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D J Nicholas

Publications and source records attributed to D J Nicholas.

At least 19 recordsLinked to original sources
PubMed1987

Inhibition of bacteriochlorophyll synthesis in Rhodobacter sphaeroides subsp. denitrificans grown in light under denitrifying conditions.

The inclusion of nitrate or nitrite in cultures of Rhodobacter spaeroides subsp. denitrificans grown heterotrophically in light depressed the formation of bacteriochlorophyll a. The pigment biosynthesis was inhibited at the stage of the reduction of chlorophyllide (chlorin) to bacteriochlorophyllide (tetrahydroporphyrin) since 3-hydroxyethylchlorophyllide a accumulated in the culture medium. The addition of exogenous 5-aminolevulinic acid to these cultures resulted in a complete restoration of bacteriochlorophyll synthesis accompanied by the accumulation of 3-vinylbacteriopheophorbide. This indicates that under these conditions bacteriochlorophyll was formed via an alternative route, in which the reduction of chlorins to tetrahydroporphyrins precedes modifications of the C-3 side chain. The multiple forms of 5-aminolevulinic acid synthase were purified from cells grown with and without nitrate. Antibodies against these proteins were raised in rabbits and used in enzyme-linked immunosorbent assays for various forms of 5-aminolevulinic acid synthase. In denitrifying cells, the amount and activity of fraction I of the enzyme was reduced by approximately 40 and 30%, respectively. Partly active enzymes from both types of cells were activated by cystine trisulfide.

5-Aminolevulinate Synthetase↗
PubMed1986

Multiple-phase equilibration headspace analysis for the determination of N2O and N2 during bacterial denitrification.

A gas-handling manifold for the preparation, introduction and analysis by gas chromatography (GC) system of the gaseous products of denitrification is described. A procedure of multiple-phase equilibration is adopted which allows the quantitative determination of the total gas present in sample vials. Assumptions of solubility coefficients are not required as these are determined during the analysis. The method is particularly suited to gases of appreciable solubilities as a significant proportion of the gas will be found in the liquid phase. This method was used for the determination of the stoichiometry of denitrification, in washed cells of Rhodopseudomonas sphaeroides f. sp. denitrificans, namely NO2-:N2 and N2O:N2, which were found to be 2:1 and 1:1, respectively.

Chromatography, Gas↗
PubMed1986

N-terminal amino acid sequence of cytochrome c-552 from Nitrosomonas europaea.

Nitrosomonas europaea is an ammonia-oxidizing bacterium which contains multiple c-type cytochromes. Few of these components have been assigned physiological roles, but on the basis of molecular weight and redox potential cytochrome c-552 has been considered to be an analogue of the mitochondrial cytochrome-c family of proteins. We present the N-terminal amino acid sequence (47 residues) of cytochrome c-552 and show that this protein is most closely related to the group of small cytochrome-c components from pseudomonads (cytochromes c-551) and is probably evolutionarily distant from the analagous protein (cytochrome c-550) from the nitrite-oxidizing bacterium Nitrobacter agilis.

Amino Acid Sequence↗
PubMed1985

Proton motive force in washed cells of Rhizobium japonicum and bacteroids from Glycine max.

The components of the proton motive force (delta p), namely the membrane potential and the transmembrane pH gradient, were measured in washed cells of Rhizobium japonicum CC705 grown in cultures (5% O2-95% N2) in the presence of 10 mM KNO3 and in bacteroids from Glycine max. The delta p and its components remained reasonably constant in cells as well as in bacteroids at various stages of growth. The effects of uncouplers and ATPase inhibitors on the delta p and its components were determined in both cultured cells and bacteroids. The data indicated that a respiration-driven H+ translocation is the source of the delta p in both cultured cells and bacteroids.

Azides↗
PubMed1984

3,3',5,5'-tetramethylbenzidine/H2O2 staining is not specific for heme proteins separated by gel electrophoresis.

Staining of sodium dodecyl sulfate or lithium dodecyl sulfate gels with 3,3',5,5'-tetramethylbenzidine (TMBZ)/H2O2 after electrophoresis has frequently been used as a specific method of detecting heme proteins. That TMBZ is an electron donor for O2 reduction by the nonheme-soluble cytochrome oxidase/nitrite reductase from Nitrosomonas europaea is now shown; this protein is detected by the TMBZ/H2O2 method. A method for the determination of TMBZ oxidase activity is given; hence, the detection of artifactual staining due to proteins of this type is possible.

Benzidines↗
PubMed1984

Na+ and K+ transport in Nitrosomonas europaea and Nitrobacter agilis.

