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.     

Catabolism of 2,6-dinitrophenol by Alcaligenes eutrophus JMP 134 and JMP 222

Both Alcaligenes eutrophus JMP 134 and its plasmid-free derivative Alcaligenes eutrophus JMP 222 utilize 2,6-dinitrophenol as sole source of carbon, energy, and nitrogen. In the presence of ammonia resting cells of these strains release two mol of nitrite per mol of 2,6-dinitrophenol. Alcaligenes eutrophus JMP 222-1D, a mutant of strain JMP 222 obtained by transposon (Tn5) mutagenesis, is able to use 2,6-dinitrophenol as nitrogen source but not as source of carbon and energy. Resting cells of this mutant liberate only one mol of nitrite per mol of 2,6-dinitrophenol. A single metabolite was detected by high-pressure liquid chromatography and identified as 2-hydroxy-5-nitropenta-2,4-dienoic acid from the mass spectrum, the ¹H-, and ¹³C-NMR spectra. Strain JMP 222-1S, a spontaneous mutant of strain JMP 222-1D, accumulates 4-nitropyrogallol which was identified as the initial metabolite of 2,6-dinitrophenol degradation.Non-standard abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2,6-DNP 2,6-dinitrophenol - HNMA 2-hydroxy-5-nitromuconic acid - HNPA 2-hydroxy-5-nitropenta-2,4-dienoic acid - NB nutrient broth - NMR nuclear magnetic resonance - NPG 4-nitropyrogallol - O.D. optical density - t_R retention time - UV/Vis ultraviolet/visible     

Characterization of a mutant of the yeast Candida maltosa defective in catabolite inactivation of gluconeogenetic enzymes

A spontaneous mutant of the yeast Candida maltosa SBUG 700 was isolated showing pseudohyphal marphology under all growth conditions tested. The C. maltosa PHM mutant takes up glucose with the kinetics of C. maltosa SBUG 700 and starved cells contain the same cyclic AMP concentration. Addition of glucose to the PHM mutant does not result in an increase of the intracellular cyclic AMP level and in catabolite inactivation of fructose-1,6-bisphosphatase, malate dehydrogenase and phosphoenolpyruvate carboxykinase. However, addition of 2,4-dinitrophenol is followed by a rapid, transient increase of the cyclic AMP level in the mutant cells, but not by catabolite inactivation. These results show that a common mechanism might be responsible for catabolite inactivation and glucose-induced cAMP signaling or that glucose-induced cAMP signaling is required for catabolite inactivation in C. maltosa.     

Studies on nitrite reductase in barley

Summary Nitrite reductase from barley seedlings was purified 50–60 fold by ammonium sulphate precipitation and gel filtration. No differences were established in the characteristics of nitrite reductases isolated in this way from either leaf or root tissues. The root enzyme accepted electrons from reduced methyl viologen, ferredoxin, or an unidentified endogenous cofactor. Enzyme activity in both tissues was markedly increased by growth on nitrate. This activity was not associated with sulphite reductase activity. Microbial contamination could not account for the presence of nitrite reductase activity in roots. Nitrite reductase assayed in vitro with reduced methyl viologen as the electron donor was inhibited by 2,4-dinitrophenol but not by arsenate.Abbreviations DNP 2,4-dinitrophenol - DEAE diethyl amino ethyl     

Naphthalene uptake by a Pseudomonas fluorescens isolate

The uptake of naphthalene has been investigated in the metabolizing cells of Pseudomonas fluorescens utilizing [1-14C]naphthalene. The uptake displayed an affinity constant (Kt) of 11 microM and a maximal velocity (Vmax) of 17 nmol.h-1.mg-1 cellular dry weight. Naphthalene uptake was not observed in a mutant strain, TG-5, which was unable to utilize naphthalene as a sole source of carbon for growth. Uptake was significantly inhibited (approximately 90%) by the presence of growth-inhibiting levels of either azide or 2,4-dinitrophenol and was sensitive to the presence of structural analogues of naphthalene. The intracellular levels of ATP were not significantly reduced by the presence of either azide or 2,4-dinitrophenol. The presence of alpha-naphthol was found to noncompetitively inhibit naphthalene uptake, displaying a Ki of 0.041 microM. It is concluded that the first step in the utilization of naphthalene by Pseudomonas fluorescens is its transport into the cell by a specific energy-linked transport system.     

