Über die Wirkung von Arsenat auf Nitratreduktion,Atmung und Photosynthese von Grünalgen

Summary The effect of sodium arsenate on the metabolism of the green alga,Ankistrodesmus braunii (strain Marburg) increases with increasing arsenate concentration and with decreasing phosphate concentration. Above 10^–4 M arsenate, in a phosphate-free medium of pH 4.1, an increase in respiratory oxygen uptake and inhibition of photosynthesis were observed. At the highest concentration used (5·10^–2 M arsenate), the rate of respiration was about 250% of that of the controls, whereas photosynthesis had dropped to about 20% of the normal value. In addition, arsenate was found to be an inhibitor of the reduction of nitrite in the dark, whereas it does not inhibit the first step of nitrate reduction,i.e. the reaction nitrate nitrite. Therefore, in a solution containing nitrate, addition of arsenate leads to accumulation of nitrite. These results further support the assumption, derived previously from experiments with 2,4-dinitrophenol, that the further reduction of nitrite requires high-energy phosphate, in contrast to the first step of nitrate reduction.

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Effect of aerobic and anaerobic conditions on the in vivo nitrate reductase assay in spinach leaves

¹⁵N-labelled nitrate was used to show that nitrate reduction by leaf discs in darkness was suppressed by oxygen, whereas nitrite present within the cell could be reduced under aerobic dark conditions. In other experiments, unlabelled nitrite, allowed to accumulate in the tissue during the dark anaerobic reduction of nitrate was shown by chemical analysis to be metabolised during a subsequent dark aerobic period. Leaves of intact plants resembled incubated leaf discs in accumulating nitrite under anaerobic conditions. Nitrate, n-propanol and several respiratory inhibitors or uncouplers partly reversed the inhibitory effect of oxygen on nitrate reduction in leaf discs in the dark. Of these nitrate and propanol acted synergistically. Reversal was usually associated with inhibition of respiration but some concentrations of 2,4-dinitrophenol (DNP) and ioxynil reversed inhibition without affecting respiratory rates. Respiratory inhibitors and uncouplers stimulated nitrate reduction in the anaerobic in vivo assay i.e. in conditions where the respiratory process is non-functional. Freezing and thawing leaf discs diminished but did not eliminate the sensitivity of nitrate reduction to oxygen inhibition.Abbreviations DNP 2,4-dinitrophenol - HOQNO 8-hydroxyquinoline-N-oxide - DCPIP 2,6-dichlorophenolindophenol - CCCP Carbonyl cyanide m-chlorophenylhydrazone - TES N-tris(hydroxymethyl)methyl-2-amino ethanesulphonic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid     

Nitrate Uptake by the Halotolerant Cyanobacterium <Emphasis Type="Italic">Aphanothece halophytica</Emphasis> Grown Under Non-Stress and Salt-Stress Conditions

We have compared the characteristics of nitrate uptake by Aphanothece halophytica grown under non-stress and salt-stress conditions. Both cell types showed essentially similar patterns of nitrate uptake toward ammonium, nitrite, and DL-glyceraldehyde. Although the affinities of nitrate to non-stress cells and salt-stress cells were not significantly different, i.e., K_s = 416 and 450 µM, respectively, the V_max value for non-stress cells was about twofold of that for salt-stress cells (9.1 vs 5.3 µmol min^–1 mg^–1 Chl). Nitrate uptake by A. halophytica was found to be dependent on Na⁺. Ammonium inhibited nitrate uptake, and the presence of methionine sulfoximine could not release the inhibition by ammonium. Nitrite appeared to competitively inhibit nitrate uptake with a K_i value of 84 µM. Both chloride and phosphate anions did not affect nitrate uptake. DL-Glyceraldehyde, an inhibitor of CO₂ fixation, caused a reduction in the uptake of nitrate.Received: 22 October 2002 / Accepted: 6 December 2002     

Interactions Between Nitrate Assimilation and 2,4-Dinitrophenol Cometabolism in Rhodobacter capsulatus E1F1

