Futile cycling of ammonium ions via the high affinity potassium uptake system (Kdp) of Escherichia coli

Escherichia coli Frag1 was grown under various nutrient limitations in chemostat culture at a fixed temperature, dilution rate and pH both in the presence and the absence of a high concentration of ammonium ions by using either ammonium chloride or dl-alanine as the sole nitrogen source. The presence of high concentrations of ammonium ions in the extracellular fluids of potassium-limited cultures of E. coli Frag1 caused an increase of the specific rate of oxygen consumption of these cultures. In contrast, under phosphate-, sulphate- or magnesium-limited growth conditions no such increase could be observed. The presence of high concentrations of ammonium ions in potassium-limited cultures of E. coli Frag5, a mutant strain of E. coli Frag1 which lacks the high affinity potassium uptake system (Kdp), did not increase the specific rate of oxygen consumption.These results indicate that ammonium ions, very similar to potassium ions both in charge and size, are transported via the K dp leading to a futile cycle of ammonium ions and ammonia molecules (plus protons) across the cytoplasmic membrane. Both the uptake of ammonium ions and the extrusion of protons would increase the energy requirement of the cells and therefore increase their specific rate of oxygen consumption. The involvement of a (methyl)ammonium transport system in this futile cycle could be excluded.     

AmtB-mediated NH3 transport in prokaryotes must be active and as a consequence regulation of transport by GlnK is mandatory to limit futile cycling of NH4(+)/NH3

The nature of the ammonium import into prokaryotes has been controversial. A systems biological approach makes us hypothesize that AmtB-mediated import must be active for intracellular NH(4)(+) concentrations to sustain growth. Revisiting experimental evidence, we find the permeability assays reporting passive NH(3) import inconclusive. As an inevitable consequence of the proposed NH(4)(+) transport, outward permeation of NH(3) constitutes a futile cycle. We hypothesize that the regulatory protein GlnK is required to fine-tune the active transport of ammonium in order to limit futile cycling whilst enabling an intracellular ammonium level sufficient for the cell's nitrogen requirements.     

Ammonium uptake by Chlorella

The preincubation of Chlorella cells with glucose caused a tenfold increase of the maximal uptake rate of ammonium without change in the K _m (2 M). A similar stimulation of ammonium uptake was found when the cells were transferred to nitrogen-free growth medium. The time-course of uptake stimulation by glucose revealed a lag period of 10–20 min. The turnover of the ammonium transport system is characterized by a half-life time of 5–10 h, but in the presence of light 30% of uptake activity stayed even after 50 h. 6-Deoxyglucose was not able to increase the ammonium uptake rate. These data together were interpreted as evidence for induction of an ammonium transport system by a metabolite of glucose. Mechanistic studies of the ammonium transport system provided evidence for the electrogenic uptake of the ammonium ion. The charge compensation for NH ₄ ⁺ entry was achieved by immediate K⁺ efflux from the cells, and this was followed after 1 min by H⁺ extrusion. Ammonium accumulated in the cells; the rate of uptake was sensitive to p-trifluoromethoxy-carbonylcyanide-phenylhydrazon and insensitive to methionine-sulfoxime. Uptake studies with methylamine revealed that methylamine transport is obviously catalyzed by the ammonium transport system and, therefore, also increased in glucose-treated Chlorella cells.Abbreviation p.c. packed cells     

Transpiration, potassium uptake and flow in tobacco as affected by nitrogen forms and nutrient levels

