Novel nirK cluster genes in Nitrosomonas europaea are required for NirK-dependent tolerance to nitrite

Nitrite reductase (NirK) of Nitrosomonas europaea confers tolerance to nitrite (NO2-). The nirK gene is clustered with three genes of unknown physiological function: ncgABC. At present, this organization is unique to nitrifying bacteria. Here we report that the ncgABC gene products facilitate NirK-dependent NO2- tolerance by reversing the negative physiological effect that is associated with the activity of NirK in their absence. We hypothesize that the ncg gene products are involved in the detoxification of nitric oxide that is produced by NirK.     

Nitrosomonas europaea expresses a nitric oxide reductase during nitrification

In this paper, we report the identification of a norCBQD gene cluster that encodes a functional nitric oxide reductase (Nor) in Nitrosomonas europaea. Disruption of the norB gene resulted in a strongly diminished nitric oxide (NO) consumption by cells and membrane protein fractions, which was restored by the introduction of an intact norCBQD gene cluster in trans. NorB-deficient cells produced amounts of nitrous oxide (N2O) equal to that of wild-type cells. NorCB-dependent activity was present during aerobic growth and was not affected by the inactivation of the putative fnr gene. The findings demonstrate the presence of an alternative site of N2O production in N. europaea.     

Revision of N2O-Producing Pathways in the Ammonia-Oxidizing Bacterium Nitrosomonas europaea ATCC 19718

Nitrite reductase (NirK) and nitric oxide reductase (NorB) have long been thought to play an essential role in nitrous oxide (N₂O) production by ammonia-oxidizing bacteria. However, essential gaps remain in our understanding of how and when NirK and NorB are active and functional, putting into question their precise roles in N₂O production by ammonia oxidizers. The growth phenotypes of the Nitrosomonas europaea ATCC 19718 wild-type and mutant strains deficient in expression of NirK, NorB, and both gene products were compared under atmospheric and reduced O₂ tensions. Anoxic resting-cell assays and instantaneous nitrite (NO₂⁻) reduction experiments were done to assess the ability of the wild-type and mutant N. europaea strains to produce N₂O through the nitrifier denitrification pathway. Results confirmed the role of NirK for efficient substrate oxidation of N. europaea and showed that NorB is involved in N₂O production during growth at both atmospheric and reduced O₂ tensions. Anoxic resting-cell assays and measurements of instantaneous NO₂⁻ reduction using hydrazine as an electron donor revealed that an alternate nitrite reductase to NirK is present and active. These experiments also clearly demonstrated that NorB was the sole nitric oxide reductase for nitrifier denitrification. The results of this study expand the enzymology for nitrogen metabolism and N₂O production by N. europaea and will be useful to interpret pathways in other ammonia oxidizers that lack NirK and/or NorB genes.     

Role of nitrite reductase in the ammonia-oxidizing pathway of <Emphasis Type="Italic">Nitrosomonas europaea</Emphasis>

Metabolism of ammonia (NH₃) and hydroxylamine (NH₂OH) by wild-type and a nitrite reductase (nirK) deficient mutant of Nitrosomonas europaea was investigated to clarify the role of NirK in the NH₃ oxidation pathway. NirK-deficient N. europaea grew more slowly, consumed less NH₃, had a lower rate of nitrite (NO₂ ⁻) production, and a significantly higher rate of nitrous oxide (N₂O) production than the wild-type when incubated with NH₃ under high O₂ tension. In incubations with NH₃ under low O₂ tension, NirK-deficient N. europaea grew more slowly, but had only modest differences in NH₃ oxidation and product formation rates relative to the wild-type. In contrast, the nirK mutant oxidized NH₂OH to NO₂ ⁻ at consistently slower rates than the wild-type, especially under low O₂ tension, and lost a significant pool of NH₂OH–N to products other than NO₂ ⁻ and N₂O. The rate of N₂O production by the nirK mutant was ca. three times higher than the wild-type during hydrazine-dependent NO₂ ⁻ reduction under both high and low O₂ tension. Together, the results indicate that NirK activity supports growth of N. europaea by supporting the oxidation of NH₃ to NO₂ ⁻ via NH₂OH, and stimulation of hydrazine-dependent NO₂ ⁻ reduction by NirK-deficient N. europaea indicated the presence of an alternative, enzymatic pathway for N₂O production.     

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.     

