Utilization of formate as an additional energy source by glucose-limited chemostat cultures ofCandida utilis CBS 621 andSaccharomyces cerevisiae CBS 8066

The oxidation of benzene to phenol by whole cells of Nitrosomonas europaea is catalysed by ammonia monooxygenase, and therefore requires a source of reducing power. Endogenous substrates, hydrazine, hydroxylamine and ammonium ions were compared as reductants. The highest rates of benzene oxidation were obtained with 4 mM benzene and hydrazine as reductant, and equalled 6 mol· h^-1·mg protein^-1. The specificity of ammonia monooxygenase for benzene as a substrate was determined by measuring k _cat/K _m for benzene relative to k _cat/K _m for uncharged ammonia, a value of 0.4 being obtained. Phenol was found to be further hydroxylated to yield hydroquinone. This reaction, like benzene oxidation, was sensitive to the ammonia monooxygenase inhibitor allylthiourea. Catechol and resorcinol were not detected as products of phenol oxidation, implying that at least 88% of the hydroxylation is para-directed.     

Three years of continuous measurements of atmospheric ammonia concentrations over a forest stand at the Höglwald site in southern Bavaria

Over a period of 3 years (1995 – 1997), atmospheric ammonia (NH₃) concentrations were measured 3 m above a spruce stand using a continuous-flow annular denuder at the Höglwald site near Munich, Bavaria. The annual average ammonia concentration was between 2.2 and 2.9 g NH₃ m^–3. The highest hourly average values occurred at the end of each year. In December 1995 the peak value was achieved with 183 g NH₃ m^–3. More than 50% of the hourly average means of the ammonia concentration were lower than 2 g NH₃ m^–3 and only fewer than 5% of the hourly average concentrations higher than 10 g NH₃ m^–3. The ammonia concentration course indicated a pronounced diurnal variation, with higher concentrations in the late morning and lower concentrations during the night. Often a sudden increase of the ammonia concentration was detected in the early morning with first sun exposure of the spruce crown and sinking humidity, indicating a reemission of ammonia from the canopy to the atmosphere.     

Permeability of ammonia,methylamine and ethylamine in the cyanobacterium,Synechococcus R-2 (Anacystis nidulans) PCC 7942

Summary Permeabilities of ammonia (NH₃), methylamine (CH₃NH₂) and ethylamine (CH₃CH₂NH₂) in the cyanobacterium (cyanophyte)Synechococcus R-2 (Anacystis nidulans) have been measured. Based on net uptake rates of DCMU (dichlorophenyldimethylurea) treated cells, the permeability of ammonia was 6.44±1.22 m sec^–1 (n=13). The permeabilities of methylamine and ethylamine, based on steady-state¹⁴C labeling were more than ten times that of ammonia (P _methylamine=84.6±9.47 m sec^–1 (76),P _ethylamine=109±11 m sec^–1 (55)). The apparent permeabilities based on net uptake rates of methylamine and ethylamine uptake were significantly lower, but this effect was partially reversible by ammonia, suggesting that net amine fluxes are rate limited by proton fluxes to an upper limit of about 700 nmol m^–2 sec^–1. Increasing concentrations of amines in alkaline conditions partially dissipated the pH gradient across the cell membrane, and this property could be used to calculate the relative permeabilities of different amines. The ratio of ethylamine to methylamine permeabilities was not significantly different from that calculated from the direct measurements of permeabilities; ammonia was much less effective in dissipating the pH gradient across the cell membrane than methylamine or ethylamine. An apparent permeability of ammonia of 5.7±0.9 m sec^–1 could be calculated from the permeability ratio of ammonia to methylamine and the experimentally measured permeability of methylamine. The permeability properties of ammonia and methylamine are very different; this poses problems in the interpretation of experiments where¹⁴C-methylamine is used as an ammonia analogue.     

