Fungal denitrification and nitric oxide reductase cytochrome P450nor

We have shown that many fungi (eukaryotes) exhibit distinct denitrifying activities, although occurrence of denitrification was previously thought to be restricted to bacteria (prokaryotes), and have characterized the fungal denitrification system. It comprises NirK (copper-containing nitrite reductase) and P450nor (a cytochrome P450 nitric oxide (NO) reductase (Nor)) to reduce nitrite to nitrous oxide (N(2)O). The system is localized in mitochondria functioning during anaerobic respiration. Some fungal systems further contain and use dissimilatory and assimilatory nitrate reductases to denitrify nitrate. Phylogenetic analysis of nirK genes showed that the fungal-denitrifying system has the same ancestor as the bacterial counterpart and suggested a possibility of its proto-mitochondrial origin. By contrast, fungi that have acquired a P450 from bacteria by horizontal transfer of the gene, modulated its function to give a Nor activity replacing the original Nor with P450nor. P450nor receives electrons directly from nicotinamide adenine dinucleotide to reduce NO to N(2)O. The mechanism of this unprecedented electron transfer has been extensively studied and thoroughly elucidated. Fungal denitrification is often accompanied by a unique phenomenon, co-denitrification, in which a hybrid N(2) or N(2)O species is formed upon the combination of nitrogen atoms of nitrite with a nitrogen donor (amines and imines). Possible involvement of NirK and P450nor is suggested.     

Denitrification by the fungus Cylindrocarpon tonkinense: anaerobic cell growth and two isozyme forms of cytochrome P-450nor.

We examined the denitrification system of the fungus Cylindrocapon tonkinense and found several properties distinct from those of the denitrification system of Fusarium oxysporum. C. tonkinense could form N2O from nitrite under restricted aeration but could not reduce nitrate by dissimilatory metabolism. Nitrite-dependent N2O formation and/or cell growth during the anaerobic culture was not affected by further addition of ammonium ions but was suppressed by respiration inhibitors such as rotenone or antimycin, suggesting that denitrification plays a physiological role in respiration. Dissimilatory nitrite reductase and nitric oxide reductase (Nor) activities could not be detected in cell extracts of the denitrifying cells. The Nor activity was purified and found to depend upon two isoenzymes of Cytochrome P-450nor (P-450nor), which were designated P-450nor1 and P-450nor2. These isozymes differed in the N-terminal amino acid sequence, isoelectric point, specificity to the reduced pyridine nucleotide (NADH or NADPH), and the reactivity to the antibody to P-450nor of F. oxysporum. the difference between the specificities to NADH and NADPH suggests that P-450nor1 and P-450nor2 play different roles in anaerobic energy acquisition.     

Nitrate reductase-formate dehydrogenase couple involved in the fungal denitrification by Fusarium oxysporum

Dissimilatory nitrate reductase (Nar) was solubilized and partially purified from the large particle (mitochondrial) fraction of the denitrifying fungus Fusarium oxysporum and characterized. Many lines of evidence showed that the membrane-bound Nar is distinct from the soluble, assimilatory nitrate reductase. Further, the spectral and other properties of the fungal Nar were similar to those of dissimilatory Nars of Escherichia coli and denitrifying bacteria, which are comprised of a molybdoprotein, a cytochrome b, and an iron-sulfur protein. Formate-nitrate oxidoreductase activity was also detected in the mitochondrial fraction, which was shown to arise from the coupling of formate dehydrogenase (Fdh), Nar, and a ubiquinone/ubiquinol pool. This is the first report of the occurrence in a eukaryote of Fdh that is associated with the respiratory chain. The coupling with Fdh showed that the fungal Nar system is more similar to that involved in the nitrate respiration by Escherichia coli than that in the bacterial denitrifying system. Analyses of the mutant species of F. oxysporum that were defective in Nar and/or assimilatory nitrate reductase conclusively showed that Nar is essential for the fungal denitrification.     

Nitrate reduction to ammonia: a dissimilatory process in Enterobacter amnigenus

Thirty-four bacterial isolates from an agricultural soil anaerobically preincubated in the presence of glucose were tested for their ability to reduce nitrate to ammonia or to denitrify in two different media: nitrate broth and a minimal medium enriched with glucose. Ten isolates were considered denitrifying bacteria and 7 were dissimilatory ammonia producers. Ammonia production by the isolate identified as Enterobacter amnigenus was quantified and attained 50% of 138?mg?L(-1) of added NO(3)(-) N. The dissimilatory character of this reduction was clearly confirmed by culturing this (15)N-labeled bacterium in the presence of unlabeled nitrite. Nitrous oxide was produced at the same time as nitrite was reduced to ammonia. Increasing nitrate N levels from 48 to 553?mg?L(-1) in culture medium resulted in an increase in the level of nitrite produced and simultaneously a decrease in ammonia and nitrous oxide production. Key words: dissimilatory nitrate reduction, dissimilatory ammonia production, denitrification, Enterobacter amnigenus, (15)N.     