BACKGROUND AND AIMS: Ammonium can result in toxicity symptoms in many plants when it is supplied as the sole source of N. In this work, influences of different nitrogen forms at two levels (2 and 15 mm N) on growth, water relations and uptake and flow of potassium were studied in plants of Nicotiana tabacum 'K 326'. METHODS: Xylem sap from different leaves was collected from 106-d-old tobacco plants cultured in quartz sand by application of pressure to the root system. Whole-shoot transpiration for each of the treatments was measured on a daily basis by weight determination. KEY RESULTS: Total replacement of NO(3)(-)N by NH(4)(+)-N caused a substantial decrease in dry weight gain, even when plants grew under nutrient deficiency. Increasing nutrient concentration resulted in a greater net dry weight gain when nitrogen was supplied as NO(3)(-) or NH(4)NO(3), but resulted in little change when nitrogen was supplied as NH(4)(+). NH(4)(+)-N as the sole N-source also caused reduction in transpiration rate, changes in plant WUE (which depended on the nutrient levels) and a decrease in potassium uptake. However, the amount of xylem-transported potassium in the plants fed with NH(4)(+) was not reduced: it was 457 % or 596 % of the potassium currently taken up at low or high nutrient level, respectively, indicating a massive export from leaves and cycling of potassium in the phloem. CONCLUSIONS: Ammonium reduces leaf stomatal conductance of tobacco plants. The flow and partitioning of potassium in tobacco plants can be changed, depending on the nitrogen forms and nutrient levels.     

Mechanisms of ammonia and ammonium ion toxicity in animal cells: Transport across cell membranes

A model for transport of ammonia and ammonium ions across cell membranes is presented. The model suggests that ammonium ions compete with potassium ions for inward transport, over the cytoplasmic membrane, via potassium transport proteins like the Na⁺/K⁺-ATPase and the Na⁺K⁺2Cl⁻-cotransporter. It also explains the difference between the ammonia/ammonium that is added to the cells and which is formed by the cells during metabolism of amino acids, especially glutamine and glutamate. The ammonium transport and subsequent events lead to predictable intracellular and extracellular pH (pH_e) changes. Experiments which verified the model and the predicted consequences were performed by measurements of the pH_e in concentrated cell suspensions. Addition of ammonium ions caused a time-dependent pH_e increase which was inhibited by potassium ions. The test system is not per se specific for transport measurements but the effect of potassium ions on the pH_e strongly favors our suggested model. Simple diffusion of ammonium ions would not be counteracted by potassium ions. The results show that ammonium ion transport in the murine myeloma cell line (Sp2/0-Ag14) used is inhibited by an excess of potassium ions. Results from experiments with specific inhibitors of suggested transport proteins were not conclusive. It is postulated that one important toxic effect of ammonia/ammonium is an increased demand for maintenance energy, caused by the need to maintain ion gradients over the cytoplasmic membrane. The results also suggest that potassium ions can be used to detoxify ammonia/ammonium in animal cell cultivations.     

Uptake and long-distance transport of phosphate,potassium and chloride in relation to internal ion concentrations in barley: evidence of non-allosteric regulation

The extent to which uptake and transport of either phosphate, potassium or chloride are controlled by the concentration of these ions within the root, perhaps through an allosteric mechanism, was investigated with young barley plants in nutrient solution culture. Plants were grown with their roots divided between two containers, such that a single seminal root was continuously supplied with all the required nutrient ions, while the remaining four or five seminal roots were either supplied with the same solution (controls) or, temporarily, a solution lacking a particular nutrient ion (nutrient-deficient treatment). Compared with controls, there was a marked stimulation of uptake and transport of labelled ions by the single root following 24 h or more of nutrient dificiency to the remainder of the root system. This stimulation, which comprised an increased transport to the shoot and, for all ions except Cl^-, increased transport to the remainder of the root system, took place without appreciable change in the concentration of particular ions within the single root. However, nutrient deficiency quickly caused a lower concentration of ions in the shoot and the remaining roots. The results are discussed in relation to various mechanisms, proposed in the literature, by which the coordination of ion uptake and transport may be maintained within the plant. We suggest that under our conditions any putative allosteric control of uptake and transport by root cortical cells was masked by an alternative mechanism, in which ion influx appears to be regulated by ion efflux to the xylem, perhaps controlled by the concentration of particular ions recycled in the phloem to the root from the shoot.     