The impact of organic matter on nitric oxide formation by Nitrosomonas europaea

Chemolithoautotrophically growing cells of Nitrosomonas europaea quantitatively oxidized ammonia to nitrite under aerobic conditions with no loss of inorganic nitrogen. Significant inorganic nitrogen losses occurred when cells were growing mixotrophically with ammonium, pyruvate, yeast extract and peptone. Under oxygen limitation the nitrogen losses were even higher. In the absence of oxygen pyruvate was metabolized slowly while nitrite was consumed concomitantly. Nitrogen losses were due to the production of nitric oxide and nitrous oxide. In mixed cultures of Nitrosomonas and Nitrobacter, strong inhibition of nitrite oxidation was reproducibly measured. NO and ammonium were not inhibitory to Nitrobacter. First evidence is given that hydroxylamine, the intermediate of the Nitrosomonas monooxygenase-reaction, is formed. 0.2 to 1.7 M NH₂OH were produced by mixotrophically growing cells of Nitrosomonas and Nitrosovibrio. Hydroxylamine was both a selective inhibitory agent to Nitrobacter cells and a strong reductant which reduced nitrite to NO and N₂O. It is discussed whether chemodenitrification or denitrification is the most abundant process for NO and N₂O production of Nitrosomonas.     

Role of nitrite reductase in the ammonia-oxidizing pathway of <Emphasis Type="Italic">Nitrosomonas europaea</Emphasis>

Metabolism of ammonia (NH₃) and hydroxylamine (NH₂OH) by wild-type and a nitrite reductase (nirK) deficient mutant of Nitrosomonas europaea was investigated to clarify the role of NirK in the NH₃ oxidation pathway. NirK-deficient N. europaea grew more slowly, consumed less NH₃, had a lower rate of nitrite (NO₂ ⁻) production, and a significantly higher rate of nitrous oxide (N₂O) production than the wild-type when incubated with NH₃ under high O₂ tension. In incubations with NH₃ under low O₂ tension, NirK-deficient N. europaea grew more slowly, but had only modest differences in NH₃ oxidation and product formation rates relative to the wild-type. In contrast, the nirK mutant oxidized NH₂OH to NO₂ ⁻ at consistently slower rates than the wild-type, especially under low O₂ tension, and lost a significant pool of NH₂OH–N to products other than NO₂ ⁻ and N₂O. The rate of N₂O production by the nirK mutant was ca. three times higher than the wild-type during hydrazine-dependent NO₂ ⁻ reduction under both high and low O₂ tension. Together, the results indicate that NirK activity supports growth of N. europaea by supporting the oxidation of NH₃ to NO₂ ⁻ via NH₂OH, and stimulation of hydrazine-dependent NO₂ ⁻ reduction by NirK-deficient N. europaea indicated the presence of an alternative, enzymatic pathway for N₂O production.     

Ethylene oxidation by Nitrosomonas europaea

Incubation of whole cells of the nitrifying bacterium Nitrosomonas europaea with ethylene led to the formation of ethylene oxide. Ethylene oxide production was prevented by inhibitors of ammonium ion oxidation, and showed properties implying that ethylene is a substrate for the ammonia oxidising enzyme, ammonia monooxygenase. Endogenous substrates, hydroxylamine, hydrazine and ammonium ions were compared as sources of reducing power in terms of rates and stoichiometries of ethylene oxidation. The highest rates of ethylene oxide formation (15 mol h^-1 mg protein^-1) were obtained with hydrazine as donor. The data suggest that at high concentrations of ethylene the rate of oxidation is limited by the rate at which reducing power can be supplied to the monooxygenase, not by an intrinsic V _max. Ethylene had an inhibitory effect on the rate of ammonium ion utilisation; an approximate K _i of 80 M was derived, but the results deviated from simple competitive behaviour. Measurement of relative rates of ethylene oxide formation and ammonium ion utilization led to a k _cat/K _m value for ethylene of 1.1 relative to NH ₄ ⁺ , or 0.04 relative to the true natural substrate, NH₃. The effects of higher concentrations of ethylene oxide on oxygen uptake rates were also investigated. The results imply that ethylene oxide is also a substrate for the monooxygenase, but with a much lower affinity than ethylene.     