Identification of the sources of nitrous oxide produced by oxidative and reductive processes in Nitrosomonas europaea

1. Cells of Nitrosomonas europaea produced N(2)O during the oxidation of ammonia and hydroxylamine. 2. The end-product of ammonia oxidation, nitrite, was the predominant source of N(2)O in cells. 3. Cells also produced N(2)O, but not N(2) gas, by the reduction of nitrite under anaerobic conditions. 4. Hydroxylamine was oxidized by cell-free extracts to yield nitrite and N(2)O aerobically, but to yield N(2)O and NO anaerobically. 5. Cell extracts reduced nitrite both aerobically and anaerobically to NO and N(2)O with hydroxylamine as an electron donor. 6. The relative amounts of NO and N(2)O produced during hydroxylamine oxidation and/or nitrite reduction are dependent on the type of artificial electron acceptor utilized. 7. Partially purified hydroxylamine oxidase retained nitrite reductase activity but cytochrome oxidase was absent. 8. There is a close association of hydroxylamine oxidase and nitrite reductase activities in purified preparations.     

Production of NO and N(inf2)O by Pure Cultures of Nitrifying and Denitrifying Bacteria during Changes in Aeration

Peak emissions of NO and N(inf2)O are often observed after wetting of soil. The reactions to sudden changes in the aeration of cultures of nitrifying and denitrifying bacteria with respect to NO and N(inf2)O emissions were compared to obtain more information about the microbiological aspects of peak emissions. In continuous culture, the nitrifier Nitrosomonas europaea and the denitrifiers Alcaligenes eutrophus and Pseudomonas stutzeri were cultured at different levels of aeration (80 to 0% air saturation) and subjected to changes in aeration. The relative production of NO and N(inf2)O by N. europaea, as a percentage of the ammonium conversion, increased from 0.87 and 0.17%, respectively, at 80% air saturation to 2.32 and 0.78%, respectively, at 1% air saturation. At 0% air saturation, ammonium oxidation and N(inf2)O production ceased but NO production was enhanced. Coculturing of N. europaea with the nitrite oxidizer Nitrobacter winogradskyi strongly reduced the relative levels of NO and N(inf2)O production, probably as an effect of the lowered nitrite concentration. After lowering the aeration, N. europaea produced large short-lasting peaks of NO and N(inf2)O emissions in the presence but not in the absence of nitrite. A. eutrophus and P. stutzeri began to denitrify below 1% air saturation, with the former accumulating nitrite and N(inf2)O and the latter reducing nitrate almost completely to N(inf2). Transition of A. eutrophus and P. stutzeri from 80 to 0% air saturation resulted in transient maxima of denitrification intermediates. Such transient maxima were not observed after transition from 1 to 0%. Reduction of nitrate by A. eutrophus continued 48 h after the onset of the aeration, whereas N(inf2)O emission by P. stutzeri increased for only a short period. It was concluded that only in the presence of nitrite are nitrifiers able to dominate the NO and N(inf2)O emissions of soils shortly after a rainfall event.     

Nitrite and nitrous oxide production by Methylosinus trichosporium

Conditions for the production of nitrite and nitrous oxide by an obligate methanotroph, Methylosinus trichosporium (OB 3b), were studied. The rate of nitrite production (V NO2-) was correlated with the concentration of ammonia up to 20 mM in the presence of sufficient amounts of oxygen and inversely correlated with the amounts of methane in the system. The rate of nitrous oxide (N2O) production (V N2O) was correlated positively with V NO2- and the amount of nitrite produced and inversely with the oxygen concentration in the system. Nitrite started to disappear when either oxygen or methane or both were depleted, but only a part of the loss could be accounted for by an increase in N2O. Maximum rates of nitrite and N2O production by Ms. trichosporium were 6.9 X 10(-16) and 2.2 X 10(-17) mol . cell-1 X day-1, respectively. These values are about 0.2 and 1.6% of the values for Nitrosomonas europaea. Therefore, production of nitrite and N2O by methanotrophs in aquatic environments may not be as significant as that of Nitrosomonas.     

A comparison of NO and N2O production by the autotrophic nitrifier Nitrosomonas europaea and the heterotrophic nitrifier Alcaligenes faecalis.

Soil microorganisms are important sources of the nitrogen trace gases NO and N2O for the atmosphere. Present evidence suggests that autotrophic nitrifiers such as Nitrosomonas europaea are the primary producers of NO and N2O in aerobic soils, whereas denitrifiers such as Pseudomonas spp. or Alcaligenes spp. are responsible for most of the NO and N2O emissions from anaerobic soils. It has been shown that Alcaligenes faecalis, a bacterium common in both soil and water, is capable of concomitant heterotrophic nitrification and denitrification. This study was undertaken to determine whether heterotrophic nitrification might be as important a source of NO and N2O as autotrophic nitrification. We compared the responses of N. europaea and A. faecalis to changes in partial O2 pressure (pO2) and to the presence of typical nitrification inhibitors. Maximal production of NO and N2O occurred at low pO2 values in cultures of both N. europaea (pO2, 0.3 kPa) and A. faecalis (pO2, 2 to 4 kPa). With N. europaea most of the NH4+ oxidized was converted to NO2-, with NO and N2O accounting for 2.6 and 1% of the end product, respectively. With A. faecalis maximal production of NO occurred at a pO2 of 2 kPa, and maximal production of N2O occurred at a pO2 of 4 kPa. At these low pO2 values there was net nitrite consumption. Aerobically, A. faecalis produced approximately the same amount of NO but 10-fold more N2O per cell than N. europaea did. Typical nitrification inhibitors were far less effective for reducing emissions of NO and N2O by A. faecalis than for reducing emissions of NO and N2O by N. europaea.(ABSTRACT TRUNCATED AT 250 WORDS)     

Marker exchange of the structural genes for nitric oxide reductase blocks the denitrification pathway of Pseudomonas stutzeri at nitric oxide.