Effect of benzoic acid on the ammonium transport system of Zygosaccharomyces bailii

Summary A study of the ammonium transport system of Zygosaccharomyces bailii was carried out using methylammonium as a non-metabolizable analogue. Benzoic acid in the growth medium decreased the capacity of the transport system from 1.46 ± 0.11 mmol.g^–1.h^–1 to 0.41±0.04 mmol.g^–1.h^–1, while the affinity for ammonium was not significatively affected. Although ammonium uptake was inhibited by benzoic acid, the ammonium transport system was still active at preservative concentrations which fully inhibited growth suggesting that inhibition of growth was not governed by the uptake of this nutrient.     

Alleviation of rapid, futile ammonium cycling at the plasma membrane by potassium reveals K+-sensitive and -insensitive components of NH4+ transport

Futile plasma membrane cycling of ammonium (NH4+) is characteristic of low-affinity NH4+ transport, and has been proposed to be a critical factor in NH4+ toxicity. Using unidirectional flux analysis with the positron-emitting tracer 13N in intact seedlings of barley (Hordeum vulgare L.), it is shown that rapid, futile NH4+ cycling is alleviated by elevated K+ supply, and that low-affinity NH4+ transport is mediated by a K+-sensitive component, and by a second component that is independent of K+. At low external [K+] (0.1 mM), NH4+ influx (at an external [NH4+] of 10 mM) of 92 micromol g(-1) h(-1) was observed, with an efflux:influx ratio of 0.75, indicative of rapid, futile NH4+ cycling. Elevating K+ supply into the low-affinity K+ transport range (1.5-40 mM) reduced both influx and efflux of NH4+ by as much as 75%, and substantially reduced the efflux:influx ratio. The reduction of NH4+ fluxes was achieved rapidly upon exposure to elevated K+, within 1 min for influx and within 5 min for efflux. The channel inhibitor La3+ decreased high-capacity NH4+ influx only at low K+ concentrations, suggesting that the K+-sensitive component of NH4+ influx may be mediated by non-selective cation channels. Using respiratory measurements and current models of ion flux energetics, the energy cost of concomitant NH4+ and K+ transport at the root plasma membrane, and its consequences for plant growth are discussed. The study presents the first demonstration of the parallel operation of K+-sensitive and -insensitive NH4+ flux mechanisms in plants.     

Methylamine and ammonia transport in Saccharomyces cerevisiae.

Methylamine (methylammonium ion) entered Saccharomyces cerevisiae X2180-A by means of a specific active transport system. Methylamine uptake was pH dependent (maximum rate between pH 6.0 and 6.5) and temperature dependent (increasing up to 35 C) and required the presence of a fermentable or oxidizable energy source in the growth medium. At 23 C the vmax for methylamine transport was similar 17 nmol/min per mg of cells (dry weight) and the apparent Km was 220 muM. The transport system exhibited maximal activity in ammonia-grown cells and was repressed 60 to 70 percent when glutamine or asparagine was added to the growth medium. There was no significant derepression of the transport system during nitrogen starvation. Ammonia (ammonium ion) was a strong competitive inhibitor of methylamine uptake, whereas other amines inhibited to a much lesser extent. Mutants selected on the basis of their reduced ability to transport methylamine (Mea-R) simultaneously exhibited a decreased ability to transport ammonia.     

Futile cycling at the plasma membrane: a hallmark of low-affinity nutrient transport

Plant nutrient acquisition from concentrated soil solutions is governed by low-affinity transport systems in the plasma membranes of root cells. In this Opinion article, we illustrate that for six major nutrient ions, in addition to influx mediation by low-affinity transporters, high rates of ion cycling at the plasma membrane are a hallmark of nutrient transport at elevated external concentrations. This phenomenon is characterized by high rates of ion influx and concurrent high efflux of the same ion, resulting in efflux:influx ratios close to 1. Prolonged maintenance of futile cellular ion cycling can be energetically unfavorable and detrimental to plant growth and survival. We discuss how ion cycling can contribute to the toxicities of ions such as Na(+) and NH(4)(+) in the low-affinity range of ion provision. We also argue that cellular ion cycling makes the reliable measurement of ion influxes in the low-affinity range a formidable challenge.     

