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.     

Distinguishing Nitrous Oxide Production from Nitrification and Denitrification on the Basis of Isotopomer Abundances

The intramolecular distribution of nitrogen isotopes in N₂O is an emerging tool for defining the relative importance of microbial sources of this greenhouse gas. The application of intramolecular isotopic distributions to evaluate the origins of N₂O, however, requires a foundation in laboratory experiments in which individual production pathways can be isolated. Here we evaluate the site preferences of N₂O produced during hydroxylamine oxidation by ammonia oxidizers and by a methanotroph, ammonia oxidation by a nitrifier, nitrite reduction during nitrifier denitrification, and nitrate and nitrite reduction by denitrifiers. The site preferences produced during hydroxylamine oxidation were 33.5 ± 1.2‰, 32.5 ± 0.6‰, and 35.6 ± 1.4‰ for Nitrosomonas europaea, Nitrosospira multiformis, and Methylosinus trichosporium, respectively, indicating similar site preferences for methane and ammonia oxidizers. The site preference of N₂O from ammonia oxidation by N. europaea (31.4 ± 4.2‰) was similar to that produced during hydroxylamine oxidation (33.5 ± 1.2‰) and distinct from that produced during nitrifier denitrification by N. multiformis (0.1 ± 1.7‰), indicating that isotopomers differentiate between nitrification and nitrifier denitrification. The site preferences of N₂O produced during nitrite reduction by the denitrifiers Pseudomonas chlororaphis and Pseudomonas aureofaciens (−0.6 ± 1.9‰ and −0.5 ± 1.9‰, respectively) were similar to those during nitrate reduction (−0.5 ± 1.9‰ and −0.5 ± 0.6‰, respectively), indicating no influence of either substrate on site preference. Site preferences of ~33‰ and ~0‰ are characteristic of nitrification and denitrification, respectively, and provide a basis to quantitatively apportion N₂O.     

High cell density cultivation of the chemolithoautotrophic bacterium <Emphasis Type="Italic">Nitrosomonas europaea</Emphasis>

Nitrosomonas europaea is a chemolithoautotrophic nitrifier, a gram-negative bacterium that can obtain all energy required for growth from the oxidation of ammonia to nitrite, and this may be beneficial for various biotechnological and environmental applications. However, compared to other bacteria, growth of ammonia oxidizing bacteria is very slow. A prerequisite to produce high cell density N. europaea cultures is to minimize the concentrations of inhibitory metabolic by-products. During growth on ammonia nitrite accumulates, as a consequence, N. europaea cannot grow to high cell concentrations under conventional batch conditions. Here, we show that single-vessel dialysis membrane bioreactors can be used to obtain substantially increased N. europaea biomasses and substantially reduced nitrite levels in media initially containing high amounts of the substrate. Dialysis membrane bioreactor fermentations were run in batch as well as in continuous mode. Growth was monitored with cell concentration determinations, by assessing dry cell mass and by monitoring ammonium consumption as well as nitrite formation. In addition, metabolic activity was probed with in vivo acridine orange staining. Under continuous substrate feed, the maximal cell concentration (2.79?×?10¹²/L) and maximal dry cell mass (0.895 g/L) achieved more than doubled the highest values reported for N. europaea cultivations to date.     

15N Kinetic Analysis of N2O Production by Nitrosomonas europaea: an Examination of Nitrifier Denitrification

A series of ¹⁵N isotope tracer experiments showed that Nitrosomonas europaea produces nitrous oxide only under oxygen-limiting conditions and that the labeled N from nitrite, but not nitrate, is incorporated into nitrous oxide, indicating the presence of the “denitrifying enzyme” nitrite reductase. A kinetic analysis of the m/z 44, 45, and 46 nitrous oxide produced by washed cell suspensions of N. europaea when incubated with 4 mM ammonium (99% ¹⁴N) and 0.4 mM nitrite (99% ¹⁵N) was performed. No labeled nitrite was reduced to ammonium. All labeled material added was accounted for as either nitrite or nitrous oxide. The hypothesis that nitrous oxide is produced directly from nitrification was rejected since (i) it does not allow for the large amounts of double-labeled (m/z 46) nitrous oxide observed; (ii) the observed patterns of m/z 44, 45, and 46 nitrous oxide were completely consistent with a kinetic analysis based on denitrification as the sole mechanism of nitrous oxide production but not with a kinetic analysis based on both mechanisms; (iii) the asymptotic ratio of m/z 45 to m/z 46 nitrous oxide was consistent with denitrification kinetics but inconsistent with nitrification kinetics, which predicted no limit to m/z 45 production. It is concluded that N. europaea is a denitrifier which, under conditions of oxygen stress, uses nitrite as a terminal electron acceptor and produces nitrous oxide.     

