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Functional and physiological evidence for a Rhesus-type ammonia transporter in Nitrosomonas europaea

Kerstin Weidinger, Benjamin Neuhäuser, Stefan Gilch, Uwe Ludewig, Ortwin Meyer, Ingo Schmidt
DOI: http://dx.doi.org/10.1111/j.1574-6968.2007.00805.x 260-267 First published online: 1 August 2007


Ammonium transporters form a conserved family of transport proteins and are widely distributed among all domains of life. The genome of Nitrosomonas europaea codes for a single gene (rh1) that belongs to the family of the AMT/Rh ammonium transporters. For the first time, this study provides functional and physiological evidence for a Rhesus-type ammonia transporter in bacteria (N. europaea). The methylammonium (MA) transport activity of N. europaea correlated with the Rh1 expression. The Km value for the MA uptake of N. europaea was 1.8±0.2 mM (pH 7.25), and the uptake was competitively inhibited by ammonium [Ki(NH4+) 0.3±0.1 mM at pH 7.25]. The MA uptake rate was pH dependent, indicating that the uncharged form of MA is transported by Rh1. An effect of the glutamine synthetase on the MA uptake was not observed. When expressed in Saccharomyces cerevisiae, the function of Rh1 from N. europaea as an ammonia/MA transporter was confirmed. The results suggest that Rh1 equilibrates the uncharged substrate species. A low pH value in the periplasmic space during ammonia oxidation seems to be responsible for the ammonium accumulation functioning as an acid NH4+ trap.

  • Nitrosomonas europaea
  • ammonium transport
  • Rh-type transporter
  • Rh1
  • methylammonium
  • nitrogen metabolism


Ammonia-oxidizing bacteria are a versatile group of microorganisms found in many ecosystems. Under oxic or anoxic conditions, Nitrosomonas europaea is able to gain energy via ammonia oxidation with oxygen or nitrogen dioxide (Dua et al., 1979; Anderson & Hooper, 1983; Hyman & Wood, 1985; Schmidt & Bock, 1997). As an alternative, ammonia oxidizers grow under anoxic conditions via denitrification. Pyruvate, acetate, or hydrogen are used as substrates (Bock et al., 1995; Schmidt & Jetten, 2004; Schmidt et al., 2004b). Denitrifying cells are not able to use ammonium as their energy source, but use ammonium as a nitrogen source (Bock et al., 1995).

The current assumption that free ammonia (NH3) rather than ammonium (NH4+) is the substrate for the ammonia oxidation in Nitrosomonas is based on the observation that the Km value of the AMO is pH dependent when determined for the substrate ammonium, but the effect of the pH value virtually disappears when ammonia is assumed to be the substrate (Suzuki et al., 1974; Schmidt & Bock, 1998). Throughout this paper, the term ‘ammonium’ is used for the sum of NH3 and NH4+, and ‘ammonia’ is used for NH3. It was suggested that NH3 is transported via passive diffusion through the cell membrane (Kleiner, 1981). On the other hand, the occurrence of highly active ammonia oxidizers at low pH values (Tarre & Green, 2004) and the high ammonium uptake rates of N. europaea (Schmidt et al., 2004a) suggest that the ammonium transport might be facilitated by a transport protein.