Potassium-depleted cells of Nitrosomonas europaea and Nitrobacter agilis were prepared by diethanolamine treatment and contained less than 5 mM intracellular K+. The addition of K+ to K+-depleted cells of N. europaea and N. agilis resulted in a depolarization of membrane potential (delta psi) by about 5 and 10 mV, respectively. This depolarization was, however, compensated by an equivalent increase in transmembrane pH gradient (delta pH), so that the total proton-motive force (delta p) remained constant, indicating that K+ transport was electrogenic in both bacteria. Using 22Na+-loaded cells, it is shown that both bacteria lack a respiration-dependent Na+ pump; however, antiporters for Na+/H+, K+/Na+ and K+/H+ were detected. Of these, at least the K+/Na+ antiporter required an electrochemical gradient for its operation. It is also shown that the unprotonated form of NH+4 is transported into these bacteria by a simple diffusion mechanism.

Ammonium Chloride↗
PubMed1983

Proton electrochemical gradients in washed cells of Nitrosomonas europaea and Nitrobacter agilis.

The components of the proton motive force (Deltap), namely, membrane potential (Deltapsi) and transmembrane pH gradient (DeltapH), were determined in the nitrifying bacteria Nitrosomonas europaea and Nitrobacter agilis. In these bacteria both Deltapsi and DeltapH were dependent on external pH. Thus at pH 8.0, Nitrosomonas europaea and Nitrobacter agilis had Deltapsi values of 173 mV and 125 mV (inside negative), respectively, as determined by the distribution of the lipophilic cation [(3)H]tetraphenyl phosphonium. Intracellular pH was determined by the distribution of two weak acids, (14)C-benzoic and (14)C-acetyl salicylic, and the weak base [(14)C]methylamine. Nitrosomonas europaea accumulated (14)C-benzoic acid and (14)C-acetyl salicylic acid when the external pH was below 7.0 and [(14)C]methylamine at alkaline pH. Similarly, Nitrobacter agilis accumulated the two weak acids below an external pH of about 7.5 and [(14)C]methylamine above this pH. As these bacteria grow best between pH 7.5 and 8.0, they do not appear to have a DeltapH (inside alkaline). Thus, above pH 7.0 for Nitrosomonas europaea and pH 7.5 for Nitrobacter agilis, Deltapsi only contributed to Deltap. In Nitrosomonas europaea the total Deltap remained almost constant (145 to 135 mV) when the external pH was varied from 6 to 8.5. In Nitrobacter agilis, Deltap decreased from 178 mV (inside negative) at pH 6.0 to 95 mV at pH 8.5. Intracellular pH in Nitrosomonas europaea varied from 6.3 at an external pH of 6.0 to 7.8 at external pH 8.5. In Nitrobacter agilis, however, intracellular pH was relatively constant (7.3 to 7.8) over an external pH range of 6 to 8.5. In Nitrosomonas europaea, Deltap and its components (Deltapsi and DeltapH) remained constant in cells at various stages of growth, so that the metabolic state of cells did not affect Deltap. Such an experiment was not possible with Nitrobacter agilis because of low cell yields. The effects of protonophores and ATPase inhibitors on DeltapH and Deltapsi in the two nitrifying bacteria are considered.

Hydrogen-Ion Concentration↗
PubMed1982

A reappraisal of antigenic determinants for nitrogenase from Azotobacter and nitrate reductase from Escherichia coli.

Previous work based on double immunodiffusion assays had shown that there are common antigenic determinants for nitrate reductase from Escherichia coli and component I of nitrogenase from Azotobacter vinelandii. Further work reported herein using a variety of immunoelectrophoretic techniques indicates that the cross-reaction between nitrate reductase and antiserum to component I of nitrogenase results from a contaminant antigen co-purified with nitrate reductase.

Antigens, Bacterial↗
PubMed1982

Respiration-dependent proton translocation in Nitrosomonas europaea and its apparent absence in Nitrobacter agilis during inorganic oxidations.

Oxygen pulse experiments were carried out with the nitrifying bacteria Nitrosomonas europaea and Nitrobacter agilis and with spheroplasts and everted vesicles prepared from Nitrobacter agilis. In addition to thiocyanate, the salting-in anions perchlorate and trichloroacetate proved to be permeant and effective in allowing respiration-dependent proton translocation with Nitrosomonas europaea. Valinomycin-K+, however, was generally ineffective in this respect with Nitrosomonas europaea. The observed leads to H+/O ratio for ammonium ion oxidation by Nitrosomonas europaea was 3.4; that for hydroxylamine and hydrazine cation oxidation was 4.4. These values, when corrected for production of stoichiometric protons and for the fact that the first step in ammonium ion oxidation (hydroxylamine production) is mediated by a monooxygenase, give effective leads to H+/O ratios of about 4 for these three substrates. This value compares favorably with those obtained with other aerobes. No convincing evidence was obtained for operation of a respiratory proton pump in Nitrobacter agilis during nitrite oxidation. Implications of this unexpected result are discussed.

Ammonium Chloride↗
PubMed1982

Fixation of dinitrogen derived from denitrification of nitrate in a photosynthetic bacterium, Rhodopseudomonas sphaeroides forma sp. denitrificans.

