Cadmium-copper antagonism in the activation of periplasmic nitrous oxide reductase of copper-deficient cells fromPseudomonas stutzeri

Summary Copper-deficient cells ofPseudomonas stutzeri strain ZoBell synthesize catalytically inactive nitrous oxide (N₂O) reductase which is activated by added Cu(II) in the absence of de novo protein synthesis. The apparentK _m for the activation process is 0.13 M. Activation is temperature-dependent and is inhibited by Cd(II)(K _i 1.27 M) and less strongly by Zn(II), Ni(II), and Co(II). The same metal ions at 20 M have little or no effect on N₂O reduction of intact cells. Apo-N₂O reductase of transposon Tn5-inducednos ^– mutants with defective Cu-chromophore biosynthesis is not reactivated by Cu(II). N₂O reductase of Cu-sufficient and Cu-deficient wild type, and ofnos ^– mutants is localized in the periplasm, the latter providing the likely site of metal incorporation into the apoenzyme.     

Role of mitochondria in ethanol tolerance of Saccharomyces cerevisiae

The presence of active mitochondria and oxidative metabolism is shown to be essential to maintain low inhibition levels by ethanol of the growth rate (), fermentation rate (v) or respiration rate () of Saccharomyces cerevisiae wild type strain S288C. Cells which have respiratory metabolism show K _i (ethanol inhibition constant) values for , v and , higher (K _i>1 M) than those of petite mutants or grande strains grown in anaerobiosis (K _i=0.7 M). In addition, the relationship between or v and ethanol concentration is linear in cells with respiratory metabolism and exponential in cells lacking respiration. When functional mitochondria are transferred to petite mutants, the resulting strain shows K _i values similar to those of the grande strain and the inhibition of and v by increasing ethanol concentrations becomes linear.     

The influence of temperature on glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and the regulation of these enzymes in a mesophilic and a thermophilic cyanobacterium

The activities and kinetics of the enzymes G6PDH (glucose-6-phosphate dehydrogenase) and 6PGDH (6-phosphogluconate dehydrogenase) from the mesophilic cyanobacterium Synechococcus 6307 and the thermophilic cyanobacterium Synechococcus 6716 are studied in relation to temperature. In Synechococcus 6307 the apparent K _m's are for G6PDH: 80M (substrate) and 20M (NADP⁺); for 6PGDH: 90M (substrate) and 25M (NADP⁺). In Synechococcus 6716 the apparent K _m's are for G6PDH: 550M (substrate) and 30M (NADP⁺); for 6PGDH: 40M (substrate) and 10M (NADP⁺). None of the K _m's is influenced by the growth temperature and only the K _m's of G6PDH for G6P are influenced by the assay temperature in both organisms. The idea that, in general, thermophilic enzymes possess a lower affinity for their substrates and co-enzymes than mesophilic enzymes is challenged.Although ATP, ribulose-1,5-bisphosphate, NADPH and pH can all influence the activities of G6PDH and 6PGDH to a certain extent (without any difference between the mesophilic and the thermophilic strain), they cannot be responsible for the total deactivation of the enzyme activities observed in the light, thus blocking the pentose phosphate pathway.Abbreviations G6PDH glucose-6-phosphate, dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase - G6P glucose-6-phosphate - 6PG 6-phosphogluconate - RUDP ribulose-1,5-bisphosphate - Tricine N-Tris (hydroxymethyl)-methylglycine     

The Biogeochemical Cycle of Methane on the Northwestern Shelf of the Black Sea

Seasonal investigations of methane distribution and rates of its oxidation and generation in the water column and sediments of the Black Sea northwestern shelf were carried out within the framework of the interdisciplinary projects European River–Ocean Systems (EROS-2000, EROS-21) and Biogenic Gases Exchange in the Black Sea (BigBlack) in August 1995, May 1997, and December 1999. Experiments that involved the addition of ¹⁴CH₃COONa and ¹⁴CO₂ to sediment samples showed the main part of methane to be formed from CO₂. Maximum values of methane production (up to 559 mol/(m² day)) were found in coastal sediments in summer time. In winter and spring, methane production in the same sediments did not exceed 3.6–4.2 mol/(m² day). The ¹³C values of methane ranged from –70.7 to –81.8, demonstrating its microbial origin and contradicting the concept of the migration of methane from cold seeps or from the oil fields located on the Black Sea shelf. Experiments that involved the addition of ¹⁴CH₄ to water and sediment samples showed that a considerable part of methane is oxidized in the upper horizons of bottom sediments and in the water column. Nevertheless, it was found that, in summer, part of the methane (from 6.8 to 320 mol/(m² day)) arrives in the atmosphere.     

