Denitrification and ammonia oxidation by Nitrosomonas europaea wild-type, and NirK- and NorB-deficient mutants

Ingo Schmidt1, Rob J. M. van Spanning2 and Mike S. M. Jetten3

1 Department of Microbiology, University of Bayreuth, Universitaetsstrasse 30, 95447 Bayreuth, Germany
2 Department of Molecular Cell Physiology, Free University, 1081 Amsterdam, The Netherlands
3 Department of Microbiology, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands

Correspondence
Ingo Schmidt
ingo.schmidt1{at}uni-bayreuth.de


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The phenotypes of three different Nitrosomonas europaea strains – wild-type, nitrite reductase (NirK)-deficient and nitric oxide reductase (NorB)-deficient strains – were characterized in chemostat cell cultures, and the effect of nitric oxide (NO) on metabolic activities was evaluated. All strains revealed similar aerobic ammonia oxidation activities, but the growth rates and yields of the knock-out mutants were significantly reduced. Dinitrogen (N2) was the main gaseous product of the wild-type, produced via its denitrification activity. The mutants were unable to reduce nitrite to N2, but excreted more hydroxylamine leading to the formation of almost equal amounts of NO, nitrous oxide (N2O) and N2 by chemical auto-oxidation and chemodenitrification of hydroxylamine. Under anoxic conditions Nsm. europaea wild-type gains energy for growth via nitrogen dioxide (NO2)-dependent ammonia oxidation or hydrogen-dependent denitrification using nitrite as electron acceptor. The mutant strains were restricted to NO and/or N2O as electron acceptor and consequently their growth rates and yields were much lower compared with the wild-type. When cells were transferred from anoxic (denitrification) to oxic conditions, the wild-type strain endogenously produced NO and recovered ammonia oxidation within 8 h. In contrast, the mutant strains remained inactive. For recovery of ammonia oxidation activity the NO concentration had to be adjusted to about 10 p.p.m. in the aeration gas.


Abbreviations: DMPS, 2,3-dimercapto-1-propane-sulfonic acid; DO, dissolved oxygen


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Nitrosomonas europaea obtains its energy for growth from aerobic and anaerobic ammonia oxidation or hydrogen-dependent denitrification (Rees & Nason, 1966; Abeliovich & Vonshak, 1992; Bock et al., 1995; Schmidt & Bock, 1997). The anaerobic ammonia oxidation is an NO2-dependent process coupled to cell growth (Schmidt & Bock, 1997). Nitrogen oxides (NO2 and NO) were also shown to be involved in the aerobic ammonia oxidation (NOx cycle, Schmidt et al., 2001a–c). Under anoxic conditions in the presence of hydrogen, Nitrosomonas species are able to grow via denitrification (Bock et al., 1995). Nitrite, but not nitrate, is used as the terminal electron acceptor. Nitric oxide, nitrous oxide and dinitrogen are well-documented gaseous products of the metabolic activity of Nitrosomonas (Hooper, 1968; Poth & Focht, 1985; Poth, 1986; Abeliovich & Vonshak, 1992; Bock et al., 1995; Bodelier et al., 1996; Kester et al., 1997; Beaumont et al., 2002). Under anoxic conditions nitrite is the only electron acceptor available, and energy generation depends on the denitrification activity. Under oxic conditions, the importance of denitrification is less obvious. Denitrification activity is induced by lowering the oxygen partial pressure (Miller & Nicholas, 1985). High denitrification rates at high oxygen concentrations (more than 4 mg l–1) have been observed in the presence of NO (more than 20 p.p.m. in the aeration gas), indicating that NO has a regulatory effect on the denitrification activity of ammonia oxidizers (Goreau et al., 1980; Miller & Nicholas, 1985; Zart & Bock, 1998; Zart et al., 2000; Schmidt et al., 2004). Genes encoding the denitrification enzymes nitrite reductase (Nir) and nitric oxide reductase (Nor) are present in the genome of Nsm. europaea (Chain et al., 2003). The mechanism of N2 formation observed in ammonia oxidizers has yet to be elucidated. Homologous genes encoding a nitrous oxide reductase (nosRZDFYL) are not present in Nsm. europaea genome (Chain et al., 2003).