Effect of copper on inhibition by nitrapyrin of growth ofNitrosomonas europaea

Exponentially growing cultures ofNitrosomonas europaea were inhibited by addition of 0.5 g nitrapyrin ml^–1. This inhibition was increased by simultaneous addition of 0.046 g Cu²⁺ ml^–1 as copper sulfate. This contradicts a previous report that copper relieves inhibition of ammonia oxidation by nitrapyrin, which report has formed the basis for hypotheses regarding the mechanism of action of this inhibitor.     

Seasonal variability of apoplastic NH4 + and pH in an intensively managed grassland

The stomatal compensation point of ammonia (_s) is a major factor controlling the exchange of atmospheric ammonia (NH₃) with vegetation. It is known to depend on the supply of nitrogen and to vary among plant species, but its seasonal variation has not yet been reported for grassland. In this study, we present the temporal variation of apoplastic NH₄ ⁺ concentration ([NH₄ ⁺]_apo) and pH (pH_apo) measured in leaves of Lolium perenne L. in a grassland, through two periods of cutting / fertilisation, followed by a livestock grazing period. The total free NH₄ ⁺ concentration measured in foliage ([NH₄ ⁺]_fol), and soil mineral NH₄ ⁺ and NO₃ ^– concentration are also presented. The value of [NH₄ ⁺]_apo varied from less than 0.01 mM to a maximum of 0.5 mM occurring just after fertilisation, whereas the apoplastic pH ranged from pH 6 to 6.5 for most of the time and increased up to pH 7.8, 9 days after the second fertilisation, when grazing started. [NH₄ ⁺]_fol varied between 20 and 50 g N-NH₄ ⁺ g^–1 f.w. The compensation point at 20°C, ranged from 0.02 g NH₃ m^–3 between the fertilisations to 10 g NH₃ m^–3 just after the second fertilisation. The reasons for these seasonal changes are discussed, with respect to plant metabolism and the concentration of ammonium and nitrate in the soil.     

Modulation of brain mitochondrial membrane permeability and synaptosomal Ca2+ transport by dopamine oxidation

Effects of dopamine on the membrane permeability transition, thioredoxin reductase activity, production of free radicals and oxidation of sulfhydryl groups in brain mitochondria and the Ca²⁺ uptake by Na⁺-Ca²⁺ exchange and sulfhydryl oxidation in brain synaptosomes were examined. The brain mitochondrial swelling and the fall of transmembrane potential were altered by pretreatment of dopamine in a dose dependent manner. Depressive effect of dopamine on mitochondrial swelling was reversed by 10 g/ml catalase, and 10 mM DMSO. The activities of thioredoxin reductase in intact or disrupted mitochondria were decreased by dopamine (1-100 M), 25 M Zn²⁺ and 50 M Mn²⁺. Dopamine-inhibited enzyme activity was reversed by 10 g/ml SOD and 10 g/ml catalase. Pretreatment of dopamine decreased Ca²⁺ transport in synaptosomes, which was restored by 10 g/ml SOD and 10 mM DMSO. Dopamine (1-100 M) in the medium containing mitochondria produced superoxide anion and hydrogen peroxide, while its effect on nitrite production was very weak. The oxidation of sulfhydryl groups in mitochondria and synaptosomes were enhanced by dopamine with increasing incubation times. Results suggest that dopamine could modulate membrane permeability in mitochondria and calcium transport at nerve terminals, which may be ascribed to the action of free radicals and the loss of reduced sulfhydryl groups.     

Kinetic studies on ammonia and methane oxidation by Nitrosococcus oceanus

The kinetics of ammonia oxidation and the ability of a marine ammonia-oxidizing bacterium, Nitrosococcus oceanus, to metabolize methane were investigated in semicontinuous batch culture. The effects of inhibitors (acetylene and nitrapyrin) and coreactants were determined in order to elucidate the behavior of the ammonia oxygenase enzyme in N. oceanus. Acetylene and nitrapyrin were potent inhibitors and their effects were not mitigated by increased ammonia concentrations. Oxygen concentration had the effect of a mixed-type inhibitor; reduced oxygen inhibited the rate or ammonia oxidation at high substrate concentration but may enhance the rate at low substrate concentrations. Substrate affinity in terms of NH ₄ ⁺ increased (K _m decreased) with increasing pH. Optimal pH was about 8. Methane inhibited ammonia oxidation; the interaction was not simple competitive inhibition and the presence of multiple active sites on the enzyme was indicated by the behaviour of the inhibited treatments. Half-saturation constants for methane (K _i=6.6 M) and ammonia (K _m=8.1 M) were similar. N. oceanus oxidized methanol and methane linearly over time, with CO₂ and cell material being produced at approximately equal rates.     