The effect of nitrate in stream water on the relationship between gentrification and nitrification in a stream-sediment microcosm

SUMMARY 1. Microcosm experiments were carried out to simulate, in the laboratory, the conditions occurring at the water-sediment interface of a stream draining agricultural land. Constant boundary conditions were attained by passing synthetic 'stream water', saturated with dissolved oxygen and containing 1 mmol NO₃?N dm^?3 (or 1 mmol Cl? dm^?3, control), once only over the sediment surface. 2. Measurements were made of inorganic-N (nitrate, nitrite, ammonium), redox potential, potential denitrification and nitrification activities, and readily mineralizable carbon sediment profiles at three incubation times up to 24 days. The peaks in denitrification and nitrification activity moved down the profile with time in the nitrate-treated sediment, but stayed relatively stationary in the control treatment. Although the zone of nitrification was restricted to the top 2–3 mm of sediment in the control treatment, high fluxes of both dissolved oxygen and NH₄?N maintained a high nitrifier activity within this zone for the duration of the experiment. 3. Increases in denitrifier activity immediately below the nitrifier activity peak indicated that a coupled nitrification-denitrification sequence was operating in both the control and nitrate-treated sediment. The greater depth of nitrification when nitrate was present in the ‘stream water’ was attributed to a feedback mechanism in which enhanced denitrification in the sediment reduced the local demand for oxygen and permitted dissolved oxygen to diffuse further into the sediment. The progressively greater depth to which oxygen penetrated caused the contiguous peaks of potential nitrifier and denitrifier enzyme activity to migrate farther from the interface. However, diffusion rates of the reactants limited the depth to which these coupled reactions could extend. 4. The possible effect of this feedback mechanism on the nitrate status of natural sediment-stream water systems is briefly discussed.     

Distinguishing nitrous oxide production from nitrification and denitrification on the basis of isotopomer abundances

The intramolecular distribution of nitrogen isotopes in N2O is an emerging tool for defining the relative importance of microbial sources of this greenhouse gas. The application of intramolecular isotopic distributions to evaluate the origins of N2O, however, requires a foundation in laboratory experiments in which individual production pathways can be isolated. Here we evaluate the site preferences of N2O produced during hydroxylamine oxidation by ammonia oxidizers and by a methanotroph, ammonia oxidation by a nitrifier, nitrite reduction during nitrifier denitrification, and nitrate and nitrite reduction by denitrifiers. The site preferences produced during hydroxylamine oxidation were 33.5 +/- 1.2 per thousand, 32.5 +/- 0.6 per thousand, and 35.6 +/- 1.4 per thousand for Nitrosomonas europaea, Nitrosospira multiformis, and Methylosinus trichosporium, respectively, indicating similar site preferences for methane and ammonia oxidizers. The site preference of N2O from ammonia oxidation by N. europaea (31.4 +/- 4.2 per thousand) was similar to that produced during hydroxylamine oxidation (33.5 +/- 1.2 per thousand) and distinct from that produced during nitrifier denitrification by N. multiformis (0.1 +/- 1.7 per thousand), indicating that isotopomers differentiate between nitrification and nitrifier denitrification. The site preferences of N2O produced during nitrite reduction by the denitrifiers Pseudomonas chlororaphis and Pseudomonas aureofaciens (-0.6 +/- 1.9 per thousand and -0.5 +/- 1.9 per thousand, respectively) were similar to those during nitrate reduction (-0.5 +/- 1.9 per thousand and -0.5 +/- 0.6 per thousand, respectively), indicating no influence of either substrate on site preference. Site preferences of approximately 33 per thousand and approximately 0 per thousand are characteristic of nitrification and denitrification, respectively, and provide a basis to quantitatively apportion N2O.     