The phototrophic, nitrate-photoassimilating bacterium Rhodobacter capsulatus E1F1 cometabolizes 2,4-dinitrophenol (DNP) by photoreducing it to 2-amino-4-nitrophenol under anaerobic conditions. DNP uptake and nitrate metabolism share some biochemical features, and in this article we show that both processes are influenced by each other. Thus, as was demonstrated for nitrate assimilation, DNP uptake requires a thermolabile periplasmic component. Nitrate assimilation is inhibited by DNP, which probably affects the nitrite reduction step because neither nitrate reductase activity nor the transport of nitrate or nitrite is inhibited. On the other hand, DNP uptake is competitively inhibited by nitrate, probably at the transport level, because the nitroreductase activity is not inhibited in vitro by nitrate, nitrite, or ammonium. In addition, the decrease in the intracellular DNP concentration in the presence of nitrate probably inactivates the nitroreductase. These results allow prediction of a negative environmental effect if nitrate and DNP are released together to natural habitats, because it may lead to a lower rate of DNP metabolism and to nitrite accumulation.     

Effect of xenobiotics on inorganic nitrogen assimilation by the phototrophic bacteriumRhodobacter capsulatus E1F1

In the presence of nitroaromatic and haloaromatic derivatives,Rhodobacter capsulatus E1F1 growth was affected in different degrees depending on the nitrogen source used. Phototrophic growth on glutamate or ammonium was not inhibited by 2,4-dinitrophenol (2,4-DNP), 4-nitrophenol (4-NP), 2-amino-4-nitrophenol (2,4-ANP), 4-aminophenol (4-AP), or 4-chlorophenol (4-CIP), whereas 2,4-dinitrophenol and 4-chlorophenol partially inhibited bacterial growth in nitrate, nitrite, and dinitrogen. On the other hand, although photosynthetic nitrate uptake was significantly inhibited by 2,4-dinitrophenol, 4-chlorophenol inhibited it to a lesser extent. Nitrogen fixation was severely inactivated in vivo by 2,4-dinitrophenol, but nitrate reductase activity was activated in vivo by 2,4-dinitrophenol, 4-nitrophenol, and 4-chlorophenol. Similar effects were found in cells growing with nitrate and 2,4-dinitrophenol under dark and aerobiosis conditions. None of the enzymatic activities related to inorganic nitrogen assimilation were affected by xenobiotics in vitro.     

Short-term ammonium inhibition of nitrate uptake by Azotobacter chroococcum

Addition of NH₄Cl at low concentrations to Azotobacter chroococcum cells caused an immediate cessation of nitrate uptake activity, which was restored when the added NH ₄ ⁺ was exhausted from the medium or by adding an NH ₄ ⁺ assimilation inhibitor, l-methionine-dl-sulfoximine (MSX) or l-methionine sulfone (MSF). In the presence of such inhibitors the newly-reduced nitrate was released into the medium as NH ₄ ⁺ . When the artificial electron donor system ascorbate/N-methylphenazinium methylsulfate (PMS), which is a respiratory substrate that was known to support nitrate uptake by A. chroococcum while inhibiting glutamine synthetase activity, was the energy source, externally added NH ₄ ⁺ had no effect on nitrate uptake. It is concluded that, in A. chroococcum cells, NH ₄ ⁺ must be assimilated to exert its short-term inhibitory effect on nitrate uptake. A similar proposal was previously made to explain the short-term ammonium inhibition of N₂ fixation in this bacterium.Abbreviations MOPS morpholinopropanesulfonic acid - MSX l-methionine-dl-sulfoximine - PMS N-methylphenazinium methylsulfate - MSF l-methionine sulfone     

3-Nitroadipate, a Metabolic Intermediate for Mineralization of 2,4-Dinitrophenol by a New Strain of a Rhodococcus Species

The bacterial strain RB1 has been isolated by enrichment cultivation with 2,4-dinitrophenol as the sole nitrogen, carbon, and energy source and characterized, on the basis of 16S rRNA gene sequence comparison, as a Rhodococcus species closely related to Rhodococcus opacus. Rhodococcus sp. strain RB1 degrades 2,4-dinitrophenol, releasing the two nitro groups from the compound as nitrite. The release of nitro groups from 2,4-dinitrophenol occurs in two steps. First, the 2-nitro group is removed as nitrite, with the production of an aliphatic nitro compound identified by ¹H nuclear magnetic resonance and mass spectrometry as 3-nitroadipate. Then, this metabolic derivative is further metabolized, releasing its nitro group as nitrite. Full nitrite assimilation upon reduction to ammonia requires that an additional carbon source be supplied to the medium.     

