Properties of nitrate reductase from Fusarium oxysporum 11dn1 fungi grown under aerobic and anaerobic conditions

Production of nitrate reductase was studied in 15 species of microscopic fungi grown on a nitrate-containing medium. Experiments were performed with Fusarium oxysporum 11dn1, a fungus capable of producing nitrous oxide as the end product of denitrification. Moreover, a shift from aerobic to anaerobic conditions of growth was accompanied by a sharp increase in the activity of nitrate reductase. Studies of nitrate reductase from the mycelium of Fusarium oxysporum 11dn1, grown under aerobic and anaerobic conditions, showed that this enzyme belongs to molybdenum-containing nitrate reductases. The enzymes under study differed in the molecular weight, temperature optimum, and other properties. Nitrate reductase from the mycelium grown under aerobic conditions was shown to belong to the class of assimilatory enzymes. However, nitrate reductase from the mycelium grown anaerobically had a dissimilatory function. An increase in the activity of dissimilatory nitrate reductase, observed under anaerobic conditions, was associated with de novo synthesis of the enzyme.     

Nitrate Reductase and Nitrous Oxide Production by Fusarium oxysporum 11dn1 Under Aerobic and Anaerobic Conditions

The fungus Fusarium oxysporum 11dn1 was found to be able to grow and produce nitrous oxide on nitrate-containing medium in anaerobic conditions. The rate of nitrous oxide formation was three to six orders of magnitude lower than the rates of molecular nitrogen production by common denitrifying bacteria. Acetylene and ammonia did not affect the release of nitrous oxide release. It was shown that under anaerobic conditions fast increase of nitrate reductase activity occurred, caused by the synthesis of enzyme de novo and protein dephosphorylation. Reverse transfer of the mycelium to aerobic conditions led to a decline in nitrate reductase activity and stopped nitrous oxide production. The presence of two nitrate reductases was shown, which differed in molecular mass, location, temperature optima, and activity in nitrate- and ammonium-containing media. Two enzymes represent assimilatory and dissimilatory nitrate reductases, which are active in aerobic and anaerobic conditions, respectively. Received: 2 February 2000 / Accepted: 28 February 2000     

Anaerobic expression of nitric oxide reductase from denitrifying Pseudomonas stutzeri

NO reductase synthesis was investigated immunochemically and by activity assays in cells of Pseudomonas stutzeri ZoBell grown in continuous culture at discrete aeration levels, or in O₂-limited batch cultures supplemented with N oxides as respiratory substrate. Under aerobic conditions, NO reductase was not expressed in P. stutzeri. Oxygen limitation in combination with the presence of nitrate or nitrite derepressed NO reductase synthesis. On transition from aerobic to anaerobic conditions in continuous culture, NO reductase was synthesized below 3% air saturation and reached maximum expression under anaerobic conditions. By use of mutant strains defective in nitrate respiration or nitrite respiration, the inducing effect of individual N oxides on NO reductase synthesis could be discriminated. Nitrite caused definite, concentration-dependent induction, while nitrate promoted moderate enzyme synthesis or amplified effects of nitrite. Exogenous nitric oxide (NO) in concentrations 25 M induced trace amounts of NO reductase; in higher concentrations it arrested cell growth. Nitrite reductase or NO reductase were not detected immunochemically under these conditions. NO generated as an intermediate appeared not to induce NO reductase significantly. Antiserum raised against the P. stutzeri NO reductase showed crossreaction with cell extracts from P. stutzeri JM300, but not with several other denitrifying pseudomonads or Paracoccus denitrificans.     