The nirK gene cluster in the genome of Nsm. europaea contains three further ORFs. These genes code for a soluble blue copper oxidase and two periplasmic c-haem-containing polypeptides (Whittaker et al., 2000; Beaumont et al., 2002). The nor genes norC, norB, norQ and norD are encoded in one operon. The putative nirK gene (Beaumont et al., 2002) and the norB gene (Beaumont et al., 2004) of Nsm. europaea were disrupted, and the phenotype of the NirK-deficient strain was characterized in aerobic batch cultures. This mutant had a lower tolerance against nitrite than the wild-type cells. The denitrification activity of ammonia oxidizers has already been discussed to serve as a protection mechanism against negative effects of high nitrite concentrations (Poth & Focht, 1985; Stein & Arp, 1998). Surprisingly, the NirK-deficient strain produced more N2O than the wild-type strain. Since the denitrification pathway was inactivated (NirK deficiency), it was speculated that the hydroxylamine oxidoreductase (HAO) might be responsible for the emission of nitrogen oxides (Beaumont et al., 2002).

The present study aimed to investigate and characterize the phenotype of the Nsm. europaea wild-type, the NirK-deficient and the NorB-deficient strain with regard to their combined aerobic and anaerobic ammonia oxidation and denitrification capabilities. Furthermore, the high N2O and NO production of the NirK- and NorB-deficient strains were examined in detail.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Organism.
Cultures of Nsm. europaea (ATCC 19718), the Nsm. europaea NirK-deficient strain (Beaumont et al., 2002) and the Nsm. europaea NorB-deficient strain (Beaumont et al. 2004) were grown aerobically in 1 l Erlenmeyer flasks containing 400 ml mineral medium (Schmidt & Bock, 1997). The medium for both mutants was supplemented with 20 mg kanamycin l–1. The cultures were grown for 1–2 weeks in the dark at 28 °C without stirring or shaking.

Experimental design (chemostat).
All strains were grown in 5 l laboratory scale reactors with 3·5 l medium. To maintain oxygen concentrations between 0 and 5 mg l–1 the reactor was aerated (0·1–2 l min–1) with variable mixtures of oxygen, carbon dioxide and argon using mass-flow controllers. The NO concentration in the off-gas (outlet) was permanently measured, and the N2O and N2 concentration was measured offline via gas chromatography (GC). Medium level, temperature, dissolved oxygen (DO) and pH value were continuously measured and controlled. The medium contained 20 mM {mic1504107E001} (nitrification) or 1 mM nitrite (denitrification) and the medium for the mutants was supplemented with 20 mg kanamycin l–1. Temperature was maintained at 28 °C. The pH value was kept at 7·4 by means of a 20 % Na2CO3 solution. Samples for offline determination of ammonium ({mic1504107E002}), hydroxylamine (NH2OH), nitrite (NO2), nitrate (NO3) and cell numbers were taken regularly. The reactor was inoculated with 400 ml of a Nsm. europaea cell suspension. The phenotypes of the three Nsm. europaea strains (wild-type, NirK- and NorB-deficient strain) were characterized under three growth conditions: (i) Cells were grown with ammonia as energy source under oxic conditions. (ii) They were grown with ammonia as energy source under anoxic conditions with NO2 (N2O4) as oxidizing agent. Under these conditions, nitrite is used as terminal electron acceptor (Schmidt & Bock, 1997). (iii) Cells were grown under anoxic conditions with hydrogen (gas atmosphere with 80 % H2 and 20 % CO2) as electron donor and nitrite (medium contained 1 mM nitrite), NO (1000 p.p.m. in the gas atmosphere) or N2O (1000 p.p.m. in the gas atmosphere) as terminal electron acceptor. The redox potential was adjusted between –300 and –200 mV by adding sodium sulfide (Na2S) or titanium(III) chloride (TiCl3) (Bock et al., 1995).