Nitrate transport system in Neurospora crassa

Nitrate uptake in Neurospora crassa has been investigated under various conditions of nitrogen nutrition by measuring the rate of disappearance of nitrate from the medium and by determining mycelial nitrate accumulation. The nitrate transport system is induced by either nitrate or nitrite, but is not present in mycelia grown on ammonia or Casamino Acids. The appearance of nitrate uptake activity is prevented by cycloheximide, puromycin, or 6-methyl purine. The induced nitrate transport system displays a K_m for nitrate of 0.25 mM. Nitrate uptake is inhibited by metabolic poisons such as 2,4-dinitrophenol, cyanide, and antimycin A. Furthermore, mycelia can concentrate nitrate 50-fold. Ammonia and nitrite are non-competitive inhibitors with respect to nitrate, with K_i values of 0.13 and 0.17 mM, respectively. Ammonia does not repress the formation of the nitrate transport system. In contrast, the nitrate uptake system is repressed by Casamino Acids. All amino acids individually prevent nitrate accumulation, with the exception of methionine, glutamine, and alanine. The influence of nitrate reduction and the nitrate reductase protein on nitrate transport was investigated in wild-type Neurospora lacking a functional nitrate reductase and in nitrate non-utilizing mutants, nit-1, nit-2, and nit-3. These mycelia contain an inducible nitrate transport system which displays the same characteristics as those found in the wild-type mycelia having the functional nitrate reductase. These findings suggest that nitrate transport is not dependent upon nitrate reduction and that these two processes are separate events in the assimilation of nitrate.     

Evidence for the nitrate assimilation-dependent nitrite excretion in cyanobacterium Nostoc MAC

Nitrate uptake and nitrite efflux patterns in Nostoc MAC showed a rapid phase followed by their saturation. Nitrite efflux was maximum in nitrate medium whereas the cells incubated in N₂ and NH ₄ ⁺ media exhibited a decreased nitrite efflux activity. The simultaneous presence of NH ₄ ⁺ and nitrate significantly decreased nitrite efflux. L-Methionine-Dl-sulphoximine (MSX) prevented inhibition of nitrite efflux by NH ₄ ⁺ . In the dark there was negligible nitrite efflux, whereas illumination increased the rate of nitrite efflux significantly. The nitrite efflux system was maximally operative at pH 8.0, 30°C and a photon fluence rate of 50 mol m^-2. s^-1. These results confirm that (i) the nitrite efflux system in Nostoc MAC is dependent upon nitrate uptake and assimilation and is repressible by NH ₄ ⁺ ; (ii) NH ₄ ⁺ itself is not the actual repressor of nitrite efflux; a product of NH ₄ ⁺ assimilation via glutamine synthetase (GS) is required for repression to occur; (iii) the catalytic function of GS does not appear to be involved in nitrate assimilation-dependent nitrite efflux, and (iv) the optimum pH, temperature and illumination for maximum nitrite efflux were found to be 8.0, 30°C and 50mol m^-2. s respectively.B.B. Singh, P.K. Pandey and P.S. Bisen are with the Department of Microbiology, Barkatullah University. Bhopal 462026, India. S.Singh is with the Department of Microbiology, School of Life Sciences, North Maharashtra University, Jalgaon, India     

Na+-stimulated nitrate uptake with increased activity under osmotic upshift in Synechocystis sp. strain PCC 6803