Bacterial denitrification reverses nitrogen fixation in the global N-cycle by transforming nitrate or nitrite to dinitrogen. Both nitrite and nitric oxide (NO) are considered as the chemical species within the denitrification pathway, that precede nitrous oxide (N2O), the first recognized intermediate with N,N-bonds antecedent to N2. Molecular cloning of the structural genes for NO reductase from Pseudomonas stutzeri has allowed us to generate the first mutants defective in NO utilization (Nor- phenotype) by marker exchange of the norCB genes with a gene cassette for gentamicin resistance. Nitric oxide reductase was found to be an indispensable component for denitrification; its loss constituted a conditionally lethal mutation. NO as the sole product accumulated from nitrite by mutant cells induced for nitrite respiration (denitrification). The Nor- mutant lost the capability to reduce NO and did not grow anymore anaerobically on nitrate. A Nir-Nor- double mutation, that inactivated also the respiratory nitrite reductase cytochrome cd1 rendered the bacterium again viable under anaerobiosis. Our observations provide evidence for a denitrification pathway in vivo of NO2(-)----NO----N2O, and N,N-bond formation catalyzed by NO reductase and not by cytochrome cd1.     

Characterization of Tn5 mutants deficient in dissimilatory nitrite reduction in Pseudomonas sp. strain G-179, which contains a copper nitrite reductase.

Tn5 was used to generate mutants that were deficient in the dissimilatory reduction of nitrite for Pseudomonas sp. strain G-179, which contains a copper nitrite reductase. Three types of mutants were isolated. The first type showed a lack of growth on nitrate, nitrite, and nitrous oxide. The second type grew on nitrate and nitrous oxide but not on nitrite (Nir-). The two mutants of this type accumulated nitrite, showed no nitrite reductase activity, and had no detectable nitrite reductase protein bands in a Western blot (immunoblot). Tn5 insertions in these two mutants were clustered in the same region and were within the structural gene for nitrite reductase. The third type of mutant grew on nitrate but not on nitrite or nitrous oxide (N2O). The mutant of this type accumulated significant amounts of nitrite, NO, and N2O during anaerobic growth on nitrate and showed a slower growth rate than the wild type. Diethyldithiocarbamic acid, which inhibited nitrite reductase activity in the wild type, did not affect NO reductase activity, indicating that nitrite reductase did not participate in NO reduction. NO reductase activity in Nir- mutants was lower than that in the wild type when the strains were grown on nitrate but was the same as that in the wild type when the strains were grown on nitrous oxide. These results suggest that the reduction of NO and N2O was carried out by two distinct processes and that mutations affecting nitrite reduction resulted in reduced NO reductase activity following anaerobic growth with nitrate.     

Nitrogen 15 tracer studies on the pathway of denitrification in Pseudomonas aeruginosa.

The pathway of anaerobic reduction of nitrite to nitrogen gas (N2) by cell suspensions of the denitrifier, Pseudomonas aeruginosa, was studied using the techniques of gas chromatography and mass spectrometry. While release of nitrous oxide (N2O) is not normally detected during the reduction of nitrite to N2 by this organism, 15N from [15N]nitrite nevertheless can be trapped quantitatively as 15N2O in a pool of added N2O. In such experiments the abundance of 15N in N2O always exceeds that in product N2, consistent with the absence of a major reductive route from nitrite to N2 which by-passes N2O. During the reduction of a mixture of [15N]nitrite and nitric oxide (NO), 15NO produced at most only in trace amounts. The final products are chiefly 15N2 and 14N2 with only a small fraction of the scrambled product, 14N15N. Much of the 14N15N can be accounted for as an artifact caused by traces of molecular oxygen, which promote the conversion of NO to nitrite by autooxidation and thereby degrade slightly the isotopic purity of [15N]nitrite. Nitrous oxide shows all the properties of a free obligatory intermediate during the denitrification of nitrite to N2 by P. aeruginosa, whereas NO does not. The inability to trap 15NO in a pool of NO indicates that NO is not a free obligatory intermediate in the reduction of nitrite. The small mole fractions of 14N15N produced from a mixture of [15N]nitrite and NO require that the main reductive pathways for these nitrogen oxides cannot share any freely diffusible mono-nitrogen intermediate in common. The simplest interpretation is that nitrite and NO are denitrified by separate pathways, at least prior to the formation of the first bi-nitrogen compound.     