Aerobic and anaerobic growth of Paracoccus denitrificans on methanol

1. The dye-linked methanol dehydrogenase from Paracoccus denitrificans grown aerobically on methanol has been purified and its properties compared with similar enzymes from other bacteria. It was shown to be specific and to have high affinity for primary alcohols and formaldehyde as substrate, ammonia was the best activator and the enzyme could be linked to reduction of phenazine methosulphate.
2. Paracoccus denitrificans could be grown anaerobically on methanol, using nitrate or nitrite as electron acceptor. The methanol dehydrogenase synthesized under these conditions could not be differentiated from the aerobically-synthesized enzyme.
3. Activities of methanol dehydrogenase, formaldehyde dehydrogenase, formate dehydrogenase, nitrate reductase and nitrite reductase were measured under aerobic and anaerobic growth conditions.
4. Difference spectra of reduced and oxidized cytochromes in membrane and supernatant fractions of methanol-grown P. denitrificans were measured.
5. From the results of the spectral and enzymatic analyses it has been suggested that anaerobic growth on methanol/nitrate is made possible by reduction of nitrate to nitrite using electrons derived from the pyridine nucleotide-linked dehydrogenations of formaldehyde and formate, the nitrite so produced then functioning as electron acceptor for methanol dehydrogenase via cytochrome c and nitrite reductase.

     

Nitrate reductase and respiratory adaptation in Bacillus stearothermophilus

Downey, R. J. (University of Notre Dame, Notre Dame, Ind.). Nitrate reductase and respiratory adaptation in Bacillus stearothermophilus. J. Bacteriol. 91:634-641. 1966.-Bacillus stearothermophilus 2184 required nitrate to grow in the absence of oxygen. Like many facultative microorganisms, the growth obtained anaerobically was considerably less than that obtained aerobically, even though the dissimilatory reduction of nitrate is, in effect, anaerobic respiration. The ability to reduce nitrate depended on the induction of nitrate reductase. Although oxygen at low levels did not retard induction of the enzyme, enzyme synthesis was considerably lessened by aeration. A semisynthetic medium containing nitrate supported aerobic growth of the thermophile but did not support anaerobic growth. The adaptation to nitrate resulted in a decrease in the level of cytochrome oxidase normally present in aerobically grown cells. Although the aerobic oxidation of succinate by the respiratory enzymes from aerobically grown cells was inhibited by 2-N-heptyl-4-hydroxyquinoline-N-oxide, the anaerobic oxidation of succinate by nitrate in a similar preparation from nitrate-adapted cells was not. The nitrate reductase in the bacillus was strongly inhibited by cyanide and azide but not by carbon monoxide. The nitrate reductase catalyzed the anaerobic oxidation of reduced nicotinamide adenine dinucleotide, and appeared to transfer electrons from cytochrome b(1) to nitrate. Cytochrome c(1) did not appear to be involved in the transfer.     

Characterization of diverse heterocyclic amine-degrading denitrifying bacteria from various environments

Although, there have been many published bacterial strains aerobically degrading the heterocyclic amine compounds, only one strain to date has been reported to degrade pyrrolidine under denitrifying conditions. In this study, denitrifying bacteria degrading pyrrolidine and piperidine were isolated from diverse geological and ecological origins through selective enrichment procedures. Based on the comparative sequence results of 16S rRNA genes, 30 heterocyclic amine-degrading isolates were grouped into ten distinct phylotypes belonging to the genera Thauera, Castellaniella, Rhizobium, or Paracoccus of the phylum Proteobacteria. The representative isolates of individual phylotypes were characterized by phylogenetic, phenotypic and chemotaxonomical traits, and dissimilatory nitrite reductase gene (nirK and nirS). All isolates completely degraded pyrrolidine and piperidine under both aerobic and anaerobic conditions. The anaerobic degradations were coupled to nitrate reduction. A metabolic pathway for the anaerobic degradation of pyrrolidine was proposed on the basis of enzyme activities implicated in pyrrolidine metabolism from three isolates. The three key pyrrolidine-metabolizing enzymes pyrrolidine dehydrogenase, γ-aminobutyrate/α-ketoglutarate aminotransferase, and succinic semialdehyde dehydrogenase, were induced by heterocyclic amines under denitrifying conditions. They were also induced in cells grown aerobically on heterocyclic amines, suggesting that the anaerobic degradation of pyrrolidine shares the pathway with aerobic degradation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Medium pH in submerged cultivation modulates differences in the intracellular protein profile of Fusarium oxysporum