Loss of Ammonia Monooxygenase Activity in Nitrosomonas europaea upon Exposure to Nitrite

Nitrosomonas europaea, an obligate ammonia-oxidizing bacterium, lost an increasing amount of ammonia oxidation activity upon exposure to increasing concentrations of nitrite, the primary product of ammonia-oxidizing metabolism. The loss of activity was specific to the ammonia monooxygenase (AMO) enzyme, as confirmed by a decreased rate of NH₄⁺-dependent O₂ consumption, some loss of active AMO molecules observed by polypeptide labeling with ¹⁴C₂H₂, the protection of activity by substrates of AMO, and the requirement for copper. The loss of AMO activity via nitrite occurred under both aerobic and anaerobic conditions, and more activity was lost under alkaline than under acidic conditions except in the presence of large concentrations (20 mM) of nitrite. These results indicate that nitrite toxicity in N. europaea is mediated by a unique mechanism that is specific for AMO.     

The effect of copper toxicity on the contents of nitrogen compounds in Silene vulgaris (Moench) Garcke

The effect of copper on the uptake of nitrogen and the tissue contents of inorganic nitrogen, amino acids and proteins were studied in cooper-sensitive Silene vulgaris (Moench) Garcke, grown at different nitrogen sources (NH₄ ⁺ or NO₃ ^-). All the toxic copper levels tested, i.e. 4, 8, 16 M Cu²⁺, strongly inhibited the uptake of nitrogen, especially of NO₃ ^-, and decreased the content of NO₃ ^-, amino acids and proteins. Especially at 4 and 8 M Cu²⁺, NH₄ ⁺ accumulated in the plants, suggesting that the conversion of NH₄ ^- into amino acids was inhibited.     

Isolation and characterization of a toxic intracellular protein from Vibrio parahaemolyticus

A toxic factor released from disrupted cells of Vibrio parahaemolyticus was partially purified by gel filtration after precipitation with (NH₄)₂SO₄ at 40% saturation. The factor, which was a thermostable protein of 63 kDa, lysed human erythrocytes at a concentration of 0.15 g ml^-1. Its LD₅₀ by intravenous injection into mice was 6.4 g. Fluid accumulated in suckling mice force-fed with the toxic material (1 to 25 g). Haemolytic activity, which occurred maximall at 37°C and pH 7.0 was enhanced by Ca²⁺, Cu²⁺ and Zn²⁺, each at 1 mm. Anti-toxic-factor serum agglutinated V. parahaemolyticus cells. The factor may play a role in the pathogenesis of V. parahaemolyticus infections and in the host's defence mechanisms against infection by the microorganism.     

Effect of light intensity on ammonia assimilation in maize leaves

The effect of light on the metabolism of ammonia was studied by subjecting detached maize leaves to 150 or 1350 mol m^–2 s^–1 PAR during incubation with the leaf base in 2 mM ¹⁵NH₄Cl. After up to 60 min, leaves were extracted. Ammonia, glutamine, glycine, serine, alanine, and aspartate were separated by isothermal distillation and ion exchange chromatography. ¹⁵N enrichments were analyzed by emission spectroscopy. The uptake of ammonium chloride did not influence CO₂ assimilation (8.3 and 17.4 mol m^–1 s^–1 at 150 and 1350 mol m^–2 s^–1 PAR, respectively). Leaves kept at high light intensity contained more serine and less alanine than leaves from low light treatments. Within 1 h of incubation the enrichment of ammonia extracted from leaves rose to approximately 20% ¹⁵N. In the high light regime the amino acids contained up to 15% ¹⁵N, whereas in low light ¹⁵N enrichments were small (up to 6%). The kinetics of ¹⁵N incorporation indicated that NH₃ was firstly assimilated into glutamine and then into glutamate. After 15 min ¹⁵N was also found in glycine, serine and alanine. At high light intensity nearly half of the ¹⁵N was incorporated in glycine. On the other hand, at low light intensity alanine was the predominant ¹⁵N sink. It is concluded that light influences ammonia assimilation at the glutamine synthetase reaction.     