Oxygen requirement for denitrification by the fungus Fusarium oxysporum

The effects of dioxygen (O2) on the denitrification activity of the fungus Fusarium oxysporum MT-811 in fed-batch culture in a stirred jar fermentor were examined. The results revealed that fungal denitrifying activity requires a minimal amount of O2 for induction, which is repressed by excess O2. The optimal O2 supply differed between the denitrification substrates : 690 micromol O2 x h(-1) (g dry cell wt.)(-1) for nitrate (NO3-) and about 250 micromol O2 x h(-1) (g dry cell wt.)(-1) for nitrite (NO2-). The reduction of NO3- required more O2 than that of NO2- . With an optimal O2 supply, 80% and 52% of nitrogen atoms in NO3- and NO2-, respectively, were recovered as the denitrification product N2O. These features of F. oxysporum differ from those of bacterial denitrifiers that work exclusively under anoxic conditions. The denitrification activity of F. oxysporum MT-811 mutants with impaired NO3- assimilation was about double that of the wild-type strain, suggesting competition for the substrate between assimilatory and dissimilatory types of NO3- reduction. These results showed that denitrification by F. oxysporum has unique features, namely, a minimal O2 requirement and competition with assimilatory NO3-.     

A cytochrome c containing nitrate reductase plays a role in electron transport for denitrification in Thermus thermophilus without involvement of the bc respiratory complex

The bc(1) respiratory complex III constitutes a key energy-conserving respiratory electron transporter between complex I (type I NADH dehydrogenase) and II (succinate dehydrogenase) and the final nitrogen oxide reductases (Nir, Nor and Nos) in most denitrifying bacteria. However, we show that the expression of complex III from Thermus thermophilus is repressed under denitrification, and that its role as electron transporter is replaced by an unusual nitrate reductase (Nar) that contains a periplasmic cytochrome c (NarC). Several lines of evidence support this conclusion: (i) nitrite and NO are as effective signals as nitrate for the induction of Nar; (ii) narC mutants are defective in anaerobic growth with nitrite, NO and N2O; (iii) such mutants present decreased NADH oxidation coupled to these electron acceptors; and (iv) complementation assays of the mutants reveal that the membrane-distal heme c of NarC was necessary for anaerobic growth with nitrite, whereas the membrane-proximal heme c was not. Finally, we show evidence to support that Nrc, the main NADH oxidative activity in denitrification, interacts with Nar through their respective membrane subunits. Thus, we propose the existence of a Nrc-Nar respiratory super-complex that is required for the development of the whole denitrification pathway in T. thermophilus.     

Denitrification under various aeration conditions in Comamonas sp., strain SGLY2

Abstract A number of bacteria were isolated from different anoxic reactors. Those having denitrifying potential were tested for their ability to denitrify under aerobic conditions. The activity of their denitrifying enzymes varied from partial inactivation by oxygen (strains NO2B9 and TCET1) to oxygen-independent activity in a strain named SGLY2 which was tentatively identified as Comamonas sp. The effect of different aeration conditions on growth and on denitrification of SGLY2 was studied more extensively. This strain was able to consume oxygen and nitrate simultaneously with the production of nitrogen and without build-up of nitrite. The dissimilatory nitrate-reductase of nitrate-adapted cells was found to be more active in the presence of oxygen than in micro-aerobic or strictly anaerobic conditions.     

真菌异化硝酸盐还原机理的研究进展

真菌异化硝酸盐还原途径的发现打破了反硝化仅存在于原核细胞这一传统观念。真菌异化硝酸盐还原途径是在环境中氧供给受限的情况下发生的, 包括反硝化和氨的发酵。硝酸盐能诱导产生反硝化作用的酶, 其中, 硝酸盐还原酶与亚硝酸还原酶位于线粒体中, 它们所催化的酶促反应能偶联呼吸链ATP合成酶合成ATP, 同时产生NO。与参与反硝化作用前两个酶不同, 真菌NO还原酶能以NADH为直接电子供体将NO还原为N2O, 在NAD+的再生和自由基NO的脱毒中起着重要作用。氨发酵则将硝酸盐还原成NH4+, 同时偶联乙酸的生成和底物水平磷酸化。此文从参与该过程的关键酶、关键酶的表达调节、真菌与细菌异化硝酸盐还原的比较等角度综述了真菌异化硝酸盐还原的最新研究进展。     