Loss of Ammonia Monooxygenase Activity in Nitrosomonas europaea upon Exposure to Nitrite

Nitrosomonas europaea, an obligate ammonia-oxidizing bacterium, lost an increasing amount of ammonia oxidation activity upon exposure to increasing concentrations of nitrite, the primary product of ammonia-oxidizing metabolism. The loss of activity was specific to the ammonia monooxygenase (AMO) enzyme, as confirmed by a decreased rate of NH₄⁺-dependent O₂ consumption, some loss of active AMO molecules observed by polypeptide labeling with ¹⁴C₂H₂, the protection of activity by substrates of AMO, and the requirement for copper. The loss of AMO activity via nitrite occurred under both aerobic and anaerobic conditions, and more activity was lost under alkaline than under acidic conditions except in the presence of large concentrations (20 mM) of nitrite. These results indicate that nitrite toxicity in N. europaea is mediated by a unique mechanism that is specific for AMO.     

Nitrogen loss caused by denitrifying Nitrosomonas cells using ammonium or hydrogen as electron donors and nitrite as electron acceptor

Cells of the obligately lithotrophic species Nitrosomonas europaea and Nitrosomonas eutropha were able to nitrify and denitrify at the same time when grown under oxygen limitation. In addition to oxygen, nitrite was used as an electron acceptor. The simultaneous nitrification and denitrification resulted in significant formation of the gaseous N-compounds nitrous oxide and dinitrogen, causing significant nitrogen loss. In mixed cultures of N. europaea and various chemoorganotrophic bacteria, the nitrogen loss was strongly influenced by the partners growing under oxygen limitation. Under anoxic conditions, pure cultures of N. eutropha were able to denitrify with molecular hydrogen as electron donor and nitrite as the only electron acceptor in a sulfide-reduced complex medium. The increase of cell numbers was directly coupled to nitrite reduction. Nitrous oxide and dinitrogen were the only detectable end products. In pure cultures of N. eutropha and mixed cultures of N. eutropha and Enterobacter aerogenes, ammonium and nitrite disappeared slowly at a molar ratio of about one when oxygen was absent. However, under these conditions cell growth was not measurable.     

Biodegradation of Halogenated Hydrocarbon Fumigants by Nitrifying Bacteria

Three species of nitrifying bacteria were tested for the ability to degrade the halocarbon fumigants methyl bromide, 1,2-dichloropropane, and 1,2-dibromo-3-chloropropane. The soil nitrifiers Nitrosomonas europaea and Nitrosolobus multiformis degraded all three fumigants, while the marine nitrifier Nitrosococcus oceanus degraded only methyl bromide under the conditions tested. Inhibition of biodegradation by allylthiourea and acetylene, specific inhibitors of ammonia monooxygenase, suggests that ammonia monooxygenase is the enzyme which catalyzes fumigant degradation.     

Kinetic Explanation for Accumulation of Nitrite, Nitric Oxide, and Nitrous Oxide During Bacterial Denitrification

The kinetics of denitrification and the causes of nitrite and nitrous oxide accumulation were examined in resting cell suspensions of three denitrifiers. An Alcaligenes species and a Pseudomonas fluorescens isolate characteristically accumulated nitrite when reducing nitrate; a Flavobacterium isolate did not. Nitrate did not inhibit nitrite reduction in cultures grown with tungstate to prevent formation of an active nitrate reductase; rather, accumulation of nitrite seemed to depend on the relative rates of nitrate and nitrite reduction. Each isolate rapidly reduced nitrous oxide even when nitrate or nitrite had been included in the incubation mixture. Nitrate also did not inhibit nitrous oxide reduction in Alcaligenes odorans, an organism incapable of nitrate reduction. Thus, added nitrate or nitrite does not always cause nitrous oxide accumulation, as has often been reported for denitrifying soils. All strains produced small amounts of nitric oxide during denitrification in a pattern suggesting that nitric oxide was also under kinetic control similar to that of nitrite and nitrous oxide. Apparent K_m values for nitrate and nitrite reduction were 15 μM or less for each isolate. The K_m value for nitrous oxide reduction by Flavobacterium sp. was 0.5 μM. Numerical solutions to a mathematical model of denitrification based on Michaelis-Menten kinetics showed that differences in reduction rates of the nitrogenous compounds were sufficient to account for the observed patterns of nitrite, nitric oxide, and nitrous oxide accumulation. Addition of oxygen inhibited gas production from ¹³NO₃⁻ by Alcaligenes sp. and P. fluorescens, but it did not reduce gas production by Flavobacterium sp. However, all three isolates produced higher ratios of nitrous oxide to dinitrogen as the oxygen tension increased. Inclusion of oxygen in the model as a nonspecific inhibitor of each step in denitrification resulted in decreased gas production but increased ratios of nitrous oxide to dinitrogen, as observed experimentally. The simplicity of this kinetic model of denitrification and its ability to unify disparate observations should make the model a useful guide in research on the physiology of denitrifier response to environmental effectors.     