Analytical procedures.
Ammonium was measured according to Schmidt & Bock (1998), hydroxylamine according to a modified method by Verstraete & Alexander (1972), and nitrite and nitrate according to van de Graaf et al. (1996). Nitric oxide (NO) and nitrogen dioxide (NO2) concentrations were measured online with an NOx analyser (chemiluminescence) and the N2O and N2 concentration by gas chromatography with a thermal conductivity detector (TCD) using a Poraplot Q and a molecular sieve column (5 Å, 60/80 mesh). Helium served as the carrier gas. The protein concentrations were determined according to Bradford (1976) and the cell numbers by light microscopy using a Helber chamber (SD 5 %). The intracellular pool of ATP was determined by a method according to Strehler & Trotter (1952) and the pool of NADH according to Slater & Sawyer (1962). The 15N analysis was performed by isotope-ratio mass spectrometry. The 15N-labelled ammonium and nitrite were analysed after conversion to N2 with hypobromite or urea, respectively (Risgaard-Petersen et al., 1995).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Characterization of the phenotype of Nsm. europaea wild-type, NirK-deficient and NorB-deficient strain
The experiments were designed to investigate the nitrification/denitrification activities and growth parameters of Nsm. europaea wild-type and the NirK- and NorB-deficient strains under defined growth conditions in a chemostat culture. Before the experiments were started all strains were cultured for 3 weeks under oxic conditions without NOx-supplementation at a DO of 2 mg l–1 (dilution rate 0·1, 20 mM ammonium), and the cell number stabilized at about 5x108 cells ml–1 (preconditioning). After these 3 weeks, the growth parameters were changed according to the needs of the different metabolic activities: First, the strains were examined during aerobic ammonia oxidation without supplementing NO or NO2 (DO, 5 mg l–1); second, during anaerobic NO2-dependent ammonia oxidation (NO2 concentration 150 p.p.m.). The dilution rate of the reactor system was reduced to 0·013 h–1 to compensate for the lower growth rates during anaerobic ammonia oxidation; third, during anaerobic denitrification. The dilution rate of the ammonium-free medium was 0·013 h–1. The cells were allowed to adapt to these new conditions for five volume changes before data acquisition was started.

Aerobic ammonia oxidation
The ammonia oxidation activity and the nitrogen loss were not significantly different in Nsm. europaea wild-type, the NirK- or the NorB-deficient strains (Table 1). However, clear differences were detectable in the amount of hydroxylamine released and the composition of the nitrogen gases. The hydroxylamine concentration in the medium of the two mutant strains was about seven times higher compared with the wild-type (Table 1). Further significant differences between the wild-type and the mutants were detectable by analysing the growth rates and yields. In both cases, the values for the wild-type were higher than those for the mutants (Table 1). These results were reflected by the ATP and NADH contents of the different strains. The mean values for ATP and NADH were higher in Nsm. europaea wild-type cells.


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Table 1. Characterization of the phenotype of Nsm. europaea wild-type, NirK- and NorB-deficient strain during aerobic ammonia oxidation

N-loss: share of ammonia converted via nitrite into nitrogen gases (NO, N2O and N2) in %. Values are means±SD.

 
The main product of the denitrification activity of the wild-type was N2. Only small amounts of NO and N2O were formed. In contrast, both mutants released almost equal amounts of NO, N2O and N2 (Table 1). Since in both mutants the denitrification pathway was interrupted (nitrite reductase or nitric oxide reductase), it can be speculated that the nitrogen gases might have been released by auto-oxidation and chemodenitrification of hydroxylamine (Chalk & Smith, 1983). 15N-labelling experiments were performed to evaluate the production pathway of the nitrogen gases. The experiments were started in the chemostat cultures by adding 15N- instead of 14N-ammonium as substrate. In control experiments the 14N-nitrite concentration was adjusted to 50 mM and this 14N-nitrite pool served as a trap for 15N-nitrite produced during ammonia oxidation. The results are shown in Table 2. The gaseous nitrogen compounds in Nsm. europaea wild-type were almost completely produced via the denitrification pathway. When the 15N-nitrite produced during ammonia oxidation was trapped in an 14N-nitrite pool, hardly any 15N-gases were produced. In contrast, the 14N-nitrite pool did not influence the production of 15N-gases by both mutants. Experiments with 14N-ammonium and a 15N-nitrite pool resulted in an NO, N2O and N2 production by the wild-type, but not by the mutants (Table 2).