Fusarium oxysporum is a filamentous fungus that damages a wide range of plants and thus causes severe crop losses. In fungal pathogens, the genes and proteins involved in virulence are known to be controlled by environmental pH. Here, we report the influence of culture-medium pH (5, 6, 7, and 8) on the production of degradative enzymes involved in the pathogenesis of F. oxysporum URM 7401 and on the 2D-electrophoresis profile of intracellular proteins in this fungus. F. oxysporum URM 7401 was grown in acidic, neutral, and alkaline culture media in a submerged bioprocess. After 96?hr, the crude extract was processed to enzyme activity assays, while the intracellular proteins were obtained from mycelium and analyzed using 2D electrophoresis and mass spectrometry. We note that the diversity of secreted enzymes was changed quantitatively in different culture-medium pH. Also, the highest accumulated biomass and the intracellular protein profile of F. oxysporum URM 7401 indicate an increase in metabolism in neutral–alkaline conditions. The differential profiles of secreted enzymes and intracellular proteins under the evaluated conditions indicate that the global protein content in F. oxysporum URM 7401 is modulated by extracellular pH.     

The anaerobic life of Bacillus subtilis: Cloning of the genes encoding the respiratory nitrate reductase system

Abstract The Gram-positive soil bacterium Bacillus subtilis , generally regarded as an aerobe, grows under strict anaerobic conditions using nitrate as an electron acceptor and should be designated as a facultative anaerobe. Growth experiments demonstrated a lag phase of 24 to 36 hours after the shift from aerobic, to the onset of anaerobic respiratory growth. Anaerobically adapted cells grew without further lag phase after their transfer to fresh anaerobic growth medium. The cells change their morphology from rods to longer filament-like structures when moved from aerobic to anaerobic respiratory growth conditions. Surprisingly, anaerobically grown B. subtilis lost the capacity for sporulation. An investigation of the molecular basis of the switch between aerobic and anaerobic growth was initiated by the cloning of the genes encoding the respiratory nitrate reductase from B. subtilis . Oligonucleotides deduced from conserved amino acid sequence regions of eubacterial respiratory nitrate reductases and related enzymes were used for the isolation of the genes. Four open reading frames with significant homology to the E. coli respiratory nitrate reductase opérons ( narGHIJ, narZYWV ) were isolated and termed narGHJI . A chromosomal knock-out mutation of the B. subtilis nar operon totally abolished nitrate respiration.     

Regulation of Nitrate Assimilation and Nitrate Respiration in Aerobacter aerogenes

The influence of growth conditions on assimilatory and respiratory nitrate reduction in Aerobacter aerogenes was studied. The level of nitrate reductase activity in cells, growing in minimal medium with nitrate as the sole nitrogen source, was much lower under aerobic than anaerobic conditions. Further, the enzyme of the aerobic cultures was very sensitive to sonic disintegration, as distinct from the enzyme of anaerobic cultures. When a culture of A. aerogenes was shifted from anaerobic growth in minimal medium with nitrate and NH(4) (+) to aerobiosis in the same medium, but without NH(4) (+), the production of nitrite stopped instantaneously and the total activity of nitrate reductase decreased sharply. Moreover, there was a lag in growth of about 3 hr after such a shift. After resumption of growth, the total enzymatic activity increased again slowly and simultaneously became gradually sensitive to sonic disintegration. These findings show that oxygen inactivates the anaerobic nitrate reductase and represses its further formation; only after a de novo synthesis of nitrate reductase with an assimilatory function will growth be resumed. The enzyme in aerobic cultures was not significantly inactivated by air, only by pure oxygen. The formation of the assimilatory enzyme complex was repressed, however, by NH(4) (+), under both aerobic and anaerobic conditions. The results indicate that the formation of the assimilatory enzyme complex and that of the respiratory enzyme complex are regulated differently. We suggest that both complexes have a different composition, but that the nitrate reductase in both cases is the same protein.     

Respiration and NADH-Oxidation of the Roots of Flood-Tolerant and Flood-Intolerant Senecio Species as Affected by Anaerobiosis