Carbohydrate Uptake and Metabolism of Ophiobolus graminis

The carbohydrate nutrition of Ophiobolus graminis, the cause of the take-all disease of wheat, was investigated in growth and respiration experiments. In a synthetic medium, d-mannitol was the only carbohydrate of thirteen studied which the fungus could not use for growth. However, the fungus was found to take up mannitol by an active mechanism, which was stopped by 2,4-dinitrophenol. Di- and trisaccharides were hydrolyzed extracellularly, and the monosaccharides were assimilated at different rates.     

Effect of 2,4-Dinitrophenol on Auxin-induced Ethylene Production and Auxin Conjugation by Mung Bean Tissue

Auxin-induced ethylene production by mung bean (Phaseolus mungo L.) hypocotyl segments was markedly inhibited by 2,4-dinitrophenol regardless of whether or not kinetin was present. Uptake of indoleacetic acid-2-¹⁴C was also inhibited in the presence of 2,4-dinitrophenol. Segments treated only with indoleacetic acid rapidly converted indoleacetic acid into indole-3-acetylaspartic acid with time whereas kinetin suppressed indoleacetic acid conjugation. Formation of indole-3-acetylaspartic acid was significantly reduced when 2,4-dinitrophenol was present. The suppression of indoleacetic acid conjugation by kinetin and 2,4-dinitrophenol appeared to be additive, and the free indoleacetic acid level in segments treated with 2,4-dinitrophenol in the presence of indoleacetic acid or indoleacetic acid plus kinetin was remarkably higher than in corresponding segments which received no 2,4-dinitrophenol.     

Modulation of the heat shock response by one-carbon metabolism in Escherichia coli.

A genetic screen designed to isolate mutants of Escherichia coli W3110 altered in the ability to induce the heat shock response identified a strain unable to induce the heat shock proteins in a rich, defined medium lacking methionine after exposure to 2,4-dinitrophenol. This strain also grew slowly at 28 degrees C and linearly at 42 degrees C in this medium. The abnormal induction of the heat shock proteins and abnormal growth at both high and low temperatures were reversed when methionine was included in the growth medium. The mutation responsible for these phenotypes mapped to the glyA gene, a biosynthetic gene encoding the enzyme that converts serine and tetrahydrofolate to glycine and 5,10-methylenetetrahydrofolate. This reaction is the major source of glycine and one-carbon units in the cell. Because fixed one-carbon units, in the form of methionine, allowed mutant cells to induce the heat shock response after exposure to 2,4-dinitrophenol, a one-carbon restriction may be responsible for the phenotypes described above.     

Adaptation of Escherichia coli to the uncoupler of oxidative phosphorylation 2,4-dinitrophenol.

Escherichia coli was found to adapt to the uncoupler of oxidative phosphorylation 2,4-dinitrophenol. The rates of synthesis of 53 proteins were increased following exposure to 2,4-dinitrophenol. Adaptation was accelerated when the cofactor pyrroloquinoline quinone was provided in the growth medium.     

Multiple drug resistance in the fission yeast Schizosaccharomyces pombe: Evidence for the existence of pleiotropic mutations affecting energy dependent transport systems

Summary The uptake of L-tyrosine into wild type and antibiotic resistant strains of Schizosaccharomyces pombe requires an energy source, is initially linear with respect to time, is inhibited by 2,4-dinitrophenol and sodium azide and is saturable. However the initial uptake rates and the amount of L-tyrosine accummulated by antibiotic resistant strains are much less than wild type. Comparison of the kinetic constants of uptake shows that mutant strains have a reduced maximum velocity of uptake compared to wild type and a larger Km.Since the three mutant strains possess a permeability barrier to L-tyrosine as well as being drug resistant this is an indication that antibiotic resistance may be caused by a decrease in plasma membrane permeability.     