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Table 2. 15N-labelling experiments during aerobic ammonia oxidation of Nsm. europaea wild-type, NirK- and NorB-deficient strain

The specific 15N-NO2, -NO, -N2O and -N2 production rates are given in µmol (g protein)–1 h–1. SD, 4 %.

 
Control experiments with heat-inactivated Nsm. europaea biomass in a medium with 80 µM hydroxylamine (concentration of hydroxylamine in experiments with the mutants, Table 1) were performed (data not shown). Here, similar amounts of NO, N2O and N2 were released compared with the growth experiments, with both mutants.

Anaerobic NO2-dependent ammonia oxidation
The anaerobic ammonia oxidation activity was lower than the aerobic activity (Table 3, Schmidt & Bock, 1997).


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Table 3. Characterization of the phenotype of Nsm. europaea wild-type, NirK- and NorB-deficient strains during anaerobic NO2-dependent ammonia oxidation

The experiments were gassed with 150 p.p.m. NO2 in argon. Values are means±SD.

 
During anaerobic ammonia oxidation nitrite was used as terminal electron acceptor by the wild-type (Table 3). About 53 % of the produced nitrite was immediately reduced (indicated by the apparently low nitrite-production rate), leading to an N-loss of 47 %. Interestingly, the NirK-deficient strain was able to oxidize ammonia and to grow under anoxic conditions (Table 3), although this strain is not able to use nitrite as terminal electron acceptor (Tables 1 and 2). The ratio of the ammonia oxidation rate and the nitrite production rate was about 1 : 1 (Table 3). In contrast to the wild-type, significantly less NO was released by the NirK-deficient strain. For further evaluation, the NO-binding compound 2,3-dimercapto-1-propane-sulfonic acid (DMPS) was added. As a consequence, the NO produced during anaerobic ammonia oxidation was immediately removed from the system. Nsm. europaea wild-type was not affected by DMPS, but ammonia oxidation and growth of the NirK-deficient strain were completely inhibited (not shown). In further control experiments with the NirK-deficient strain, 15N-NO was added, and immediately after the ammonia oxidation started, 15N-N2O and 15N-N2 were detectable. The NorB-deficient strain remained inactive under anoxic conditions (Table 3).

Denitrification by Nsm. europaea wild-type and the mutants
Nsm. europaea wild-type cells immediately switched their metabolic activity from aerobic ammonia oxidation to anaerobic denitrification (Table 4) when the redox potential was adjusted between –300 and –200 mV, the gas atmosphere was changed to H2/CO2 (80/20 %) and nitrite (1 mM) was added as electron acceptor. Both mutants remained inactive throughout the 16 days of the experiment (five volume changes) and were washed out. In further experiments, NO or N2O were added as electron acceptor. The wild-type strain was able to denitrify with both electron acceptors, but the activities were reduced and the growth rates and yields were low. Interestingly, the NirK-deficient strain was able to grow with NO or N2O, reaching a growth yield similar to that of the wild-type. The NorB-deficient strain was only able to grow with N2O as electron acceptor (Table 4). The main product of the denitrification activity was always N2.


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Table 4. Characterization of the phenotype of Nsm. europaea wild-type, NirK- and NorB-deficient strains during anaerobic hydrogen-dependent denitrification

The experiments were performed with nitrite, NO or N2O as electron acceptor. Values are means±SD.

 
Recovery of ammonia oxidation activity
Further experiments were performed to evaluate the capability of the three strains to recover ammonia oxidation activity after they were grown via hydrogen-dependent denitrification. Therefore, the strains were grown with nitrite (wild-type), NO (NirK-deficient strain) or N2O (NorB-deficient strain) as electron acceptors for 4 weeks in a chemostat. By adding ammonium (20 mM) and oxygen (1 mg l–1) the recovery of ammonia oxidation was initiated. Nsm. europaea wild-type cells first recovered ammonia oxidation activity within 8 h (Fig. 1), and within 40 to 48 h they reached the maximum activity of 1380 µmol (g protein)–1 h–1. Schmidt et al. (2001c) showed that the recovery of an ammonia oxidation activity depends on the presence of NO or NO2. Here, throughout the recovery process an NO concentration between 5 and 8 p.p.m. was detectable in the off-gas (Fig. 1). When NO was removed by the addition of DMPS, Nsm. europaea wild-type cell did not recover ammonia oxidation activity.