Respiration was measured under anaerobiosis in the roots of two Senecio species: S. aquaticus Hill, which is flood-tolerant, and S. jacobaea L., which is flood-intolerant. NADH-oxidation under anaerobiosis was measured in roots of S. aquaticus, S. jacobaea and S. vulgaris L., which is also flood-intolerant. Protein content of S. aquaticus was about 15% higher under anaerobiosis. At 20°C respiration of the roots of S. aquaticus was 50% inhibited under anaerobiosis, while an almost complete inhibition occurred in the roots of S. jacobaea. The activities of nitrate reductase, glutamate dehydrogenase and lactate dehydrogenase were considerably higher in the roots of S. aquaticus grown under anaerobic conditions than in roots grown under aerobic conditions. In S. jacobaea glutamate dehydrogenase activity was lower and in S. vulgaris nitrate reductase was lower and glutamate dehydrogenase activity was higher in roots grown under anaerobic conditions. The possible role of these enzymes for metabolism under anaerobic conditions by oxidizing a surplus of NADH is discussed. Since oxidative phosphorylation is 50% inhibited under anaerobiosis, ATP has to be generated in a different way. It is argued that maintenance of the ATP-level may be compensated by way of the enzymes mentioned above, in combination with a modified glucose utilization.     

A Denitrifying Activity in an Aerobic Photosynthetic Bacterium, Erythrobacter sp. Strain OCh 114

An aerobic photosynthetic bacterium, Erythrobacter sp. strainOCh 114, was capable of growth under anaerobic conditions inthe dark with nitrate as a terminal electron acceptor. The optimalnitrate concentration was about 6 mM for anaerobic growth, althougha wide range of concentrations from 1 to 400 mM were effective.A large amount of N₂O gas was released during this anaerobicgrowth, indicating a denitrifying activity in this bacterium.Light had no stimulating or inhibiting effect on the rates ofanaerobic growth and gas release. The enzymes responsible forthe denitrifying activity, dissimilatory nitrate and nitritereductases, were present in aerobically grown cells. (Received February 19, 1988; Accepted May 16, 1988)     

Nitric oxide reduction, the last step in denitrification by Fusarium oxysporum, is obligatorily mediated by cytochrome P450nor

The involvement of cytochrome P450nor (P450nor) is the most striking feature of the fungal denitrifying system, and has never been shown in bacterial systems. To establish the physiological significance of the P450nor, we constructed and investigated mutants of Fusarium oxysporum that lacked the gene for P450nor. We mutated the gene by targeted integration of a disrupted gene into the chromosome of F. oxysporum. The mutants were shown to contain neither P450nor protein nor nitric oxide (NO) reductase (Nor) activity, implying that they are indeed deficient in P450nor. These mutants had apparently lost the denitrifying activity and failed to evolve nitrous oxide (N₂O) upon incubation under oxygen-limiting conditions in the presence of nitrate. Their mycelia exhibited normal levels of dissimilatory nitrite reductase (Nir) activity and were able to evolve NO under these conditions. The promoter region of the P450nor gene was fused to lacZ and introduced into the wild-type strain of F. oxysporum. The transformed strain produced β-galactosidase under denitrifying conditions as efficiently as the wild type does P450nor. These results represent unequivocal genetic evidence that P450nor is essential for the reduction of NO to N₂O, the last step in denitrification by F. oxysporum. Received: 28 June 1999 / Accepted: 22 December 1999     

Anaerobic growth and potential for amino acid production by nitrate respiration in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Oxygen limitation is a crucial problem in amino acid fermentation by Corynebacterium glutamicum. Toward this subject, our study was initiated by analysis of the oxygen-requiring properties of C. glutamicum, generally regarded as a strict aerobe. This organism formed colonies on agar plates up to relatively low oxygen concentrations (0.5% O₂), while no visible colonies were formed in the absence of O₂. However, in the presence of nitrate (), the organism exhibited limited growth anaerobically with production of nitrite (), indicating that C. glutamicum can use nitrate as a final electron acceptor. Assays of cell extracts from aerobic and hypoxic cultures yielded comparable nitrate reductase activities, irrespective of nitrate levels. Genome analysis revealed a narK2GHJI cluster potentially relevant to nitrate reductase and transport. Disruptions of narG and narJ abolished the nitrate-dependent anaerobic growth with the loss of nitrate reductase activity. Disruption of the putative nitrate/nitrite antiporter gene narK2 did not affect the enzyme activity but impaired the anaerobic growth. These indicate that this locus is responsible for nitrate respiration. Agar piece assays using l-lysine- and l-arginine-producing strains showed that production of both amino acids occurred anaerobically by nitrate respiration, indicating the potential of C. glutamicum for anaerobic amino acid production.     