In the non-diazotrophic cyanobacterium Synechocystis sp. strain PCC 6803, an osmolality of 30 and 40 mosmol/kg sorbitol and NaCl resulted in 3.5- and 4.5-fold increase of nitrate uptake, respectively. The NaCl-stimulated uptake was abolished by treatment with chloramphenicol. At 25 mosmol/kg or higher, NaCl induced higher nitrate uptake than sorbitol suggesting an ionic effect of Na⁺. The nitrate uptake in Synechocystis showed K _s and V _max values of 46 μM and 1.37 μmol/min/mg Chl, respectively. Mutants disrupted in nitrate and nitrite reductase exhibited a decreased nitrate uptake. Ammonium, chlorate, and dl-glyceraldehyde caused a reduction of nitrate uptake. Dark treatment caused a drastic reduction of uptake by 70% suggesting an energy-dependent system. Nitrate transport was sensitive to various metabolic inhibitors including those dissipating proton gradients and membrane potential. The results suggest that nitrate uptake in Synechocystis is stimulated by Na⁺ ions and requires energy provided by the functioning electron transport chain.     

Selenite uptake and incorporation by Selenomonas ruminatium

Selenite uptake and incorporation in Selenomonas ruminantium was constitutive with an inducible component. It was distinct from sulphate or selenate transport, since sulphate and selenate did not inhbit uptake, nor could sulphate or selenate uptake be demonstrated. Selenite uptake had an apparent K _m of 1.28 mM and a V _max of 148 ng Se min^-1 mg^-1 protein. Uptake was sensitive to inhibition by 2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenyl hydrazone (CCCP), azide, iodoacetic acid (IAA) and N-ethylmaleimide (NEM), but not chloropromazine (CPZ), N,N-dicyclohexyl-carbodiimide (DCCD), quinine, arsenate, or fluoride. Treatment of cells accumulating ⁷⁵[Se]-Selenite with 2,4,DNP inhibited uptake, but did not cause efflux. Transport of selenite was inhibited by sulphite and nitrite, but not by nitrate, phosphate, sulphate of selenate. ⁷⁵[Se]-Selenite was incorporated into selenocystine, selenoethionine, selenohomocysteine, and selenomethionine and was also reduced to red elemental selenium.     

Inhibition of nucleic acids synthesis in Chlorella by 2,4-dinitrophenol a unique concentration effect on DNA synthesis

Nucleic acids and protein synthesis in synchronously growing Chlorella cells were inhibited by 2,4-dinitrophenol. RNA and protein synthesis decreased gradually from about 100% at 0.1 mM to almost 0% at 10 mM dinitrophenol. DNA synthesis was strongly inhibited at 0.5 mM but less at 1 mM concentration of the inhibitor. Beyond 1 mM the inhibitory effect increased again. A transient exposure to 0.5 and 10 mM dinitrophenol was fully reversible and cell division after the inhibition proceeded normally except for a slight delay.Abbreviation DNP 2,4-dinitrophenol     

The effect of sodium nitrite on the growth of Micrococcus denitrificans

Summary Nitrite accumulates during the growth of M. denitrificans in a medium in which nitrate is the terminal oxidant. If H₂ is the electron donor for nitrate reduction, the level of nitrite produced is sufficiently high to inhibit hydrogenase; this inhibition consequently inhibits growth. Yeast extract alleviates the inhibition and permits a resumption of growth. The release from inhibition may result from the provision of a growth factor for or from a more rapid induction of, a system for nitrite dissimilation.Dedicated to Prof. C. B. van Niel on the occasion of his 70th birthday. The author is indepted to Prof. Van Niel for many helpful suggestions and discussions, and for the hospitality of his laboratory during the author's stay as a National Science Foundation Post-Doctoral Fellow.     