AmtB-type ammonium transporters are widely spread in archaea, fungi, plants, and invertebrate animals and are the usual transporters in eubacteria (Huang & Peng, 2005). Rh-type transporters belong to the same superfamily, but are extremely rare in bacteria (Chain et al., 2003; Huang & Peng, 2005). In contrast to AmtB-type proteins with eleven transmembrane helices, Rh proteins have been predicted to possess 12 transmembrane domains (Winkler, 2006). In the structure of AmtB and Rh-type proteins, a pore is formed lined by hydrophobic side chains that is supposed to channel ammonium. Trp148 and Ser219, which are important for ammonium binding in AmtB proteins, are replaced by aliphatic residues in Rh proteins. As a consequence, Rh-type transporters show saturation with apparent Km values in the millimolar range (RhAG: Km=1.6 mM). An effective inhibition of the MA uptake by ammonium at millimolar concentrations was observed (Marini et al., 2000; Ludewig, 2004). The type of physiological substrate (NH3, NH4+, or CO2) of Rh-type transporters is still under debate, but recent data suggest that net transport is as a channel-like NH3 transport or NH4+/H+ exchange (Marini et al., 2000; Westhoff et al., 2002; Ludewig, 2004; Mak et al., 2006; Mayer et al., 2006). Rh-type transporters seem to support a bi-directional ammonium transport and equilibrate ammonia across the membrane (Marini et al., 2000). A gene coding for GlnK, a PII signal transduction protein that regulates the activity of ammonium transporters in many bacteria in response to the nitrogen status, does not exist in N. europaea (Arcondeguy et al., 2001; Chain et al., 2003; Huang & Peng, 2005).

This study aimed at investigating the ammonium uptake of N. europaea, with special focus on the involvement of the rhesus-type Rh1 protein. The biochemical characterization of the ammonium transport gave evidence for ammonia being the transported substrate and for a bi-directional ammonia transport mechanism of Rh1.

Materials and methods

Organisms and growth conditions

Nitrosomonas europaea was grown in 30 L laboratory-scale reactors operated as Chemostat (Braun, Melsungen, Germany) with 20 L mineral medium (Schmidt & Bock, 1997). To produce ‘nitrifying N. europaea cells’, the reactor was aerated with 0.2–8 L air min−1. The temperature was maintained at 28°C and the dissolved oxygen at 5±0.2 mg L−1. The pH value was maintained at 7.4 [20% (w/v) Na2CO3]. Ammonium, nitrite, and nitrate concentrations were documented. The dilution rate of the Chemostat varied between 0.002 (start-up) and about 0.1 h−1 (biomass production). The ammonium concentration was adjusted at 1±0.5 mM, and the cell number was 2 × 108±3 × 107 cells mL−1.

‘Denitrifying N. europaea cells’ were grown in the same reactor system under anoxic conditions. Medium and gas atmosphere were composed as described earlier (Bock et al., 1995; Schmidt et al., 2004b), but the medium (denitrification medium) contained 5 mM sodium nitrite, 5 mM sodium pyruvate, and 0.25 mM ammonium chloride. The redox potential of the medium was adjusted between −300 and −200 mV using sodium sulfide (Na2S). The preparation of cell-free extracts of N. europaea and the determination of the ammonia oxidation activity in these have been described before (Schmidt & Bock, 1998). The ammonium transporter-deficient strain of Saccharomyces cerevisiae (31019b; ΔΔΔmep1;2;3) and the corresponding wild-type strain (23344c) (Marini et al., 1997) were grown in a YNB medium (pH 5.5) according to Mayer (2006). Nitrosomonas europaea Rh1 (gi_30248465) as well as AtAMT1;1 were subcloned into pDR199 and were expressed in a wild type and in an ammonium transporter-deficient strain of S. cerevisiae. The expression and functionality of the protein were documented as described previously (Mayer et al., 2006).

[14C]MA transport assay

Nitrosomonas europaea cells (Chemostat) were washed twice in mineral medium without ammonium (pH value as required for the following experiment). The cell number was adjusted at about 5 × 109 cell mL−1. The bacteria suspensions (28°C) were stirred (800 r.p.m.), and the experiments were started with the addition of methylammonium (MA) or ammonium. [14C]MA (Biotrend, Köln, Germany; 55 mCi mmol−1) was used as a radiotracer. The assays were designed as described previously by Thomas (2000) and Javelle (2005), and were performed as ‘unwashed assays’ (Javelle et al., 2005). As a control experiment, a [14C]MA-containing assay buffer was passed over a filter. Then, the radioactivity of the filter (polycarbonate, pore size 0.2 µm) was determined. The unspecific binding of [14C]MA to inactivated (heat treated) N. europaea cells was also evaluated. Radioactivity was measured by liquid scintillation counting (BETAmatic BASIC counter, Kontron Analytical, Münchenstein, Switzerland).