Influence of NO3 - and NH4 + nutrition on fermentation,nitrate reductase activity and adenylate energy charge of roots of Carex pseudocyperus L. and Carex sylvatica Huds. exposed to anaerobic nutrient solutions

The adenylate energy charge, production of ethanol and lactate, and nitrate reductase activity were determined in order to study the influence of different nitrogen sources on the metabolic responses of roots of Carex pseudocyperus L. and Carex sylvatica HUDS. exposed to anaerobic nutrient solutions. Determination of adenylates was carried out by means of a modified HPLC technique. Total quantity of adenylates was higher in Carex pseudocyperus than in Carex sylvatica under all conditions. In contrast, the adenylate energy charge was only slightly different between the species and decreased more or less in relation to the applied nitrogen source under oxygen deficiency. The adenylate energy charge in roots of plants under nitrate nutrition showed a smaller decrease under anaerobic environmental conditions than plants grown with ammonium or nitrate/ammonium. Roots of nitrate-fed plants showed a lower ethanol and lactate production than ammonium/nitrate- and ammonium-fed plants. Ethanol production was higher in C. pseudocyperus, formation of lactate was lower compared to that in Carex sylvatica. The activity of enzymes involved in fermentation processes (ADH, LDH and PDC) was enhanced significantly after 24 hours of exposure to anaerobic nutrient solutions in roots of both species. The induction of these enzymes was only slightly influenced by different nitrogen supply. In vivo nitrate reductase activity increased almost 3-fold compared to the aerobic treatment in both species and overcompensated loss of NADH reoxidation capacity caused by decrease of ethanol and lactate development. Induction of in vitro nitrate reductase activity was enhanced 313% in C. pseudocyperus and 349% in C. sylvatica under anaerobic environmental conditions and nitrate supply. These results indicate that nitrate may serve as an alternative electron acceptor in anaerobic plant root metabolism and that the nitrate-supported energy charge may be due to an accelerated glycolytic flux resulting from a more effective NADH reoxidation capacity by nitrate reduction plus fermentation than by fermentation alone.Abbreviations ADH alcohol dehydrogenase - AEC adenylate energy charge - DMSO dimethyl sulfoxide - EDTA ethylen diamine tetraacetic acid - HPLC high performance liquid chromatography - LDH lactate dehydrogenase - NRA nitrate reductase activity - PCA perchloric acid - PDC pyruvate decarboxylase - PVP polyvinylpyrrolidone - PVPP polyvinylpolypyrrolidone - TCA trichloroacetic acid, Tris-tris(hydroxymethyl)aminomethane     

Dissimilatory iodate reduction by marine Pseudomonas sp. strain SCT

Bacterial iodate (IO(3)(-)) reduction is poorly understood largely due to the limited number of available isolates as well as the paucity of information about key enzymes involved in the reaction. In this study, an iodate-reducing bacterium, designated strain SCT, was newly isolated from marine sediment slurry. SCT is phylogenetically closely related to the denitrifying bacterium Pseudomonas stutzeri and reduced 200 microM iodate to iodide (I(-)) within 12 h in an anaerobic culture containing 10 mM nitrate. The strain did not reduce iodate under the aerobic conditions. An anaerobic washed cell suspension of SCT reduced iodate when the cells were pregrown anaerobically with 10 mM nitrate and 200 microM iodate. However, cells pregrown without iodate did not reduce it. The cells in the former category showed methyl viologen-dependent iodate reductase activity (0.31 U mg(-1)), which was located predominantly in the periplasmic space. Furthermore, SCT was capable of anaerobic growth with 3 mM iodate as the sole electron acceptor, and the cells showed enhanced activity with respect to iodate reductase (2.46 U mg(-1)). These results suggest that SCT is a dissimilatory iodate-reducing bacterium and that its iodate reductase is induced by iodate under anaerobic growth conditions.     