Independent induction of two blue light-dependent monovalent anion transport systems in the plasma membrane of Monoraphidium braunii

In the plasma membrane of the green alga Monoraphidium braunii there are at least two monovalent anion transport systems. One of them is specific for bicarbonate. This transport system is activated by blue light and its induction is triggered by a decrease in the external CO2 concentration. The second transport system is responsible for nitrate uptake at least. This transport system is also activated by blue light and its induction occurs when there is no ammonium in the external medium. Both transport systems are synthesized independently. Hence, when M. braunii cells grow with nitrate as the only nitrogen source under high CO2, they have a nitrate transport system but lack a bicarbonate transporter. Conversely, cells grown with ammonium under low CO2, have a bicarbonate transport system but lack a nitrate transporter. Both transport systems are induced in cells irradiated with white light in the absence of a carbon source, suggesting that there may be precursors in the plasma membrane that only need the synthesis and assembly of some component(s) to become fully active. The induction of nitrate and nitrite reductases, however, only takes place when a carbon source is supplied to the cells.     

The molecular physiology of ammonium uptake and retrieval

Plants are able to take up ammonium from the soil, or through symbiotic interactions with microorganisms, via the root system. Using functional complementation of yeast mutants, it has been possible to identify a new class of membrane proteins, the ammonium transporter/methylammonium permease (AMT/MEP) family, that mediate secondary active ammonium uptake in eukaryotic and prokaryotic organisms. In plants, the AMT gene family can be subdivided according to their amino-acid sequences into three subfamilies: a large subfamily of AMT1 genes and two additional subfamilies each with single members (LeAMT1;3 from tomato and AtAMT2;1 from Arabidopsis thaliana). These transporters vary especially in their kinetic properties and regulatory mechanism. High-affinity transporters are induced in nitrogen-starved roots, whereas other transporters may be considered as the 'work horses' that are active when conditions are conducive to ammonium assimilation. The expression of several AMTs in root hairs further supports a role in nutrient acquisition. These studies provide basic information that will be needed for the dissection of nitrogen uptake by plants at the molecular level and for determining the role of individual AMTs in nutrient uptake and potentially in nutrient efficiency.     

Iron-nutritional aspects of the ionic balance of plants

Summary The effect of iron on the ionic balance of several plant species and cultivars was studied. Those plants which normally excrete relatively low amounts of hydroxyl ions respond to iron stress by lowering the pH of the nutrient medium and decreasing anion uptake. These plants may be considered as being Fe efficient. Plants which normally excrete relatively high amounts of hydroxyl ions and which continue to increase the pH of a nutrient medium when under iron stress may be considered as Fe inefficient.Iron deficiency tends to increase carboxylate accumulation and to decrease anion uptake. When cation uptake is depressed by iron deficiency this is mainly a non specific depression of potassium uptake, on the other hand when iron stress stimulates cation uptake this is mainly due to a specific stimulation of the divalent cations Ca and Mg.Additional key words: Iron, efficiency of uptake, ionic balance, hydroxyl and hydrogen ion excretion.     

Effects of Ionic Strength of Nutrient Solutions on Phosphate Uptake by Sunflower

Active phosphate uptake by the roots of young sunflower plants was stimulated nonspecifically by increasing the total salt concentration of the uptake solution. Inhibition of active uptake by DNP-treatment removed the salt stimulation. Independently of the rate of active uptake the amount of phosphate present in the free space of the roots increased as the salt concentration was raised. It is suggested that at low ionic strength of the nutrient solution the initial passive step of ion transport through the root free space can limit the overall uptake rate.     