Properties of an enzymatic complex active in sulfite and thiosulfate oxidation by Rhodotorula sp.

An enzymatic complex from Rhodotorula was characterized and it was indicated that it possessed thiosulfate-oxidizing activity, forming tetrathionate as well as sulfite oxidase activity. Both activities coupled with ferricyanide and native cytochrome c but no with mammalian cytochrome c. Activities of these enzymes were inhibited by thiol inhibitors. Chelating agents did not affect thiosulfate oxidizing activity and only moderately inhibited sulfite oxidase. Both activities disappeared after treatment with proteolytic enzymes or sodium deoxycholate which indicates an essential role played not only by protein but also by phospholipids in the enzymatic activity of the complex. Thiosulfate oxidizing enzyme had a K _m for thiosulfate of 0.16 mM with ferricyanide as electron acceptor and of 14 M with native cytochrome c and of 0.34 mM for ferricyanide. Optimum pH for this activity was 7.8. Other properties of this enzyme were similar to those of thiobacilli and heterotrophic bacteria. The activity of sulfite oxidase was inhibited by 50% with 10 M AMP. The K _m values of this enzyme were 1 mM with ferricyanide as electron acceptor and 60 M with native cytochrome c for sulfite and 0.42 mM for ferricyanide. The enzyme did not show a specific optimum pH value with ferricyanide as electron acceptor. However, with native cytochrome c optimum pH was 7.8 for its activity. In many properties the sulfite oxidase from Rhodotorula was similar to the enzyme from Thiobacillus ferrooxidans, T. concretivorus, T. thioparus and T. novellus.Abbreviations CSH reduced glutathion - APS reductase, adenosine-S-phosphosulfate reductase - pHMB p-hydroxymercuribenzoate - NEM N-ethylmalcimide - TCA trichloroacetic acid - PPO 2,5-diphenyloxazole - POPOP 2,2-p-phenylen-bis 5-phenyloxazol     

Determination of mannitol in ectomycorrhizal fungi and ectomycorrhizas by enzymatic micro-assays

Two sensitive methods for the enzymatic determination of mannitol are described which were applied to fungal and mycorrhizal extracts. Both methods are based on the oxidation of mannitol by mannitol dehydrogenase from Agaricus hortensis and the fluorometric determination of the NADPH produced in this reaction. The detection limits are 125 pmol for the direct fluorometric assay and 100 fmol, when enzymatic cycling of NADPH is included. The levels of mannitol detected were 123 pmol/g dry wt (mycelia from Cenococcum geophilum, cultivated on malt medium), below 0.3 or about 2.4 pmol/g dry wt (mycelia from Amanita muscaria, dependent on carbon source in the cultivation medium), and between 1.9 and 5.2 pmol/g dry wt in mycorrhizal short roots of Picea abies/Amanita muscaria.     

Induction of fertile male flowers in genetically female Cannabis sativa plants by silver nitrate and silver thiosulphate anionic complex

Summary Apical application of silver nitrate (AgNO₃; 50 and 100 g per plant) and silver thiosulphate anionic complex (Ag(S₂O₃) ₂ ^3– ; STS; 25, 50 and 100 g per plant) to female plants of Cannabis sativa induced the formation of reduced male, intersexual and fully altered male flowers on the newly formed primary lateral branches (PLBs); 10 g per plant of AgNO₃ was ineffective and 150 g treatment proved inhibitory. A maximum number of fully altered male flowers were formed in response to 100 g STS. The induced male flowers produced pollen grains that germinated on stigmas and effected seed set. Silver ion applied as STS was more effective than AgNO₃ in inducing flowers of altered sex. The induction of male flowers on female plants demonstrated in this work is useful for producing seeds that give rise to only female plants. This technique is also useful for maintaining gynoecious lines.     

