ± 0.1 kg N₂O‐N ha^?1 over the 2‐year measurement period was at the lower end of reported fluxes from rainforest soils. The rainforest soil functioned as a sink for atmospheric CH₄ throughout the entire 2‐year period, despite periods of substantial rainfall. A clear linear correlation between soil moisture and CH₄ uptake was found. Rates of uptake ranged from greater than 15 g CH₄‐C ha^?1 day^?1 during extended dry periods to less than 2–5 g CH₄‐C ha^?1 day^?1 when soil water content was high. The calculated annual CH₄ uptake at the site was 3.65 kg CH₄‐C ha^?1 yr^?1. This is amongst the highest reported for rainforest systems, reiterating the ability of aerated subtropical rainforests to act as substantial sinks of CH₄. The spatial study showed N₂O fluxes almost eight times higher, and CH₄ uptake reduced by over one‐third, as clay content of the rainforest soil increased from 12% to more than 23%. This demonstrates that for some rainforest ecosystems, soil texture and related water infiltration and drainage capacity constraints may play a more important role in controlling fluxes than either vegetation or seasonal variability.     

Effects of phosphate fertilization,lime amendments and inoculation with VA-mycorrhizal fungi on soybeans in an acid soil

Soybeans [Glycine max (L.) Merr. cv. Essex] were grown in nonsterile acid (pH. 5.2) infertile Wynnville silt loam (Glossic Fragiudult) in a glasshouse. The effects of P fertilization and lime were determined by inoculation with two VAM-fungi (VAMF): Glomus fasciculatum (Gf) and Glomus etunicatum (Ge). An important factor affected by the interaction between applied lime (soil acidity), applied P, and VAMF inoculation was the soil Al. Five application rates of P as KH₂PO₄ and three rates of lime were tested. Potassium was equalized with KCl (muriate of potash). P-efficiency (g seed/mg P kg^-1 soil) by vesicular-arbuscular mycorrhiza (VAM) was maximal at 20 mg P kg^-1 soil at all lime and VAMF treatments. VAMF inoculation increased plant survival and protected the soybeans from leaf scorch, thereby substituting for the effects of lime and P. The Ge inoculum was superior in ameliorating leaf scorch in the nonlimed soil. The Gf inoculum required more lime and P than the Ge inoculum to increase seed yield relative to the noninoculated controls containing only native VAMF. Both inocula increased root Al uptake and extractable soil Al in the acid soil without apparent adverse effects on root or shoot. The ability of the VAMF inocula to enhance the efficiency of applied P and decrease seed Cl concentration was increased by lime. Seed yield (Y) was negatively related to seed Cl concentration (X) where Y=aX^-b. Both VAMF inoculation and lime application reduced this negative relationship and may have increased the tolerance to both Cl and soil Al.