Production of starved and acetylene-inhibited N. europaea cells

To inactivate the AMO completely and irreversibly, acetylene was added (Hyman & Wood, 1985) to the cell suspension for about 10 min. Then, the cells were washed twice with 5 mM Hepes buffer (pH 7.25). Before the cells were used for MA uptake assays, they were prepared as follows: ‘Fresh cells’, no treatment; ‘acetylene cells’, cells were inhibited with acetylene; ‘starved cells’, cells were stored for 5 h in an ammonium-free 5 mM Hepes buffer (pH 7.25); and ‘starved acetylene cells’, cells were inhibited with acetylene and then stored for 5 h in an ammonium-free 5 mM Hepes buffer (pH 7.25). As a control, starvation of the cells was extended to 10 or 20 h. An effect on the MA uptake rates was not detectable.

Proportion between oxidized and assimilated ammonium

Mineral medium (400 mL) with 10 mM ammonium chloride was inoculated with N. europaea (107 cells mL−1). Cultures were grown at 28°C on a rotary shaker (200 r.p.m.). Twice a day, samples were taken to determine the ammonium, nitrite, and protein concentrations as well as the dry weight. Based on the data, the amount and proportion of the oxidized and assimilated ammonium were calculated.

Transcription analysis of rh1

For mRNA analysis, the total RNA was isolated from N. europaea applying the acid phenol method described by Völker (1994). After transferring the RNA onto a nylon membrane (slot-blotting, 10 µg slot−1) and hybridization with digoxigenin-labeled probes (immunochemoluminescence, CDP-Star, Roche, Mannheim), the membrane was exposed to Hyperfilm ECL (GE Healthcare Life Science, England) for 20 min. The signals on the autoradiographs were scanned, densitometrically analyzed, and the background signals and signals for unspecific binding of digoxigenin-labeled probes were subtracted. The expression of the 16S rRNA gene was documented as a reference, and the values for rh1 were normalized to corresponding values of the 16S rRNA gene.

Analytical procedures

Ammonium was measured according to Schmidt & Bock (1997) (ortho-phtaldialdehyde reagent), and nitrite according to van de Graaf (1996) (N-naphtylethylendiamine-dihydrochloride reagent). Protein was determined according to Bradford (1976). The oxygen concentration in the medium was measured with a Clark-type oxygen electrode. The intracellular concentration of ATP was determined according to Strehler & Trotter (1952) (bioluminescense assays) and the NADH concentration according to Slater & Sawyer (1962) (alcohol dehydrogenase/phenanzine methosulfate assay). To measure the dry weight of N. europaea, a 20 mL cell suspension was filtered (Nucleopore, pore size 0.2 µm), dried for 48 h at 90°C, and the weight difference of the filter was measured. The pH value in the periplasmic space was evaluated with the pH-sensitive dye LysoSensor™ Green DND-189 (Molekular Probes Europe BV, Leiden, The Netherlands; fluorescent at pH values below 6).


In the first experiments, N. europaea cells were produced with either down-regulated (denitrifying cells) or unmodified (nitrifying cells) rh1 expression. Both cell types were applied in the following experiments investigating the ammonium uptake.

Transcription of rh1

The genome of N. europaea encodes a single ammonium transporter-like gene (rh1). The expression of this gene was about 95% down-regulated in denitrifying cells compared with nitrifying cells (Fig. 1). Variations of the ammonium concentration during aerobic nitrification showed no significant effect on the rh1 expression. At 0.5, 10, and 50 mM NH4+ (pH 7.4), the relative transcription level of rh1 was at 103±16.8%, 102±8.2%, and 92±14.2%, respectively. CO2 concentrations in the aeration gas between 0.1% and 5% did not significantly affect the relative transcription level (96±4.7%).