Isolation of glucosyltransferase fromStreptomyces aureofaciens

Streptomyces aureofaciens B 96 grown on a synthetic medium glucosylated exogenous 1,2-dihydroxy-9, 10-anthraquinone (alizarin). The glucosylation was inhibited by 2,4-dinitrophenol added to the cultivation medium. A cell-free preparation was obtained from the mycelium isolated after 16 h of growth and was found to catalyze the transfer of glucose from glucosyluridyl diphosphate to 1,2-dihydroxy-9, 10-anthraquinone, giving rise to l-hydroxy-2-(β-D-glueopyranosyloxy)-9,10-anthraquinone.     

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 functional significance of glucose dehydrogenase in Klebsiella aerogenes

In order to assess the functional significance of the quinoprotein glucose dehydrogenase recently found to be present in K⁺-limited Klebsiella aerogenes, a broad study was made of the influence of specific environmental conditions on the cellular content of this enzyme. Whereas high activities were manifest in cells from glucose containing chemostat cultures that were either potassium- or phosphate-limited, only low activities were apparent in cells from similar cultures that were either glucose-, sulphate- or ammonia-limited. With these latter two cultures, a marked increase in glucose dehydrogenase activity was observed when 2,4-dinitrophenol (1 mM end concentration) was added to the growth medium. These results suggested that the synthesis of glucose dehydrogenase is not regulated by the level of glucose in the growth medium, but possibly by conditions that imposed an energetic stress upon the cells. This conclusion was further supported by a subsequent finding that K⁺-limited cells that were growing on glycerol also synthesized substantial amounts of glucose dehydrogenase.The enzyme was found to be membrane associated, and preliminary evidence has been obtained that it is located on the periplasmic side of the cytoplasmic membrane and functionally linked to the respiratory chain. This structural and functional orientation is consistent with glucose dehydrogenase serving as a low impedance energy generating system.Abbreviations D dilution rate - DNP 2,4-dinitrophenol - PQQ 2,7,9-tricarboxy-1H-pyrrolo(2,3-f)quinoline-4,5-dione - PTS phosphoenolpyruvate: glucose phosphotransferase - WB Wurster's Blue     

The glucose dehydrogenase-mediated energization of Acinetobacter calcoaceticus as a tool for evaluating its susceptibility to,and defence against,hazardous chemicals

Cells of Acinetobacter calcoaceticus 69-V could be energized by glucose oxidation after the growth on acetate, ethanol, hexanol and benzoate. The velocities of glucose oxidation-driven ATP syntheses were relatively constant in the range from pH 5.4 to 7.5. With decreasing pH values (7.0, 6.0, 5.4) ATP synthesis was inhibited more strongly by the action of 2,4-dinitrophenol and at the same pH value glucose oxidation was nearly unimpaired or inhibited more weakly. This finding is expressed by a decrease of the P/O ratios, indicating the uncoupling of the electron-transport phosphorylation by 2,4-dinitrophenol. The sensitivity towards this uncoupling effect was higher in ethanol-grown cells of Acinetobacter calcoaceticus 69-V than in hexanol- or acetate-grown cells. This increase in sensitivity was accompanied by a decrease of the ratio of saturated (mainly C16:0) to unsaturated (C16:1, C18:1) fatty acids in ethanol-grown cells compared with hexanol-grown ones. The knowledge of such differences in the susceptibility and its molecular background, e.g. possible substrate-induced changes of the fatty acid composition of the cytoplasmic membranes, should help elucidate mechanisms of poisoning by membrane-active hazardous chemicals and develop defence strategies.     

Photoinactivation of the detoxifying enzyme nitrophenol reductase from Rhodobacter capsulatus