The pathway of nitrogen and reductive enzymes of denitrification

Some recent studies on the pathway of nitrogen and the reductases of denitrification are reviewed. The available evidence suggests that while the intermediates of denitrification can remain enzyme-bound (presumably to nitrite reductase) prior to formation of N₂O, NO and nitroxyl (HNO) can be released in part by certain bacteria. Release of NO is recognized by a nitrite/NO?¹⁵N exchange reaction and isotopic scrambling in product N₂O; release of nitroxyl by Pseudomonas stutzeri is recognized by isotopic scrambling of nitrite and NO in product N₂O in absence of exchange and affords evidence that the first N?N bond forms in denitrification at the N¹⁺ redox level. The recent purification and partial characterization of nitrous oxide reductase are described. The ability of the dissimilatory nitrite reductase to activate nitrite for nitrosyl transfer affords a new chemical probe into the mechanism of action of this central enzyme. It would appear that reduction of nitrite is subject to electrophilic catalysis. ¹⁸O studies show that dissociation of nitrite from nitrite reductase can be slow relative to competing reduction or nitrosyl transfer.     

Effect of Tungstate on Nitrate Reduction by the Hyperthermophilic Archaeon Pyrobaculum aerophilum

Pyrobaculum aerophilum, a hyperthermophilic archaeon, can respire either with low amounts of oxygen or anaerobically with nitrate as the electron acceptor. Under anaerobic growth conditions, nitrate is reduced via the denitrification pathway to molecular nitrogen. This study demonstrates that P. aerophilum requires the metal oxyanion WO₄²⁻ for its anaerobic growth on yeast extract, peptone, and nitrate as carbon and energy sources. The addition of 1 μM MoO₄²⁻ did not replace WO₄²⁻ for the growth of P. aerophilum. However, cell growth was completely inhibited by the addition of 100 μM MoO₄²⁻ to the culture medium. At lower tungstate concentrations (0.3 μM and less), nitrite was accumulated in the culture medium. The accumulation of nitrite was abolished at higher WO₄²⁻ concentrations (<0.7 μM). High-temperature enzyme assays for the nitrate, nitrite, and nitric oxide reductases were performed. The majority of all three denitrification pathway enzyme activities was localized to the cytoplasmic membrane, suggesting their involvement in the energy metabolism of the cell. While nitrite and nitric oxide specific activities were relatively constant at different tungstate concentrations, the activity of nitrate reductase was decreased fourfold at WO₄²⁻ levels of 0.7 μM or higher. The high specific activity of the nitrate reductase enzyme observed at low WO₄²⁻ levels (0.3 μM or less) coincided with the accumulation of nitrite in the culture medium. This study documents the first example of the effect of tungstate on the denitrification process of an extremely thermophilic archaeon. We demonstrate here that nitrate reductase synthesis in P. aerophilum occurs in the presence of high concentrations of tungstate.     

Insights into Glycogen Metabolism in Chemolithoautotrophic Bacteria from Distinctive Kinetic and Regulatory Properties of ADP-Glucose Pyrophosphorylase from Nitrosomonas europaea