Figure 1

Relative transcription levels of rh1 during denitrification and nitrification. The highest expression signal of rh1 (0 h) was set to 100. The intensity of the other hybridization signals was calculated in relation to the signal at 0 h. The experiment was inoculated with aerobically precultured Nitrosomonas europaea cells and was started under anaerobic growth conditions (0–336 h, black bars). Then, the cell suspension was transferred to aerobic growth conditions (338–528 h, white bars). Results are mean values±SD (n=4).

[14C]MA transport in N. europaea

The MA transport activities of nitrifying and denitrifying cells (harvested after 528 and 336 h; Fig. 1) were examined to verify whether the different expression of rh1 resulted in different MA uptake characteristics. The MA uptake rate of the denitrifying cells [23.3±2.2 nmol (mg protein)−1 min−1] was 10 times lower than that of the nitrifying cells [252.7±50.9 nmol (mg protein)−1 min−1] (Fig. 2). The addition of 20 µM of the glutamine synthetase (GS) inhibitor l-methionine sulfone (MSF), which had been shown to lead to inhibition of the GS and consequently of the MA uptake by Escherichia coli (Javelle et al., 2005), did not affect the MA uptake of N. europaea (Fig. 2).

Figure 2

MA uptake of Nitrosomonas europaea. The MA concentration in the uptake assay was adjusted to 10 mM, and the cell number was 5 × 109 cells mL−1 [0.8 mg (protein mL−1)]. (•) Nitrosomonas europaea cells that were precultured under oxic conditions (nitrifying cells), (Δ) as before, but the uptake assay was supplemented with 20 µM l-methionine sulfone (MSF). Also, MSF concentrations of 10, 50, and 100 µM did not affect the MA uptake rate (not shown), (□) cells that were pre-cultured under anoxic conditions (denitrifying cells). Results are mean values±SD (n=5).

Effect of ammonium and MA concentrations on the [14C]MA uptake

These experiments aimed at measuring the Km and Ki(NH4+) value for the MA uptake of N. europaea. The MA concentration giving a half-maximum transport rate at pH 7.25 was 1.8±0.2 mM (Fig. 3a). The Km value for the uncharged MA was calculated and was almost constant at about 1 µM independent of the pH value (pH 6.75–8.25). These results suggest that the uncharged substrate is recognized by the transport system of N. europaea. A Km value for denitrifying cells could not be determined, because the MA uptake was dominated by passive diffusion of MA into the cells (Fig. 3a). The pH dependency of the MA uptake is shown in Fig. 3b. The uptake rate changed by a factor of about 3 per 0.5 pH units (about factor 10 per pH unit), except for the step from pH 7.75 to 8.25 (only factor 1.5). The uptake rate of the denitrifying cells was always about 90% lower compared with the nitrifying cells. This strongly favors the suggestion that the uncharged substrate is transported. The diffusion of ammonia, which has a significantly impact on the MA uptake of denitrifying cells, consequently did not significant contribute to the MA uptake of nitrifying N. europaea cells. In further experiments, a competition between the transport of ammonium and MA was detectable (Fig. 3c). At the Ki value, the NH3 concentration was about three times lower than the uncharged MA concentration. Hence, the affinity of the transport system for NH3 was about three times higher than that for the uncharged MA.

Figure 3

[14C]MA uptake kinetics of Nitrosomonas europaea. (a) Correlation of MA uptake rate and MA concentration at pH 7.25. (▪) Nitrifying N. europaea cells. The Km value for MA was 1.8±0.2 mM. The Km value was pH dependent: Km (pH 6.75)=5.2±0.9 mM, Km (pH 7.75)=0.61±0.3 mM, and Km (pH 8.25)=0.19±0.08 mM, but pH independent at about 1 µM when calculated for uncharged MA concentrations. (□) Denitrifying cells. (b) pH dependence of the MA uptake rate (MA concentration 0.5 mM). Black bars: nitrifying N. europaea cells, white bars: denitrifying N. europaea cells. (c) Inhibition of the MA uptake by ammonium at pH 7.25 (Dixon plot). The assays were performed at MA concentrations of: (▪) 10 mM, (Δ) 5 mM, (•) 1 mM. Results are mean values±SD (n=5).