NH4+ currents across the peribacteroid membrane of soybean. Macroscopic and microscopic properties, inhibition by Mg2+, and temperature dependence indicate a SubpicoSiemens channel finely regulated by divalent cations

The control of ammonium (NH(4)(+)) transport is critical in preventing futile cycles of NH(4)(+)/ammonia transport. An unusual nonselective cation channel with subpicoSiemens single-channel conductance permeable to NH(4)(+) had previously been identified in the peribacteroid membrane (PBM) of symbiosomes from soybean (Glycine max) nodules. Here, we investigate the proposed channel mechanism and its control by luminal magnesium. Currents carried by NH(4)(+) were measured in inside-out PBM patches by patch clamp. NH(4)(+) transport corresponding to the physiological direction of net transfer showed time-dependent activation and associated single-channel-like events. These could not be resolved to discrete conductances but had the same selectivity as the total current. The voltage dependence of the steady-state current was affected by temperature consistent with the rate constant of channel opening being reduced with decreased temperature. This resulted in steady-state currents that were more temperature sensitive at voltages where the current was only partially activated. When fully activated, the current reflected more the ion conduction through open channels and had an activation energy of 28.2 kJ mol(-1) (Q10 = 1.51, 8 degrees C-24 degrees C). Increased Mg(2+) on the symbiosome lumen side blocked the current (ID(50) = 351 microm, with 60 mm NH(4)(+)). Complete inhibition with 2 mm Mg(2+) was relieved with a small increase in NH(4)(+) on the lumen side of the membrane (shift of 60-70 mm). With Mg(2+) the selectivity of the transport for divalent cations increased. From these features, we propose a divalent-dependent feedback regulation of the PBM-nonselective cation channel that could maintain a constant NH(4)(+) gradient across the membrane.     

Uptake and transport of N,P and K in tomato supplied with nettle water and nutrient solution

Water extract of stinging nettle (Urtica dioica) has a growth stimulating effect on plants. This investigation elucidated effects of nettle water on uptake and transport of N, P and K. Tomato plants (Solanum lycopersicum L. cv. Dansk export) were grown in sand culture 6–8 weeks. Plants were supplied with nettle water and nutrient solution was used as a control medium. Uptake and transport of N, P and K⁺ were determined with isotopes (¹⁵N,³²P and⁸⁶Rb⁺ as a tracer for K⁺) and ion-selective electrodes and in exudation experiments. A 15% higher uptake of nitrogen (¹⁵N assay) was found after nettle water treatment compared with the nutrient solution control. The total amount of nitrogen was also higher in plants cultivated with nettle water. Transport of inorganic and organic nitrogen, measured in exudation experiments, was more than 50% higher for plants supplied with nettle water compared with plants supplied with nutrient solution. In contrast, nettle water had no effect on uptake, transport or total amount of phosphorus and potassium in the plants. Experiments in hydroculture showed that nettle water had a strong pH-elevating effect. Uptake of NH ₄ ⁺ was strongly stimulated by nettle water compared with nutrient solution. By holding pH at a constant level during the uptake period for 6 h, the uptake of NH ₄ ⁺ from nettle water was significantly lower when no adjustment of pH was made. Consequently a good deal of the NH ₄ ⁺ uptake enhancement by nettle water could be explained by pH-stimulation. Assays with the uncoupler/inhibitor 2,4-dinitrophenol (DNP) and dichlorophenyl-dimethyl-urea (DCMU) showed that uptake of nitrogen from nettle water was less metabolically-linked than uptake from a corresponding nutrient solution. All together, nettle water seems to stimulate the uptake of nitrogen, but not phosphorus or potassium.     

Nitrite uptake and its regulation in the cyanobacterium Anacystis nidulans

Two different components seem to participate in the uptake of nitrite by the cyanobacterium Anacystis nidulans, namely a transport system sensitive to N,N′-dicyclohexylcarbodiimide and a passive influx. The relative contribution of each component depended on the pH of the medium, that of the active system being prevalent at high pH values. The active transport of nitrite appears to be mediated by a high-affinity system, whereas the affinity for nitrite of the passive system is lower, similar to that of nitrite reductase. The utilization of nitrite was inhibited by products of the assimilation of ammonium via glutamine synthetase, apparently acting at the level of the active component involved in nitrite uptake.     