Nitrate reduction in Aerobacter aerogenes

Summary Mutants of A. aerogenes blocked in aerobic and anaerobic nitrate assimilation and deficient in the reduction of nitrate and chlorate were found to give a positive methylred reaction and no gas formation from glucose. Resting cells, grown anaerobically in minimal medium with glucose, did not show gas production from formate. These results show that these mutants are also deficient in formate hydrogenylase. Revertants could readily be obtained by plating on minimal medium with nitrate as sole nitrogen source, indicating that in these mutants a pleiotropic point mutation is present, which affects both nitrate reductase and formate hydrogeny lase. It is suggested, that these mutants are deficient in the formation of an enzyme complex or particle on which these enzymes are present.     

The nitrite reductase gene of Pseudomonas aeruginosa: Effect of growth conditions on the expression and construction of a mutant by gene disruption

Abstract The expression of nitrite reductase has been tested in a wild-type strain of Pseudomonas aeruginosa (Pao1) as a function of nitrate concentration under anaerobic and aerobic conditions. Very low levels of basal expression are shown under non-denitrifying conditions (i.e. absence of nitrate, in both aerobic and anaerobic conditions); anaerobiosis is not required for high levels of enzyme production in the presence of nitrate. A Pseudomonas aeruginosa strain, mutated in the nitrite reductase gene, has been obtained by gene replacement. This mutant, the first of this species described up to now, is unable to grow under anaerobic conditions in the presence of nitrate. The anaerobic growth can be restored by complementation with the wild-type gene.     

Ferric reductases of Legionella pneumophila

Ferric reductase enzymes requiring a reductant for maximal activity were purified from the cytoplasmic and periplasmic fractions of avirulent and virulent Legionella pneumophila. The cytoplasmic and periplasmic enzymes are inhibited by zinc sulfate, constitutive and active under aerobic or anaerobic conditions. However, the periplasmic and cytoplasmic reductases are two distinct enzymes as shown by their molecular weights, specific activities, reductant specificities and other characteristics. The molecular weights of the cytoplasmic and periplasmic ferric reductases are approximately 38 and 25 kDa, respectively. The periplasmic reductase (K _m = 7.0 m) has a greater specific activity and twice the affinity for ferric citrate as the cytoplasmic enzyme (K _m = 15.3 m). Glutathione serves as the optimum reductant for the periplasmic reductase, but is inactive for the cytoplasmic enzyme. In contrast, NADPH is the optimum reductant for the cytoplasmic enzyme. Ferric reductases of avirulent cells show a 2-fold increase in their activities when NADPH is used as a reductant in comparison with NADH. In contrast, ferric reductases from virulent cells demonstrated an equivalent activity with NADH or NADPH as reductants. With the exception of their response to NADPH, the ferric reductase at each respective location appears to be similar for avirulent and virulent cells.     

Dynamics of denitrification activity of Paracoccus denitrificans in continuous culture during aerobic-anaerobic changes.

Induction and repression of denitrification activity were studied in a continuous culture of Paracoccus denitrificans during changes from aerobic to anaerobic growth conditions and vice versa. The denitrification activity of the cells was monitored by measuring the formation of denitrification products (nitrite, nitric oxide, nitrous oxide, and dinitrogen), individual mRNA levels for the nitrate, nitrite, and nitrous oxide reductases, and the concentration of the nitrite reductase enzyme with polyclonal antibodies against the cd1-type nitrite reductase. On a change from aerobic to anaerobic respiration, the culture entered an unstable transition phase during which the denitrification pathway became induced. The onset of this phase was formed by a 15- to 45-fold increase of the mRNA levels for the individual denitrification enzymes. All mRNAs accumulated during a short period, after which their overall concentration declined to reach a stable value slightly higher than that observed under aerobic steady-state conditions. Interestingly, the first mRNAs to be formed were those for nitrate and nitrous oxide reductase. The nitrite reductase mRNA appeared significantly later, suggesting different modes of regulation for the three genes. Unlike the mRNA levels, the level of the nitrite reductase protein increased slowly during the anaerobic period, reaching a stable value about 30 h after the switch. All denitrification intermediates could be observed transiently, but when the new anaerobic steady state was reached, dinitrogen was the main product. When the anaerobic cultures were switched back to aerobic respiration, denitrification of the cells stopped at once, although sufficient nitrite reductase was still present. We could observe that the mRNA levels for the individual denitrification enzymes decreased slightly to their aerobic, uninduced levels. The nitrite reductase protein was not actively degraded during the aerobic period.