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Fig. 1. Recovery of ammonia oxidation by Nsm. europaea wild-type in continuous culture. Experiments (three replicates) were performed in chemostats. At time 0, the ammonium concentration was adjusted to 20 mM and the oxygen concentration to 1 mg l–1 (not shown). During the experiment fresh medium (ammonium concentration 20 mM) was added continuously (dilution rate 0·07 h–1), and the oxygen concentration was kept constant. The NO concentration was measured in the off-gas. Ammonium ({blacksquare}), nitrite ({blacklozenge}) and NO ({circ}).

 
The NirK- and the NorB-deficient strains were not able to recover any ammonia oxidation activity under oxic conditions within 6 weeks. The NirK-deficient strain was unable to produce NO and remained inactive. In contrast, the NorB-deficient strain released NO leading to an NO concentration of about 210±43 p.p.m. in the headspace within 8 h. After 8–10 h the NO production stopped and the cells became inactive. When the NO concentration was adjusted to a concentration of 10 p.p.m. (NO addition to the NirK-deficient strain, NO removal from the NorB-deficient strain) both strains recovered ammonia oxidation within 10–14 h.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The role of the denitrification activity by ammonia oxidizers has evolved from being recognized as a side aspect of metabolism to a process of high importance for anaerobic growth (Poth & Focht, 1985; Bock et al., 1995; Schmidt et al., 2001a) as well as a pathway supplying the cells with NO (NOx cycle) (Schmidt et al., 2001a, b, 2004). Furthermore, the denitrification activity seems to be important for energy generation under oxic conditions (Table 1). Although the ammonia oxidation activities were similar in the wild-type and the NirK- and NorB-deficient strains, the ATP- and NADH-content, as well as the growth rates and yields of the mutants, were significantly lower. Both mutants released more hydroxylamine, the electron-providing substrate, into the medium (Table 1). The increased loss of hydroxylamine by the mutants caused a loss of electrons and finally energy, leading to reduced growth rates and yields.

Aerobic denitrification might supply the NO necessary for ammonia oxidation (NOx cycle). Evidence that NO activates nitrification was given in this study and by Schmidt et al. (2001c). Denitrifying Nsm. europaea wild-type cells were unable to recover ammonia oxidation if the NO produced was removed from the system. As a key element of the NOx cycle and ammonia oxidation, NO might be necessary to start-up this metabolic function. Whether NO further acts as a transcription factor has yet to be examined. The NirK-deficient strain was unable to denitrify and to produce NO and had to be supplied with a catalytic amount of NO to recover its ammonia oxidation. In contrast, the NorB-deficient strain produced high amounts of NO. The nitrite reductase in this strain might be active, leading to the formation of NO, but the cells were not able to further reduce NO. As a consequence NO accumulated and, most probably, the toxicity of NO caused cell damage (Wink & Mitchell, 1998) and inactivated the recovery process. When the NO concentration was lowered to a non-toxic level of about 10 p.p.m., the NorB-deficient cells recovered an ammonia oxidation activity almost as quickly as the Nsm. europaea wild-type.

Beaumont et al. (2002) characterized the NirK-deficient strain during aerobic ammonia oxidation in batch culture. The high N2O production by this strain, which was three to four times higher than in Nsm. europaea wild-type, was confirmed in this study in chemostat cultures (Table 1). The wild-type mainly produced N2, while the mutants released almost equal amounts of N2, NO and N2O. Experiments with 15N-ammonium and a 14N-nitrite pool (trap for 15N-nitrite produced by 15N-ammonia oxidation) demonstrated that in Nsm. europaea wild-type, ammonia is converted via nitrite, NO and N2O to N2 (Table 2). Here, denitrification is the major source of gaseous N-compounds. In contrast, the two mutants did not produce N-gases via denitrification: First, when 15N-nitrite was added during ammonia oxidation, the cells did not produce 15N-gases (Table 2). Second, a 14N-nitrite pool did not prevent the formation of 15N-gases. These results do not only demonstrate that the mutants lack a denitrification activity, but also show that they release NO and N2O during the oxidation of ammonia to nitrite. Most probably, the auto-oxidation and chemodenitrification of hydroxylamine is responsible for the emission of nitrogen gases. The NorB-deficient strain should be able to produce NO with its nitrite reductase, leading to an increased NO production (Fig. 2), but the results presented in Table 2 did not indicate NO formation from nitrite. Why the nitrite reductase remained inactive in these experiments although other experiments indicate an active nitrite reductase (high NO concentration during the recovery of ammonia oxidation) has yet to be determined.