Nitrosomonas europaea is a chemolithoautotroph that obtains energy by oxidizing ammonia in the presence of oxygen and fixes CO₂ via the Benson-Calvin cycle. Despite its environmental and evolutionary importance, very little is known about the regulation and metabolism of glycogen, a source of carbon and energy storage. Here, we cloned and heterologously expressed the genes coding for two major putative enzymes of the glycogen synthetic pathway in N. europaea, ADP-glucose pyrophosphorylase and glycogen synthase. In other bacteria, ADP-glucose pyrophosphorylase catalyzes the regulatory step of the synthetic pathway and glycogen synthase elongates the polymer. In starch synthesis in plants, homologous enzymes play similar roles. We purified to homogeneity the recombinant ADP-glucose pyrophosphorylase from N. europaea and characterized its kinetic, regulatory, and oligomeric properties. The enzyme was allosterically activated by pyruvate, oxaloacetate, and phosphoenolpyruvate and inhibited by AMP. It had a broad thermal and pH stability and used different divalent metal ions as cofactors. Depending on the cofactor, the enzyme was able to accept different nucleotides and sugar phosphates as alternative substrates. However, characterization of the recombinant glycogen synthase showed that only ADP-Glc elongates the polysaccharide, indicating that ATP and glucose-1-phosphate are the physiological substrates of the ADP-glucose pyrophosphorylase. The distinctive properties with respect to selectivity for substrates and activators of the ADP-glucose pyrophosphorylase were in good agreement with the metabolic routes operating in N. europaea, indicating an evolutionary adaptation. These unique properties place the enzyme in a category of its own within the family, highlighting the unique regulation in these organisms.     

Pyruvic Oxime Dioxygenase from the Heterotrophic Nitrifier Alcaligenes faecalis: Purification, and Molecular and Enzymatic Properties

From the heterotrophic nitrifier Alcaligenes faecalis IFO 13111,an enzyme was purified which oxidized pyruvic oxime to nitrite.The molecular mass of the enzyme was 115 kDa and its moleculewas composed of three molecules of subunits with the same molecularmass of 40 kDa. The enzyme contained 3 atoms of nonheme ironin the molecule and was active when the iron atoms were in aferrous state. The enzyme consumed one mol of O₂ to form onemol each of nitrite and pyruvate from one mol of pyruvic oxime.Therefore, the enzyme was thought to be a pyruvic oxime dioxygenase. (Received June 29, 1998; Accepted October 26, 1998)     

Inhibition of denitrification activity but not of mRNA induction in Paracoccus denitrificans by nitrite at a suboptimal pH

The influence of pH on the denitrification activity of a continuous culture of Paracoccus denitrificans was studied in relation to the presence of nitrite. After a transition from aerobic to anaerobic conditions at the suboptimal pH of 6.8, P. denitrificans was not able to build up a functional denitrification pathway. Nitrite accumulated in the medium as the predominant denitrification product. Although the nitrite reductase gene was induced properly, the enzyme could not be detected at sufficient amounts in the culture. These observations indicate that either translation was somehow inhibited, or once synthesized nitrite reductase was inactivated, possibly by the high concentrations of nitrous acid (HNO₂. Interestingly, when a P. denitrificans culture which was grown to steady-state under anaerobic conditions was then exposed to suboptimal pHs, cells exhibited a reduced overall denitrification activity, but neither nitrite nor any other denitrification intermediate accumulated.     

Maintenance Energy Demand and Starvation Recovery Dynamics of Nitrosomonas europaea and Nitrobacter winogradskyi Cultivated in a Retentostat with Complete Biomass Retention

Nitrosomonas europaea and Nitrobacter winogradskyi (strain “Engel”) were grown in ammonia-limited and nitrite-limited conditions, respectively, in a retentostat with complete biomass retention at 25°C and pH 8. Fitting the retentostat biomass and oxygen consumption data of N. europaea and N. winogradskyi to the linear equation for substrate utilization resulted in up to eight-times-lower maintenance requirements compared to the maintenance energy demand (m) calculated from chemostat experiments. Independent of the growth rate at different stages of such a retention culture, the maximum specific oxygen consumption rate measured by mass spectrometric analysis of inlet and outlet gas oxygen content always amounted to approximately 45 μmol of O₂ mg⁻¹ of biomass-C · h⁻¹ for both N. europaea and N. winogradskyi. When bacteria were starved for different time periods (up to 3 months), the spontaneous respiratory activity after an ammonia or nitrite pulse decreased with increasing duration of the previous starvation time period, but the observed decrease was many times faster for N. winogradskyi than for N. europaea. Likewise, the velocity of resuscitation decreased with extended time periods of starvation. The increase in oxygen consumption rates during resuscitation referred to the reviving population only, since in parallel no significant increase in the cell concentrations was detectable. N. europaea more readily recovers from starvation than N. winogradskyi, explaining the occasionally observed nitrite accumulation in the environment after ammonia becomes available. From chloramphenicol (100 μg · ml⁻¹) inhibition experiments with N. winogradskyi, it has been concluded that energy-starved cells must have a lower protein turnover rate than nonstarved cells. As pointed out by Stein and Arp (L. Y. Stein and D. J. Arp, Appl. Environ. Microbiol. 64:1514–1521, 1998), nitrifying bacteria in soil have to cope with extremely low nutrient concentrations. Therefore, a chemostat is probably not a suitable tool for studying their physiological properties during a long-lasting nutrient shortage. In comparison with chemostats, retentostats offer a more realistic approach with respect to substrate provision and availability.     