Growth and rosmarinic acid accumulation in callus,cell suspension,and root cultures of wild Salvia fruticosa

The accumulation of rosmarinic acid (RA) in Salvia fruticosa callus, cell suspension, and root cultures was studied. For callus induction, leaves excised from microshoots were cultured on MS medium containing thidiazuron (TDZ) (0, 2.3, 4.6, 6.9, 9.2, or 11.5 M) and indole-3-acetic acid (IAA) (0 or 3 M). For root culture, hairy roots were cultured in B5 medium containing 2.7 M -naphthaleneacetic acid (NAA) and different concentrations of sucrose or phenylalanine. Induction of callus was completely inhibited in the absence of both TDZ and IAA and the largest callus (0.79 g) was obtained with a combination of 6.9 M TDZ and 3 M IAA. Culture duration of 5 weeks resulted in maximum callus growth and RA yield (2.12 mg/ 100 mg dry weight). Cell suspension growth and RA yield (5.1 mg/ 100 mg dry weight) were maximum after 20 days of culture. The highest root growth and RA yield (2.62 mg/ 100 mg dry weight) was obtained with 4% (w/ v) sucrose. Incorporation of 10 mg l^–1 phenylalanine in the medium increased RA yield in the roots to 4.68 mg/ 100 mg dry weight after 4 weeks of culture. Amounts of RA extracted from in vivo leaves and roots were 0.21 and 0.72 mg/ 100 mg dry weight, respectively.     

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.     

Responses of nitrogen-fixing and nitrate-supplied alfalfa (Medicago sativa L.) to iron chelates in an alkaline hydroponic medium

Ferric ethylenediamine di-(o-hydroxyphenylacetate) (FeEDDHA) and ferric hydroxyethylethylenediaminetriacetic acid (FeHEDTA) were evaluated as Fe sources for hydroponic growth of alfalfa (Medicago sativa L., cv. Mesilla), either dependent on N₂ fixation or supplied with NO₃. The hydroponic medium was maintained at pH 7.5 by addition of CaCO₃. Nitrogen-fixing cultures were inoculated with Rhizobium meliloti 102 F51 and grown in medium without added nitrogen. After five to seven weeks of growth under greenhouse conditions, plants were harvested. Nitrogen fixation was measured by the acetylene reduction method.When FeEDDHA was supplied, growth of alfalfa, whether dependent on N₂ fixation or supplied with NO₃, was severely limited at concentrations typically used in hydroponic medium (10 or 20 M). Maximum yield of NO₃-supplied alfalfa was obtained at 100 M while maximum yield of N₂-fixing alfalfa was obtained in the range of 33 to 200 M FeEDDHA. Nodule fresh weights and N₂ fixation rates increased with FeEDDHA concentration up to 33 M and remained essentially constant up to 200 M. With FeHEDTA, maximum yields of both NO₃-grown and N₂-fixing alfalfa were obtained at 10 M. Growth of NO₃-supplied plants was inhibited at 200 M FeHEDTA while growth of N₂-fixing plants was inhibited at 100 M FeHEDTA. The numbers of nodules per plant increased between 3.3 and 10 M FeHEDTA; however, inhibition of nodule formation occurred at a concentration of 33 M or higher. Nodule weights per plant and N₂ fixation rates were depressed at 3.3 M as well as at 100 M FeHEDTA. The results suggest that alfalfa dependent on N₂ fixation is more sensitive to limited Fe availability than alfalfa supplied with NO₃.     

Kinetics of ammonia oxidation by a marine nitrifying bacterium: Methane as a substrate analogue

In pure culture, the marine ammonia oxidizer,Nitrosococcus oceanus, exhibits normal Michaelis Menten kinetics with respect to its primary substrate, ammonia.N. oceanus also exhibits a kinetic response to methane. In the absence of methane, oxidation of ammonia is first order with respect to ammonia concentration under atmospheric oxygen concentrations at seawater pH. In the presence of methane, ammonia oxidation is inhibited, and the amount of inhibition is related to the relative concentrations of methane and ammonia. Using semicontinuous batch cultures as a source of organisms for short-term kinetic experiments, I investigated the relationship between ammonia and methane oxidation inN. oceanus by varying the absolute and relative concentration of both substrates. Methane appeared to act as a substrate analogue, and its effect on ammonia oxidation was modeled as a permutation of competitive inhibition involving a cooperative enzyme system. Methane was oxidized byN. oceanus, even in the absence of measurable ammonia oxidation, but the process was inhibited at increasing methane concentrations. Of the two product pools analyzed, an average of 37% of methane oxidized was detected in particulate (cell) material and the remainder was detected in¹⁴CO₂. The contribution of methane to total carbon assimilation varied with the ratio [CH₄]/[NH₃] and may be significant under substrate concentrations typical of a dilute aquatic environment.     