Transport and metabolism of glucose and arabinose in Bifidobacterium breve

Glucose was required for the transport of arabinose into Bifidobacterium breve. The non-metabolisable glucose analogue 2-deoxy-d-glucose (2-DG) did not facilitate assimilation of arabinose. Studies using d-[U-¹⁴C]-labelled arabinose showed that it was fermented to pyruvate, formate, lactate and acetate, whereas the principal metabolic products of d-[U-¹⁴C]-labelled glucose were acetate and formate. In contrast to glucose, arabinose was not incorporated into cellular macromolecules. A variety of metabolic inhibitors and inhibitors of sugar transport (proton ionophores, metal ionophores, compounds associated with electron transport) were used to investigate the mechanisms of sugar uptake. Only NaF, an inhibitor of substrate level phosphorylation, and 2-DG inhibited glucose assimilation. 2-DG had no effect on arabinose uptake, but NaF was stimulatory. High levels of phosphorylation of glucose and 2-DG by PEP and to a lesser degree, ATP were seen in phosphoenolpyruvate: phosphotransferase (PEP:PTS) assays. These data together with strong inhibition of glucose uptake by NaF suggest a role for phosphorylation in the transport process. Arabinose uptake in B. breve was not directly dependent on phosphorylation or any other energy-linked form of transport but may be assimilated by glucose-dependent facilitated diffusion.Abbreviations (2,4-DNP) 2,4-dinitrophenol - (2,4-DNP) carbonylcyanide m-chlorophenylhydrazone - (CCCP) (phosphoenolpyruvate phosphotransferase system) - PEP: PTS trichloroacetic acid - (TCA) 2-deoxy-d-glucose - (2-DG) 2-deoxy-d-glucose     

Über die energetische Kopplung der Nitritassimilation von Grünalgen an Atmung und Photosynthese

Summary Inhibitors and uncouplers of phosphorylation, i.e., arsenate, 2.4-dinitrophenol (DNP), pentachlorophenol (PCP), and carbonyl cyanide m-chlorophenylhydrazone (CCCP), inhibit the assimilation of nitrite by the green alga Ankistrodesmus braunii in the dark and in the light. In a medium containing nitrate, these inhibitors interrupt nitrate reduction at the level of nitrite. In phosphatedeficient algae, the assimilation of nitrite can be decreased by a concomitant, energy-dependent uptake of chloride and phosphate ions. These results support the assumption that high-energy phosphate is required for the assimilation of nitrite.CO₂ and glucose (after pre-illumination) increase nitrite assimilation in the light. Photosynthetic nitrite reduction is inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), an inhibitor of oxygen evolution, and by disalicylidene-propanediamine-(1,3) (DSPD), an inhibitor of the photosynthetic reduction of ferredoxin.

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Abkürzungen CCCP Carbonylcyanid-m-chlorphenylhydrazon - DCMU 3-(3,4-Dichlorphenyl)-1,1-dimethylharnstoff - DNP 2,4-Dinitrophenol - DSPD Disalicylidenpropandiamin-(1,3) - PCP Pentachlorphenol - JAA Jodacetamid     

Ferrous iron dependent nitric oxide production in nitrate reducing cultures of Escherichia coli

l-Lactate-driven ferric and nitrate reduction was studied in Escherichia coli E4. Ferric iron reduction activity in E. coli E4 was found to be constitutive. Contrary to nitrate, ferric iron could not be used as electron acceptor for growth. Ferric iron reductase activity of 9 nmol Fe²⁺ mg^-1 protein min^-1 could not be inhibited by inhibitors for the respiratory chain, like Rotenone, quinacrine, Actinomycin A, or potassium cyanide. Active cells and l-lactate-driven nitrate respiration in E. coli E4 leading to the production of nitrite, was reduced to about 20% of its maximum activity with 5 mM ferric iron, or to about 50% in presence of 5 mM ferrous iron. The inhibition was caused by nitric oxide formed by a purely chemical reduction of nitrite by ferrous iron. Nitric oxide was further chemically reduced by ferrous iron to nitrous oxide. With electron paramagnetic resonance spectroscopy, the presence of a free [Fe²⁺-NO] complex was shown. In presence of ferrous or ferric iron and l-lactate, nitrate was anaerobically converted to nitric oxide and nitrous oxide by the combined action of E. coli E4 and chemical reduction reactions (chemodenitrification).     