Effects of Soil on Ammonia, Ethylene, Chloroethane, and 1,1,1-Trichloroethane Oxidation by Nitrosomonas europaea

Ammonia monooxygenase (AMO) from Nitrosomonas europaea catalyzes the oxidation of ammonia to hydroxylamine and has been shown to oxidize a variety of halogenated and nonhalogenated hydrocarbons. As part of a program focused upon extending these observations to natural systems, a study was conducted to examine the influence of soil upon the cooxidative abilities of N. europaea. Small quantities of Willamette silt loam (organic carbon content, 1.8%; cation-exchange capacity, 15 cmol/kg of soil) were suspended with N. europaea cells in a soil-slurry-type reaction mixture. The oxidations of ammonia and three different hydrocarbons (ethylene, chloroethane, and 1,1,1-trichloroethane) were compared to results for controls in which no soil was added. The soil significantly inhibited nitrite production from 10 mM ammonium by N. europaea. Inhibition resulted from a combination of ammonium adsorption onto soil colloids and the exchangeable acidity of the soil lowering the pH of the reaction mixture. These phenomena resulted in a substantial drop in the concentration of NH₄⁺ in solution (10 to 4.5 mM) and, depending upon the pH, in a reduction in the amount of available NH₃ to concentrations (8 to 80 μM) similar to the K_s value of AMO for NH₃ (~29 μM). At a fixed initial pH (7.8), the presence of soil also modified the rates of oxidation of ethylene and chloroethane and changed the concentrations at which their maximal rates of oxidation occurred. The modifying effects of soil on nitrite production and on the cooxidation of ethylene and chloroethane could be circumvented by raising the ammonium concentration in the reaction mixture from 10 to 50 mM. Soil had virtually no effect on the oxidation of 1,1,1-trichloroethane.     

Purification of glycollate oxidase from greening cucumber cotyledons

Glycollate oxidase (glycollate: oxygen oxidoreductase, EC 1.1.3.1) was purified to apparent homogeneity from crude extracts of greening cucumber cotyledons (Cucumis sat vus). Molecular sieving and chromatofocusing resulted in 700-fold purification and specific activity of 1 kat mg^-1 protein. The enzyme exhibited a M_r of 180,000, or 700,000, respectively, and is a tetramer or 16-mer made of identical subunits of M_r 43,000. Monospecific antibodies were raised against the homogeneous protein.     

Physiological responses of Pinus sylvestris to atmospheric ammonia

Summary Young saplings of Pinus sylvestris were fumigated for 3 months with ammonia in concentrations ranging from 0 to 240 g m^-3. Despite the much higher concentrations than normal in the field, no visible damage occurred. Photosynthesis, dark respiration, transpiration and biomass production were stimulated. At 240 g m^-3 with high irradiance (PAR: 950 mol m^-2 s^-1), net photosynthesis was stimulated by 24% and dark respiration by 76%. Intitial light use efficiency was not significantly affected. Transpiration increased, both in the dark and at 950 mol m^-2 s^-1 by 40% and 57%, respectively. In the presence of ammonia, stomatal control was less efficient. Though growth of roots was not affected by NH₃, that of current year needles was stimulated, resulting in an increased mass ratio of needles to roots. The nitrogen content of the needles increased, but the contents of other mineral components did not change significantly. Due to increased transpiration per unit of needle area and increased mass of needles per tree, water loss per tree was about twice as high in the treatment with 240 g m^-3 as in the control. Towards the end of fumigation, a 10-day period without water supply followed and then the water potential of the shoots was measured as an indicator of water demand. This demand was higher with higher concentrations of NH₃, suggesting a higher risk of injury from drought.     