Deciphering the oxygen isotope composition of nitrous oxide produced by nitrification

The ability to use δ¹⁸O values of nitrous oxide (N₂O) to apportion environmental emissions is currently hindered by a poor understanding of the controls on δ¹⁸O–N₂O from nitrification (hydroxylamine oxidation to N₂O and nitrite reduction to N₂O). In this study fertilized agricultural soils and unfertilized temperate forest soils were aerobically incubated with different ¹⁸O/¹⁶O waters, and conceptual and mathematical models were developed to systematically explain the δ¹⁸O–N₂O formed by nitrification. Modeling exercises used a set of defined input parameters to emulate the measured soil δ¹⁸O–N₂O data (Monte Carlo approach). The Monte Carlo simulations implied that abiotic oxygen (O) exchange between nitrite (NO₂^?) and H₂O is important in all soils, but that biological, enzyme‐controlled O‐exchange does not occur during the reduction of NO₂^? to N₂O (nitrifier‐denitrification). Similarly, the results of the model simulations indicated that N₂O consumption is not characteristic of aerobic N₂O formation. The results of this study and a synthesis of the published literature data indicate that δ¹⁸O–N₂O formed in aerobic environments is constrained between +13‰ and +35‰ relative to Vienna Standard Mean Ocean Water (VSMOW). N₂O formed via hydroxylamine oxidation and nitrifier‐denitrification cannot be separated using δ¹⁸O unless ¹⁸O tracers are employed. The natural range of nitrifier δ¹⁸O–N₂O is discussed and explained in terms of our conceptual model, and the major and minor controls that define aerobically produced δ¹⁸O–N₂O are identified. Despite the highly complex nature of δ¹⁸O–N₂O produced by nitrification this δ¹⁸O range is narrow. As a result, in many situations δ¹⁸O values may be used in conjunction with δ¹⁵N–N₂O data to apportion nitrifier‐ and denitrifier‐derived N₂O. However, when biological O‐exchange during denitrification is high and N₂O consumption is low, there may be too much overlap in δ¹⁸O values to distinguish N₂O formed by these pathways.     

Expression of a fully functional cd1 nitrite reductase from Pseudomonas aeruginosa in Pseudomonas stutzeri

Nitrite reductases are redox enzymes catalysing the one electron reduction of nitrite to nitrogen monoxide (NO) within the bacterial denitrification process. We have cloned the gene for cd(1) nitrite reductase (Pa-nirS) from Pseudomonas aeruginosa into the NiRS(-) strain MK202 of Pseudomonas stutzeri and expressed the enzyme under denitrifying conditions. In the MK202 strain, denitrification is abolished by the disruption of the endogenous nitrite reductase gene; thus, cells can be grown only in the presence of oxygen. After complementation with Pa-nirS gene, cells supplemented with nitrate can be grown in the absence of oxygen. The presence of nitrite reductase was proven in vivo by the demonstration of NO production, showing that the enzyme was expressed in the active form, containing both heme c and d(1). A purification procedure for the recombinant PaNir has been developed, based on the P. aeruginosa purification protocol; spectroscopic analysis of the purified protein fully confirms the presence of the d(1) heme cofactor. Moreover, the functional characterisation of the recombinant NiR has been carried out by monitoring the production of NO by the purified NiR enzyme in the presence of nitrite by an NO electrode. The full recovery of the denitrification properties in the P. stutzeri MK202 strain by genetic complementation with Pa-NiR underlines the high homology between enzymes of nitrogen oxianion respiration. Our work provides an expression system for cd(1) nitrite reductase and its site-directed mutants in a non-pathogenic strain and is a starting point for the in vivo study of recombinant enzyme variants.