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     

Disruption of narG, the Gene Encoding the Catalytic Subunit of Respiratory Nitrate Reductase, Also Affects Nitrite Respiration in Pseudomonas fluorescens YT101

The Pseudomonas fluorescens YT101 gene narG, which encodes the catalytic alpha subunit of the respiratory nitrate reductase, was disrupted by insertion of a gentamicin resistance cassette. In the Nar(-) mutants, nitrate reductase activity was not detectable under all the conditions tested, suggesting that P. fluorescens YT101 contains only one membrane-bound nitrate reductase and no periplasmic nitrate reductase. Whereas N(2)O respiration was not affected, anaerobic growth with NO(2) as the sole electron acceptor was delayed for all of the Nar(-) mutants following a transfer from oxic to anoxic conditions. These results provide the first demonstration of a regulatory link between nitrate and nitrite respiration in the denitrifying pathway.     

Gas chromatographic assay for in vitro complementation of Pseudomonas aeruginosa mutants deficient in nitrate reduction.

An electron capture gas-chromatographic technique was developed to continuously measure nitrate (NO3-) reduction during in vitro complementation tests with extracts from Pseudomonas aeruginosa mutants deficient in both assimilatory and dissimilatory nitrate reduction as a result of a single genetic mutation. The procedure involves coupling nitrate reduction to nitrous oxide (N2O) evolution via a series of reactions specific to the denitrification pathway. The assay was dependent on nitrate concentration, and optimal activity was obtained with a final concentration of 0.2% potassium nitrate. The reduction exhibited a stoichiometry of 2:1 (NO3-/N2O), and succinate was the best electron source for the reaction, followed by glucose, pyruvate, and malate. The technique can also be used for continuously monitoring nitrate reduction. The optimal nitrite concentration in the nitrite reductase assay was 0.025%. The initial complementation studies of mutant extracts demonstrated that at least two genes are shared between the two nitrate reduction pathways in P. aeruginosa.     

Gas chromatographic assay for in vitro complementation of Pseudomonas aeruginosa mutants deficient in nitrate reduction

An electron capture gas-chromatographic technique was developed to continuously measure nitrate (NO3-) reduction during in vitro complementation tests with extracts from Pseudomonas aeruginosa mutants deficient in both assimilatory and dissimilatory nitrate reduction as a result of a single genetic mutation. The procedure involves coupling nitrate reduction to nitrous oxide (N2O) evolution via a series of reactions specific to the denitrification pathway. The assay was dependent on nitrate concentration, and optimal activity was obtained with a final concentration of 0.2% potassium nitrate. The reduction exhibited a stoichiometry of 2:1 (NO3-/N2O), and succinate was the best electron source for the reaction, followed by glucose, pyruvate, and malate. The technique can also be used for continuously monitoring nitrate reduction. The optimal nitrite concentration in the nitrite reductase assay was 0.025%. The initial complementation studies of mutant extracts demonstrated that at least two genes are shared between the two nitrate reduction pathways in P. aeruginosa.     

Nitric oxide metabolism in Neisseria meningitidis

Neisseria meningitidis, the causative agent of meningococcal disease in humans, is likely to be exposed to nitrosative stress during natural colonization and disease. The genome of N. meningitidis includes the genes aniA and norB, predicted to encode nitrite reductase and nitric oxide (NO) reductase, respectively. These gene products should allow the bacterium to denitrify nitrite to nitrous oxide. We show that N. meningitidis can support growth microaerobically by the denitrification of nitrite via NO and that norB is required for anaerobic growth with nitrite. NorB and, to a lesser extent, the cycP gene product cytochrome c' are able to counteract toxicity due to exogenously added NO. Expression of these genes by N. meningitidis during colonization and disease may confer protection against exogenous or endogenous nitrosative stress.     

Heterotrophic nitrification by Alcaligenes faecalis: NO2-, NO3-, N2O, and NO production in exponentially growing cultures.