Active and Passive Components of Sulfate Uptake in Sunflower Plants

The aim of the investigation was to identify components of active and passive ion uptake and transport in roots of plants and to assess their quantitative relations under different external and internal conditions. The uptake of radiosulfate and water by young sunflower plants from complete nutrient solutions labelled with ³⁵S was studied. The metabolism-linked nature of the sulfate uptake in the root following the passive migration into the apparent free space (AFS) was demonstrated by the addition of sodium. selenate, 2,4-dinitrophenol, potassium cyanide, and sodium azide to the nutrient solutions. The magnitude of the AFS measured on a root volume basis varied between 14 and 57 per cent depending on the pretreatment of the plants and the sulfate concentration of the nutrient solution. The variations were supposed to be due to different capacity to bind sulfate by exchange-adsorption within the AFS. The amounts of sulfate in different fractions of the total AFS-uptake were computed under certain theoretical assumptions. A quantitative connection was proposed between the magnitude of the adsorbed sulfate fraction in the AFS and the rate of active uptake into the symplasm. The exchange-adsorption probably constitutes the initial stage of active ion uptake. The stimulating effect by water on ion uptake would be an increase of the speed of transporting ions to, from, or along the adsorption sites in the AFS. Experiments conducted at temperatures in the nutrient solution between 5 and 35 C elucidated the multistep nature of ion transport within a root.     

Cation/proton antiport systems in Escherichia coli. Solubilization and reconstitution of delta pH-driven sodium/proton and calcium/proton antiporters

Uptake of 22Na+ and 45Ca2+ into everted membrane vesicles from Escherichia coli was measured with imposed transmembrane pH gradients, acid interior, as driving force. Vesicles loaded with 0.5 M KCl were diluted into 0.5 M choline chloride to create a potassium gradient. Addition of nigericin to produce K+/H+ exchange resulted in formation of a pH gradient. This imposed gradient was capable of driving 45Ca2+ accumulation. In another method vesicles loaded with 0.5 M NH4Cl were diluted into 0.5 M choline chloride, creating an ammonium diffusion potential. A gradient of H+ was produced by passive efflux of NH3. With an ammonium gradient as driving force, everted vesicles accumulated both 45Ca2+ and 22Na+. The data suggest that 22Na+ uptake was via the sodium/proton antiporter and 45Ca2+ via the calcium/proton antiporter. Uptake of both cations required alkaline pHout. A minimum pH gradient of 0.9 unit was needed for transport of either ion, suggesting gating of the antiporters. Octyl glucoside extracts of inner membrane were reconstituted with E. coli phospholipids in 0.5 M NH4Cl. NH4+-loaded proteoliposomes accumulated both 22Na+ and 45Ca2+, demonstrating that the sodium/proton and calcium/proton antiporters could be solubilized and reconstituted in a functional form.     

Ammonium ion transport—a cause of cell death

Ammonium can be transported into the cell by ion pumps in the cytoplasmic membrane. Ammonia then diffuse out through the cell membrane. A futile cycle is created that results in cytoplasmic acidification and extracellular alkalinisation. Ammonium transport can be quantified by measuring the extracellular pH changes occurring in a cell suspension (in PBS) after addition of ammonium. By using this technique, in combination with specific inhibitors of various ion pumps, it was shown that ammonium ions are transported across the cytoplasmic membrane by the Na⁺K⁺2Cl^--cotransporter in both hybridoma and myeloma cells. Further, the Na⁺/H⁺ exchanger, which regulates intracellular pH by pumping out protons, was shown to be active during ammonium exposure. The viability of hybridoma cells suspended in PBS and exposed to NH _inf4 ^sup+ for only 90 min, was reduced by 11% (50% necrosis and 50% apoptosis). A control cell suspension did not loose viability during this time. Turning off the activity of the Na⁺/H⁺ exchanger (by amiloride) during ammonium exposure decreased viability further, while inhibiting transport itself (by bumetanide) restored viability to the same level as for the control experiment with bumetanide alone. These results show that one effect of ammonia/ammonium on cell physiology is specifically related to the inward transport of ammonium ions by membrane bound ion pumps.Abbreviations q _pH specific rate of pH increase (pH units per min and 10⁶ cells per ml)