Indigo production in hairy root cultures of Polygonum tinctorium Lour.

The transformed root culture of Polygonum tinctorium Lour. was established by infecting leaf explants with Agrobacterium rhizogenes A4. These cultures were examined for their growth and indigo content under various culture conditions. Among the four different culture media tested, SH medium showed the highest yield for root growth (28 mg dry wt/30 ml) and indigo production (152 g/dry wt). In SH medium, 30 g sucrose l^–1, 2500 mg KNO₃ l^–1, 300 mg NH₄H₂PO₄ l^–1 were the best conditions for indigo production at pH 5.7. The production of indigo in hairy roots slightly increased with the addition of 200 mg chitosan l^–1 (186 g/dry wt) and 20 U pectinase l^–1 (181 g/dry wt).     

Kinetic Studies of Ammonia Monooxygenase Inhibition in Nitrosomonas europaea by Hydrocarbons and Halogenated Hydrocarbons in an Optimized Whole-Cell Assay

The inhibitory effects of 15 hydrocarbons and halogenated hydrocarbons on NH₃ oxidation by ammonia monooxygenase (AMO) in intact cells of the nitrifying bacterium Nitrosomonas europaea were determined. Determination of AMO activity, measured as NO₂^- production, required coupling of hydroxylamine oxidoreductase (HAO) activity with NH₃-dependent NH₂OH production by AMO. Hydrazine, an alternate substrate for HAO, was added to the reaction mixtures as a source of reductant for AMO. Most inhibitors exhibited competitive or noncompetitive inhibition patterns. The competitive character generally decreased (K_iE/K_iES increased) as the molecular size of the inhibitors increased. For example, CH₄ and C₂H₄ were competitive inhibitors of NH₃ oxidation, whereas the remaining alkanes (up to C₄) and monohalogenated (Cl, Br, I) alkanes were noncompetitive. Oxidation of C₂H₅Br (noncompetitive) increased as the NH₄⁺ concentration increased up to 40 mM, whereas oxidations of inhibitors with competitive character (K_iE K_iES) were diminished at 40 mM NH₄⁺. Multichlorinated compounds produced nonlinear Lineweaver-Burk plots. Iodinated alkanes (CH₃I, C₂H₅I) and C₂Cl₄ were potent inhibitors of NH₃ oxidation. Maximum rates of NH₃, C₂H₄, and C₂H₆ oxidations were approximately equivalent, suggesting a common rate-determining step. These data support an active-site model for AMO consisting of an NH₃-binding site and a second site that binds noncompetitive inhibitors, with oxidation occurring at either site.     

The bioenergetics of ammonia and hydroxylamine oxidation in Nitrosomonas europaea at acid and alkaline pH

Autotrophic ammonia oxidizers depend on alkaline or neutral conditions for optimal activity. Below pH 7 growth and metabolic activity decrease dramatically. Actively oxidizing cells of Nitrosomonas europaea do not maintain a constant internal pH when the external pH is varied from 5 to 8. Studies of the kinetics and pH-dependency of ammonia and hydroxylamine oxidation by N. europaea revealed that hydroxylamine oxidation is moderately pH-sensitive, while ammonia oxidation decreases strongly with decreasing pH. Oxidation of these oxogenous substrates results in the generation of higher proton motive force which is mainly composed of a . Hydroxylamine, but not ammonia, is oxidized at pH 5, which leads to the generation of a high proton motive force which drives energy-dependent processes such as ATP-synthesis and secondary transport of amino acids.Endogenoussubstrates can be oxidized between pH 5 to 8 and this results in the generation of a considerable proton motive force which is mainly composed of a . Inhibition of ammonia-mono-oxygenase or cytochrome aa3 does not influence the magnitude of this gradient or the oxygen consumption rate, indicating that endogenous respiration and ammonia oxidation are two distinct systems for energytransduction.The results indicate that the first step in ammonia oxidation is acid sensitive while the subsequent steps can take place and generate a proton motive force at acid pH.