Regulatory aspects of inorganic nitrogen metabolism in the Rhodospirillaceae

Regulatory aspects of the assimilation of inorganic nitrogen compounds (ammonia, nitrate, nitrogen) were studied in 12 strains belonging to the Rhodospirillaceae. All strains possessed an ammonium transport system, as demonstrated by ¹⁴C-methylammonium uptake. This uptake showed saturation kinetics (K _m between 50–150 M), and was competitively inhibited by ammonium (K _i between 5–18 M). The ammonium transport systems were repressed by ammonium in the growth medium. The nitrogenase activity of all strains was reversibly inhibited by ammonium (switch-off). This effect was not shown under nitrogen starvation conditions with the exception of some strains of Rhodopseudomonas capsulata, the nitrogenase of which was always susceptible to switch-off by ammonium. Assimilation of nitrate was confined to some strains of Rhodopseudomonas capsulata.Abbreviations ATCC American Type culture Collection, Rockville, MD, USA - DSM Deutsche Sammlung von Mikroorganismen, Göttingen, FRG     

Methane in sulfate-rich and sulfate-poor wetland sediments

Wetlands of northern Belize provide a unique opportunity to study methane production and emissions from marshes dominated by identical species (Typha domingensis, Cladium jamaicense) and genus (Eleocharis spp.), but differing substantially in the amount of sulfates present in the sediments. Some marshes occur on limestone marls rich in gypsum (CaSO₄) while others are underlain by alluvial sands poor in sulfates. Concentrations of methane and sulfates in the sediment interstitial water are one or more orders of magnitude different for these two geological substrata averaging 139.2 and 14.9 M of CH₄, and 0.08 and 11.53 mM of SO₄ ^–2 2 on alluvial sands and limestone respectively. The amount of methane found in the internal atmosphere of plants from alluvial sands is significantly higher (6.3 M) than in plants from limestone (0.19 M). The average methane emissions measured in wetlands located on alluvial sands were 25.2 mg m^–2 h^–1 while emissions from marshes on limestone were only 2.4 mg m^–2 h^–1. These values extrapolated for the entire year and the respective wetland areas resulted in the estimate of total CH4 emissions from northern Belize of 0.066 Tg per year.     

Competition between sulfate-reducing and methanogenic bacteria for H2 under resting and growing conditions

The basis for the outcome of competition between sulfidogens and methanogens for H₂ was examined by comparing the kinetic parameters of representatives of each group separately and in co-culture. Michaelis-Menten parameters (V _max and K _m) for four methanogens and five sulfate-reducing bacteria were determined from H₂-depletion data. Further, Monod growth parameters (_max, K _s, Y _H2) for Desulfovibrio sp. G11 and Methanospirillum hungatei JF-1 were similarly estimated. H₂ K _m values for the methanogenic bacteria ranged from 2.5 M (Methanospirillum PM1) to 13 M for Methanosarcina barkeri MS; Methanospirillum hungatei JF-1 and Methanobacterium PM2 had intermediate H₂ K _m estimates of 5 M. Average H₂ K _m estimates for the five sulfidogens was 1.2 M. No consistent difference among the V _max estimates for the above sulfidogens (mean=100 nmol H₂ min^-1 mg^-1 protein) and methanogens (mean=110 nmol H₂ min^-1 mg^-1 protein) was found. A two-term Michaelis-Menten equation accurately predicted the apparent H₂ K _m values and the fate of H₂ by resting co-cultures of sulfate-reducers and methanogens. Half-saturation coefficients (K _s) for H₂-limited growth of Desulfovibrio sp. G11 (2–4 M) and Methanospirillum JF-1 (6–7 M) were comparable to H₂ K _m estimates obtained for these organisms. Maximum specific growth rates for Desulfovibrio sp. G11 (0.05 h^-1) were similar to those of Methanospirillum JF-1 (0.05–0.06 h^-1); whereas G11 had an average yield coefficient 4 x that of JF-1. Calculated _max and V _max/K _m values for the methanogens and sulfidogens studied predict that the latter bacterial group will process more H₂ whether these organisms are in a growing or resting state, when the H₂ concentration is in the first-order region.     