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.     

Growth of Nitrosomonas europaea on hydroxylamine

Abstract Hydroxylamine is an intermediate in the oxidation of ammonia to nitrite, but until now it has not been possible to grow Nitrosomonas europaea on hydroxylamine. This study demonstrates that cells of N. europaea are capable of growing mixotrophically on ammonia and hydroxylamine. The molar growth yield on hydroxylamine (4.74 g mol⁻¹ at a growth rate of 0.03 h⁻¹) was higher than expected. Aerobically growing cells of N. europaea oxidized ammonia to nitrite with little loss of inorganic nitrogen, while significant inorganic nitrogen losses occurred when cells were growing mixotrophically on ammonia and hydroxylamine. In the absence of oxygen, hydroxylamine was oxidized with nitrite as electron acceptor, while nitrous oxide was produced. Anaerobic growth of N. europaea on ammonium, hydroxylamine and nitrite could not be observed at growth rates of 0.03 h⁻¹ and 0.01 h⁻¹.     

Effects of nitrite on ammonia-oxidizing activity and gene regulation in three ammonia-oxidizing bacteria

Nitrite is the highly toxic end product of ammonia oxidation that accumulates in the absence of a nitrite-consuming process and is inhibitory to nitrifying and other bacteria. The effects of nitrite on ammonia oxidation rates and regulation of a common gene set were compared in three ammonia-oxidizing bacteria (AOB) to determine whether responses to this toxic metabolite were uniform. Mid-exponential-phase cells of Nitrosomonas europaea ATCC 19718, Nitrosospira multiformis ATCC 25196, and Nitrosomonas eutropha C-91 were incubated for 6 h in mineral medium supplemented with 0, 10, or 20 mM NaNO(2) . The rates of ammonia oxidation (nitrite production) decreased significantly only in NaNO(2) -supplemented incubations of N. eutropha; no significant effect on the rates was observed for N. europaea or N. multiformis. The levels of norB (nitric oxide reductases), cytL (cytochrome P460), and cytS (cytochrome c'-β) mRNA were unaffected by nitrite in all strains. The levels of nirK (nitrite reductase) mRNA increased only in N. europaea in response to nitrite (10 and 20 mM). Nitrite (20 mM) significantly reduced the mRNA levels of amoA (ammonia monooxygenase) in N. multiformis and norS (nitric oxide reductase) in the two Nitrosomonas spp. Differences in response to nitrite indicated nonuniform adaptive and regulatory strategies of AOB, even between closely related species.     

Production of nitrous oxide from nitrite in Klebsiella pneumoniae: mutants altered in nitrogen metabolism.

Under anaerobic conditions, Klebsiella pneumoniae reduced nitrite (NO2-), yielding nitrous oxide (N2O) and ammonium ions (NH4+) as products. Nitrous oxide formation accounted for about 5% of the total NO2- reduced, and NH4+ production accounted for the remainder. Glucose and pyruvate were the electron donors for NO2- reduction to N2O by whole cells, whereas glucose, NADH, and NADPH were found to be the electron donors when cell extracts were used. On the one hand, formate failed to serve as an electron donor for NO2- reduction to N2O and NH4+, whereas on the other hand, formate was the best electron donor for nitrate reduction in either whole cells or cell extracts. Mutants that are defective in the reduction of NO2- to NH4+ were isolated, and these strains were found to produce N2O at rates comparable to that of the parent strain. These results suggest that the nitrite reductase producing N2O is distinct from that producing NH4+. Nitrous oxide production from nitric oxide (NO) occurred in all mutants tested, at rates comparable to that of the parent strain. This result suggests that NO reduction to N2O, which also uses NADH as the electron donor, is independent of the protein(s) catalyzing the reduction of NO2- to N2O.     

Physiologic and proteomic evidence for a role of nitric oxide in biofilm formation by Nitrosomonas europaea and other ammonia oxidizers

NO, a free radical gas, is the signal for Nitrosomonas europaea cells to switch between different growth modes. At an NO concentration of more than 30 ppm, biofilm formation by N. europaea was induced. NO concentrations below 5 ppm led to a reversal of the biofilm formation, and the numbers of motile and planktonic (motile-planktonic) cells increased. In a proteomics approach, the proteins expressed by N. europaea were identified. Comparison studies of the protein patterns of motile-planktonic and attached (biofilm) cells revealed several clear differences. Eleven proteins were found to be up or down regulated. Concentrations of other compounds such as ammonium, nitrite, and oxygen as well as different temperatures and pH values had no significant effect on the growth mode of and the proteins expressed by N. europaea.