The phototrophic bacterium Rhodobacter capsulatus E1F1 detoxifies 2,4-dinitrophenol by inducing an NAD(P)H-dependent iron flavoprotein that reduces this compound to the less toxic end product 2-amino-4-nitrophenol. This nitrophenol reductase was stable in crude extracts containing carotenes, but it became rapidly inactivated when purified protein was exposed to intense white light or moderate blue light intensities, especially in the presence of exogenous flavins. Red light irradiation had no effect on nitrophenol reductase activity. Photoinactivation of the enzyme was irreversible and increased under anoxic conditions. This photoinactivation was prevented by reductants such as NAD(P)H and EDTA and by the excited flavin quencher iodide. Addition of superoxide dismutase, catalase, tryptophan or histidine did not affect photoinactivation of nitrophenol reductase, thus excluding these reactive dioxygen species as the inactivating agent. Substantial protection by 2,4-dinitrophenol also took place when the enzyme was irradiated at a wavelength coinciding with one of the absorption peaks of this compound (365nm). These results suggest that the lability of nitrophenol reductase was due to the absorption of blue light by the flavin prosthetic group, thus producing an excited flavin that might irreversibly oxidize some functional group(s) necessary for enzyme catalysis. Nitrophenol reductase may be preserved in vivo from blue light photoinactivation by the high content of carotenes and excess of reducing equivalents in phototrophic growing cells.Abbreviations 2,4-DNP 2,4-dinitrophenol - ANP 2-amino-4-nitrophenol - EDTA ethylenediamine tetraacetic acid - MES 2-(N-Morpholino) ethanesulfonic acid - NPR nitrophenol reductase     

Transformation of nitroaromatic compounds by a methanogenic bacterium,Methanococcus sp. (strain B)

The transformation of several nitroaromatic compounds by a newly isolated methanogenic bacterium, Methanococcus sp. (strain B) was studied. The presence of nitroaromatic compounds (0.5 mM) viz., nitrobenzene, 2,4-dinitrobenzene, 2,4,6-trinitrobenzene, 2,4-dinitrophenol, 2,4-dinitrobenzene, and 2,6-dinitrotoluene in the culture medium did not inhibit growth of the isolate. The bacteria grew rapidly and reached stationary phase within seven days of incubation. All the nitroaromatic compounds tested were 80 to 100% transformed by the bacterium to amino compounds by a reduction process. The isolate did not use the nitroaromatic compounds as the sole source of carbon or nitrogen. The transformation of nitroaromatic compounds by this isolate was compared to that of other methanogenic bacteria. Out of five methanogens studied, only Methanococcus deltae and Methanococcus thermolithotrophicus could transform the nitroaromatic compounds; however, the transformation rates were significantly less than that of the new isolate Methanococcus sp. (strain B). The nitroaromatic compounds were not transformed by Methanosarcina barkeri, Methanobacterium thermoautotrophicum, and Methanobrevibacter ruminantium.Abbreviations NB Nitrobenzene - DNB 2,4-Dinitrobenzene - TNB 2,4,6-Trinitrobenzene - DNP 2,4-Dinitrophenol - 2,4-DNT 2,4-Dinitrotoluene - 2,6-DNT 2,6-Dinitrotoluene     

Acceleration of proton exchange between octanol and water by 2,4-dinitrophenol determined by 1H-NMR spectrometry a new model for proton transfer in a hydrophobic environment

The rate of proton transfer between the octanol -OH group and water dissolved in octanol after partition equilibrium was determined by ¹H-NMR spectrometry. The rate was found to depend on the pH of the aqueous phase, being minimal at about pH 11. The uncoupler of oxidative phosphorylation 2,4-dinitrophenol at about 10^?3 M accelerated proton transfer several-fold. Its effect was shown to depend on the concentration of the neutral form of 2,4-dinitrophenol in the octanol phase, irrespective of the pH of the aqueous phase. This effect is suggested to be based on the catalytic action of the phenolic -OH group in 2,4-dinitrophenol. The importance of this effect in the uncoupling action of 2,4-dinitrophenol is discussed.     

Photoinduced conidiation inTrichoderma viride: A study with inhibitors

The influence of a number of inhibitors affecting respiration, oxidative phosphorylation, cAMP-phosphodiesterase and of the antioxidant 1,4-dithiothreitol on growth and photoinduced conidiation ofTrichoderma viride were investigated. In all cases, growth and conidiation were influenced to a different extent. Among the first group of compounds, antimycin A was the most potent inhibitor of conidiation while it influenced growth much less. A similar effect was obtained with 2,4-dinitrophenol and 1,4-dithiothreitol. On the other hand, 3-isobutyl-1-methylxanthine (inhibitor of phosphodiesterase) greatly stimulated the conidiation induced by light without affecting growth. It is concluded that the redox reactions represent a vital component of the differentiation pathway and that cAMP may play a regulatory role in this process.