Euglena gracilis: Phenotypic resistance to 2,4-dinitrophenol

Euglena gracilis, when grown on a medium containing 10^?5m 2,4-dinitrophenol, will initially bleach, cease to divide, and about one-half will die off. After a prolonged lag period of 6–8 days, the remaining cells green and begin to multiply. The resultant cells are resistant to dinitrophenol and will grow in its presence at rates close to those in normal medium. The resistant cells do not differ greatly from the nonresistant ones, except that they show no sign of respiratory control, their photosynthetic activity is somewhat reduced, and their size is larger. The resistant cells appear less motile, their flagellar movement is slower, and their motility appears disturbed (they tend to swim in circles). The resistance is lost when the cells are grown for 2–3 generations on medium lacking dinitrophenol.     

Ammonium regulation of urate uptake in Chlamydomonas reinhardtii

Urate was taken up at a negligible rate by Chlamydomonas reinhardtii cells grown on ammonium and transferred to media containing urate plus ammonium or urate plus chloral hydrate or cycloheximide. Addition of ammonium to cells actively consuming urate produced a rapid inhibition of urate uptake whereas the intracellular oxidation of urate was unaffected. Methylammonium but not glutamine or glutamate inhibited urate uptake. Addition of l-methionine-dl-sulfoximine to cells actively consuming urate provoked ammonium excretion, which was accompanied by a rapid inhibition of urate uptake. In cells growing on urate and exhibiting noticeable levels of nitrite-reductase activity, nitrite caused a sudden inhibition of urate uptake whereas nitrate required a time to induce nitrate reductase and to exert its inhibitory effect on uptake. The urate-uptake system did not require urate for induction since the urate-uptake capacity appeared in nitrogen-starved cells. From these results it is concluded that, in Chlamydomonas reinhardtii, ammonium inhibits urate uptake and also acts as co-repressor of the uptake system.     

Nitrate transport and its regulation by O2 in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an obligate respirer which can utilize nitrate as a terminal electron acceptor under anaerobic conditions (denitrification). Immediate, transient regulation of nitrate respiration is mediated by oxygen through the inhibition of nitrate uptake. In order to gain an understanding of the bioenergetics of nitrate transport and its regulation by oxygen, the effects of various metabolic inhibitors on the uptake process and on oxygen regulation were investigated. Nitrate uptake was stimulated by the protonophores carbonyl cyanide m-chlorophenylhydrazone and 2,4-dinitrophenol, indicating that nitrate uptake is not strictly energized by, but may be affected by the proton motive force. Oxygen regulation of nitrate uptake might in part be through redox-sensitive thiol groups since N-ethylmaleimide at high concentrations decreased the rate of nitrate transport. Cells grown with tungstate (deficient in nitrate reductase activity) and azide-treated cells transported nitrate at significantly lower rates than untreated cells, indicating that physiological rates of nitrate transport are dependent on nitrate reduction. Furthermore, tungstate grown cells transported nitrate only in the presence of nitrite, lending support to the nitrate/nitrite antiport model for transport. Oxygen regulation of nitrate transport was relieved (10% that of typical anaerobic rates) by the cytochrome oxygen reductase inhibitors carbon monoxide and cyanide.     

Role of ATP in nitrite reduction in roots of wheat and pea

Excised wheat (Triticum aestivum L.) and field pea (Pisum arvense L.) roots, incubated under anaerobic conditions or in the presence of uncouplers of oxidative phosphorylation [2,4-dinitrophenol (DNP), carbonylcyanide-m-chlorophenylhydrazone, pentachlorophenol] accumulated nitrite as a result of an inhibition of nitrite reduction. In isolated root plastids, nitrite reduction was dependent on a supply of glucose-6-phosphate (G6P) and did not require ATP. The estimated K_m value for glucose 6-phosphate was 1.25 mM. Glucose and fructose-1,6-diphosphate were ineffective substrates for nitrate reduction. Anaerobic conditions and treatment with DNP, which would result in a cessation of ATP production by the mitochondria and a stimulation of glycolysis via the Pasteur effect, were shown to decrease the G6P content of excised roots of wheat and pea. A negative correlation was observed between the level of G6P and nitrite accumulation on root tissues. It is proposed that an interruption in the supply of G6P to the root plastid under these conditions would result in an inhibition of nitrite reduction leading to nitrite accumulation.Abbreviation G6P glucose-6-phosphate