First survey on the natural occurrence of Fusarium mycotoxins in Bulgarian wheat

Wheat for human consumption (140 samples) was collected after harvest from all regions of Bulgaria. The 1995 crop year was characterized by heavy rainfall in the spring and summer months. The internal mycoflora of wheat samples was dominated by Fusarium spp. and Alternaria spp., and storage fungi were rarely present. The samples were analysed for contamination with Fusarium mycotoxins deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), T-2 Toxin (T-2), diacetoxyscirpenol (DAS), and zearalenone (ZEA), using enzyme immunoassay methods. DON and ZEA were the predominant toxins, with a contamination frequency of 67% and 69%, respectively. The average levels of these toxins in positive samples were 180 g/kg (DON) and 17 g/kg (ZEA), maximum concentrations were 1800 g kg^–1 and 120 g kg^–1, respectively. Acetyl derivatives of DON, namely 3-AcDON and 15-AcDON, were found in 2.1 % and 0.7% of the samples, at at maximum level of about 100 g kg^–1. Only one sample was positive for T-2 (55 g/kg), DAS was not detected. This is the first report about the natural occurrence of a range of Fusarium mycotoxins in wheat for human consumption in Bulgaria.Abbreviations 3-AcDON 3-acetyldeoxynivalenol - 15-AcDON 15-acetyldeoxynivalenol - DAS diacetoxyscirpenol - DON deoxynivalenol - EIA enzyme immunoassay - T-2 T-2 toxin - ZEA zearalenone     

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.     

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.     

Changes in phosphorus concentrations and pH in the rhizosphere of some agroforestry and crop species

The aim of this work was to assess whether agroforestry species have the ability to acquire P from pools unavailable to maize. Tithonia diversifolia(Hemsley) A. Gray, Tephrosia vogelii Hook f., Zea mays and Lupinus albusL. were grown in rhizopots and pH change and depletion of inorganic and organic P pools measured in the rhizosphere. Plants were harvested at the same growth stage, after 56 days for maize and white lupin and 70 days for tithonia and tephrosia, and the rhizosphere sampled. The rhizosphere was acidified by tithonia (pH change –0.3 units to pH 4.8) and lupins (–0.2 units to 4.9), alkalinised by tephrosia (+0.4 units to pH 5.4), and remained unchanged with maize growth. Concurrent with acidification in the rhizosphere of tithonia there was a decline in resin-P (0.8 g P g^–1). However, there was also a decline in NaOH extractable inorganic P (NaOH-P_i) (5.6 g P g^–1 at the root surface) and organic P pools (NaOH-P_o) (15.4 g P g^–1 at 1.5 mm from the root), which would not be expected without specific P acquisition mechanisms. Alkalinisation of tephrosia rhizosphere was accompanied by changes in all measured pools, although the large depletion of organic P (21.6 g P g^–1 at 5 mm from the root) suggests that mineralisation, as well as desorption of organic P, was stimulated. The size of changes of both pH and P pools varied with distance away from the rhizoplane. Decline of more recalcitrant P pools with the growth of the agroforestry species contrasted with the effect of maize growth, which was negligible on resin-P and NaOH-P_i, but led to an accumulation of P as NaOH-P_o (14.2 g P g^–1 at 5 mm from the root). Overall the depletion of recalcitrant P pools, particularly P_o, suggests that the growth of tithonia and tephrosia enhance desorption and dissolution of P, while also enhancing organic P mineralisation. Both species appear to have potential for agroforestry technologies designed to enhance the availability of P to crops, at least in the short term.     

Growth kinetics of EDTA biodegradation by Burkholderia cepacia

A pure culture of an EDTA-degrading strain was isolated from the Taiwan environment. It was identified as Burkholderia cepacia, an aerobic bacterium, elliptically shaped with a length of 5–15 m. The degradation assay showed that the degradation efficiency of Fe-EDTA by B. cepacia was approximately 91%. Evaluation of kinetic parameters showed that Fe-EDTA degradation followed substrate inhibition kinetics. This is evident from the decrease in specific growth rate with an increase in the initial substrate concentration greater than 500 mg/l. To estimate the kinetic parameters – _max, K_S and K_I, five substrate–inhibition models were used. From the results of non-linear regression, the value of _max ranged from 0.150 to 0.206 d^–1, K_S from 74 to 87 mg/l, and K_I from 890 to 2289 mg/l. The five models were found to underestimate the maximum specific growth rate by 1.5–3.7. Therefore, predictions based on these models would result in lower predicted value than those from the experimental kinetic data.     

Gallic acid: a Potent Inhibitor of Tetramethylbenzidine Peroxidation in Aqueous and Micellar Media

Gallic acid competitively inhibited 3,3",5,5"-tetramethylbenzidine peroxidation both in 0.01 M of phosphate buffer (pH 6.4) (K _I= 13.3 M) and in reversed aerosol OT micelles of different hydration degrees dispersed in heptane (K _I= 21.3 to 29.3 M). The average number of free radical particles terminated by one inhibitor molecule (f) was estimated to be 1.3 to 1.6 in aqueous buffer solutions and 1.08 to 2.72 in reversed micelles, depending on their hydration.