Heterotrophic nitrification by Alcaligenes faecalis DSM 30030 was not restricted to media containing organic forms of nitrogen. In both peptone-meat extract and defined media with ammonium and citrate as the sole nitrogen and carbon sources, respectively, NO2-, NO3-, NO, and N2O were produced under aerobic growth conditions. Heterotrophic nitrification was not attributable to old or dying cell populations. Production of NO2-, NO3-, NO, and N2O was detectable shortly after cultures started growth and proceeded exponentially during the logarithmic growth phase. NO2- and NO3- production rates were higher for cultures inoculated in media with pH values below 7 than for those in media at alkaline pH. Neither assimilatory nor dissimilatory nitrate or nitrite reductase activities were detectable in aerobic cultures.     

Heterotrophic nitrification by Alcaligenes faecalis: NO2-, NO3-, N2O, and NO production in exponentially growing cultures

Heterotrophic nitrification by Alcaligenes faecalis DSM 30030 was not restricted to media containing organic forms of nitrogen. In both peptone-meat extract and defined media with ammonium and citrate as the sole nitrogen and carbon sources, respectively, NO2-, NO3-, NO, and N2O were produced under aerobic growth conditions. Heterotrophic nitrification was not attributable to old or dying cell populations. Production of NO2-, NO3-, NO, and N2O was detectable shortly after cultures started growth and proceeded exponentially during the logarithmic growth phase. NO2- and NO3- production rates were higher for cultures inoculated in media with pH values below 7 than for those in media at alkaline pH. Neither assimilatory nor dissimilatory nitrate or nitrite reductase activities were detectable in aerobic cultures.     

Nitrous oxide-forming codenitrification catalyzed by cytochrome P450nor

Intact cells of the denitrifying fungus Fusarium oxysporum were previously shown to catalyze codenitrification to form a hybrid nitrous oxide (N2O) species from nitrite and other nitrogen compounds such as azide and ammonia. Here we show that cytochrome P450nor can catalyze the codenitrification reaction to form N2O from nitric oxide (NO) but not nitrite, and azide or ammonia. The results show that the direct substrate of the codenitrification by intact cells should not be nitrite but NO, which is formed from nitrite by the reaction of a dissimilatory nitrite reductase.     

Anaerobic activation of the entire denitrification pathway in Pseudomonas aeruginosa requires Anr, an analog of Fnr.

The Pseudomonas aeruginosa gene anr, which encodes a structural and functional analog of the anaerobic regulator Fnr in Escherichia coli, was mapped to the SpeI fragment R, which is at about 59 min on the genomic map of P. aeruginosa PAO1. Wild-type P. aeruginosa PAO1 grew under anaerobic conditions with nitrate, nitrite, and nitrous oxide as alternative electron acceptors. An anr deletion mutant, PAO6261, was constructed. It was unable to grow with these alternative electron acceptors; however, its ability to denitrify was restored upon the introduction of the wild-type anr gene. In addition, the activities of two enzymes in the denitrification pathway, nitrite reductase and nitric oxide reductase, were not detectable under oxygen-limiting conditions in strain PAO6261 but were restored when complemented with the anr+ gene. These results indicate that the anr gene product plays a key role in anaerobically activating the entire denitrification pathway.     

Denitrification in lucerne nodules is not involved in nitrite detoxification

Dissimilatory reduction of ionic nitrogen oxides to gaseous forms such as nitrous oxide or nitrogen can be carried out by free living or symbiotic forms of some strains of Rhizobium meliloti. In this paper we investigate whether bacteroid denitrification plays a role in the alleviation of the inhibitory effects of nitrate on nitrogen fixation both in bacteroid incubations as in whole nodules. The presence of a constitutive nitrate reductase (NR) activity in isolated bacteroids caused nitrite accumulation in the incubation medium, and acetylene reduction activity in these bacteroids was progressively inhibited, since nitrite reductase (NiR) activity was unable to reduce all the nitrite produced by NR and denitrification occurred slowly. Even nodules infiltrated with nitrate and nitrite failed to increase gaseous forms of nitrogen substantially, indicating that nitrite availability was not limiting denitrification by bacteroids. In spite of the low rates of bacteroidal denitrification, the effect of nodule denitrification on the inhibition of nitrogen fixation by nitrate in whole plants was tested. For that purpose, lucerne plants (Medicago sativa L. cv. Aragon) were inoculated with two Rhizobium meliloti strains: 102-F-65 (non denitrifying) and 102-F-51 (a highly denitrifying strain). After a seven days nitrate treatment, both strains showed the same pattern of inhibition, and it occurred before any nitrate or nitrite accumulation within the nodules could be detected. This observation, together with the lack of alleviation of the ARA inhibition in the denitrifying strain, and the limited activity of dissimilatory nitrogen reduction present in these bacteroids, indicate a role other than nitrite detoxification for denitrification in nodules under natural conditions.