Studies with 15N demonstrated that the phototrophic bacterium Rhodopseudomonas sphaeroides forma sp. denitrificans strain IL106 cannot assimilate NO-3 but rather denitrifies it to N2. This strain also fixed N2 into cell protein, although nitrogenase activity was partially inhibited in the presence of NO-3. Strain IL106 did not assimilate NO-3, but growing cultures and washed cell suspensions incorporated the tracer from 15NO-3 via denitrification to 15N2 and then via nitrogenase into cell nitrogen. This incorporation was inhibited in cells supplied with NH4+ or in the absence of light, thus confirming the participation of nitrogenase in the assimilation of nitrogen from nitrate. This represents a novel type of N2 recycling in a photodiazotrophic denitrifying bacterium.

Bacterial Proteins↗
PubMed1981

Oxidation of ammonia by Nitrosomonas europaea. Definite 18O-tracer evidence that hydroxylamine formation involves a monooxygenase.

NH2OH, the first intermediate in the oxidation of NH4+ to nitrite by the nitrifying bacterium, Nitrosomonas europaea, was recovered as the oxime of cyclohexanone. 15N, 18O-tracer experiments using highly enriched 15NH4Cl and 18O2 yielded oxime that was correspondingly highly enriched (greater than or equal to 92 atom %) in these isotopes. These results show that the source of NH2OH is largely or entirely NH4+, as opposed to hydrazine, which was added to inhibit the further oxidation of NH2OH to nitrite, and that NH4+ yields NH2OH by way of a monooxygenase reaction involving direct insertion of O from O2. The oxidation of NH4+ and NH2OH must be functionally linked in N. europaea, inasmuch as the reducing equivalents required by the monooxygenase to reduce the second atom of O2 to water can arise only through the concomitant oxidation of NH2OH.

Ammonia↗
PubMed1981

Inhibition of nitrogenase activity by metronidazole in rhodopseudomonas capsulata.

Inhibition of photosynthetic growth of Rhodopseudomonas capsulata by metronidazole was dependent on the nitrogen supply in culture solutions. Cultures fixing dinitrogen were more susceptible to inhibition by low concentrations than those supplied with NH4+. Light-dependent C2H2 reduction and H2 production by washed cells were inhibited by 80% and 60% respectively by 1 mM metronidazole. When this compound was first reduced with H2-palladised asbestos prior to assay, it only partially restricted C2H2 reduction in washed cells (33%) compared with unreduced inhibitor (68%). Metronidazole was without effect on other metabolic functions. Thus, even at 40 mM it did not inhibit either (a) dark or light respiration in cells grown under photo- and chemo-heterotrophic conditions; (b) H2-dependent photoreduction of 14CO2; (C) gamma-glutamyltransferase activity of glutamine synthetase in cell-free extracts (25 mM inhibitor). Metronidazole (1 mM) completely inhibited C2H2 reduction by washed cells of Azotobacter vinelandii. The dithionite-dependent C2H2 reduction of a partially purified nitrogenase was only partially inhibited (30%) by 1 mM metronidazole.

Azotobacter↗
PubMed1981

Some properties of glutamine synthetase from the nitrifying bacterium Nitrosomonas europaea.

Nitrosomonas europaea oxidizes ammonia to nitrite, thereby deriving energy for growth. Glutamate dehydrogenase (NADP+) (EC 1.4.1.4) is the main route for the incorporation of ammonia into glutamic acid, because glutamate synthase (NADPH)(EC 1.4.1.13) was not detected in cell-free extracts of N. europaea. Some properties of a partially purified glutamine synthetase (EC 6.3.1.2) have been determined, namely the effects of pH and metal ions, substrate requirements, Km and Ki values, based on biosynthetic and gamma-glutamyltransferase (EC 2.3.2.2) assays. The molecular weight of the enzyme preparation was approximately 440 000. The gamma-glutamyltransferase activity was markedly inhibited by alanine, lysine, glutamic acid, aspartic acid and serine and to a lesser extent by glycine, asparagine, arginine and histidine. Except for tryptophan and cystine, the gamma-glutamyltransferase activity was inhibited to a greater extent by these amino acids than was the biosynthetic activity. Different pairs of amino acids in various combinations resulted in a cumulative inhibition of enzyme activity determined by either method. Of the various nucleotides tested, the gamma-glutamlytransferase activity of the enzyme was inhibited to a greater extent by di- and triphosphate nucleotides--IDP, CDP, UDP, ITP, CTP, TTP and ATP (except GDP and GTP) than by monophosphate nucleotides except AMP. Saturating concentrations of pyruvate, oxalate, oxaloacetate and alpha-ketoglutarate depressed enzyme activity. Various combinations of amino acids with adenine nucleotides exerted cumulative inhibitory effects on the transferase activity.

Amino Acids↗