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Fig. 2. Pathways of NO and N2O production by Nsm. europaea wild-type and the NirK- and NorB-deficient strains during aerobic ammonia oxidation and denitrification. While the wild-type released NO/N2O during denitrification, both mutants released high quantities of hydroxylamine into the medium which subsequently converted into nitrogen gases (auto-oxidation/chemodenitrification). AMO, ammonia monooxygenase; HAO, hydroxylamine oxidoreductase; NIR, nitrite reductase; NOR, nitric oxide reductase; NOS?, not yet identified nitrous oxide reductase.

 
During anaerobic NO2-dependent ammonia oxidation only nitrite is available as the electron acceptor (Schmidt & Bock, 1997). The wild-type strain oxidized ammonia via hydroxylamine to nitrite, and about 47 % of this nitrite was reduced, mainly to N2 (Table 3). Since anaerobic ammonia oxidation depends on nitrite as the electron acceptor, both mutants were supposed to be inactive under anoxic conditions. True to this prediction, the NorB-deficient strain was unable to convert ammonia and grow (Fig. 3), although nitrite might serve as a potential electron acceptor (reduction to NO). In contrast, the NirK-deficient strain was active. The ammonia oxidation activity, growth rate and growth yield were about 25 % lower compared with the wild-type (Table 3). The mutant was unable to reduce nitrite (ammonia consumption : nitrite production, about 1 : 1), but interestingly, the apparent NO production was low (ammonia consumption : NO production, about 1 : 0·8). When 15N-NO was added the NirK-deficient strain converted it into 15N-N2, but in the absence of NO (addition of NO-binding DMPS) the cells remained inactive. These results gave evidence that the NirK-deficient strain used the NO produced during anaerobic ammonia oxidation as a terminal electron acceptor (Fig. 3).



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Fig. 3. Anaerobic NO2-dependent ammonia oxidation of Nsm. europaea wild-type and NirK-deficient strain. The wild-type was using nitrite, and the NirK-deficient strain NO, as terminal electron acceptors. The intermediates are shown in parentheses.

 
The third energy-generating metabolic activity of Nsm. europaea is hydrogen-dependent denitrification (Table 4). It is interesting to note that Nsm. europaea has the capability to utilize hydrogen as an electron donor, as reported for Nitrosomonas eutropha (Bock et al., 1995). A key element to activate this metabolic activity is a low redox potential between –300 and –200 mV. At higher redox potentials a hydrogen-dependent denitrification activity of Nsm. europaea was not observed (Uemoto & Saiki, 2000).

Denitrification of Nsm. europaea wild-type strain was detectable with nitrite, NO or N2O as electron acceptors, although the growth yields and rates differed significantly. The mutant strains were dependent on the addition of electron acceptors that compensate for the lack of nitrite reductase and nitric oxide reductase, respectively. Hence, the NirK-deficient strain could only grow with NO and N2O and the NorB-deficient strain was restricted to N2O as an electron acceptor. The mechanism of N2 formation by ammonia oxidizers has yet to be elucidated since the genes encoding a nitrous oxide reductase are missing in the genome of Nsm. europaea (Chain et al., 2003). It might be speculated whether a novel NOS is active in Nitrosomonas.


   ACKNOWLEDGEMENTS
 
We thank Drs. Hubertus J. E. Beaumont for kindly supplying the NirK- and NorB-deficient strains of Nsm. europaea and for fruitful discussion.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 7 June 2004; revised 28 July 2004; accepted 4 August 2004.



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