In addition, to the competition of ammonium and MA uptake, an inhibitory effect of MA on the ammonia oxidation activity was observed. At 2 mM NH4+ and 20 mM MA, the ammonia oxidation was completely inhibited after 142±3 min [cell number 5 × 109 cells mL−1 (0.8 mg (protein mL−1) at pH 7.25; n=3]. At an increased MA concentration of 250 mM, complete inhibition already occurred after 22±2 min. The internal [14C]MA concentrations were calculated on the basis of intracellular radioactivity. The ammonia oxidation activity was always completely inhibited at an internal MA concentration of 11.4±3.7 µmol MA (mg protein)−1. To verify these data, the inhibitory effect of MA was examined in crude cell-free extracts of N. europaea (0.8 mg protein mL−1 at pH 7.25; n=5). Applying 12 µmol MA (mg protein)−1, complete inhibition was observed within 10 min. Higher MA concentrations resulted in a faster inactivation of the ammonia oxidation activity. At 8 µmol MA (mg protein)−1, the inhibition remained incomplete at about 90% after 30 min. MA was not oxidized by the AMO, because the inactivation of the ammonia oxidation correlated with a loss of the oxygen consumption.

Ammonia transport function of N. europaea Rh1 in S. cerevisiae

The N. europaea Rh1 protein was expressed in a triple mep-deficient mutant of S. cerevisiae (31019b; ΔΔΔmep1;2;3). For comparison and as a control, the function of the electrogenic ammonium transporter AtAMT1;1 from the plant Arabidopsis thaliana was assayed in parallel (Mayer & Ludewig, 2006). Rh1 of N. europaea was able to transport ammonium in S. cerevisiae (Fig. 4). The expression of Rh1 improved the growth of the ammonium transporter-deficient S. cerevisiae strain at 5 mM ammonium (pH value 5.5), although the growth was slower compared with the AtAMT1;1-transformed control (Fig. 4b). In further experiments, a pH-dependent growth improvement by Rh1 in S. cerevisiae strain ΔΔΔmep1;2;3 was observed. At pH values of 6.25 and 7.25, but not at 5.25, the growth of S. cerevisiae transformed with Rh1 was significantly increased (not shown).

Figure 4

Effect of the expression of Rh1 from Nitrosomonas europaea and AtAMT1;1 from Arabidopsis thaliana on the growth of Saccharomyces cerevisiae. (a, b) Growth of the ammonium transporter-deficient S. cerevisiae (31019b;ΔΔΔmep1;2;3) on serial 10-fold dilutions (1–1 : 10 000) of saturated cultures transformed with AtAMT1;1 (pDR-AtAMT1;1), empty pDR199 plasmid (pDR), or Rh1 (pDR-Rh1). The YNB media (pH value 5.5) were supplemented with 1 mM ammonium (a) or 5 mM ammonium (b). (c) Growth of S. cerevisiae wild-type strain on serial 10-fold dilutions (1–1 : 10 000) of saturated cultures transformed with AtAMT1;1 (pDR-AtAMT1;1), empty pDR199 plasmid (pDR), or Rh1 (pDR-Rh1). The YNB media (pH value 5.5) were supplemented with 125 mM MA.

The expression of Rh1 in wild-type S. cerevisiae still having its endogenous ammonium transporter mediated resistance against the toxicity of MA (Fig. 4c). Saccharomyces cerevisiae wild type with N. europaea Rh1 grew significantly faster than the wild type without Rh1 or the wild type expressing AtAMT1;1. This protecting effect may reflect the MA efflux by Rh1 (bi-directional transport characteristics of Rh-type transporter) resulting in a lower internal MA concentration.

Influence of AMO inhibition on the MA uptake of N. europaea

During ammonia oxidation, N. europaea maintains a membrane potential of about −140 mV (Kumar & Nicholas, 1983), and a pH value between 4.5 and 6 was observed in the periplasmic space (Schmidt et al., 2004a). In inactive N. europaea cells (without ammonia oxidation activity), the pH value of the periplasmic space equals the pH value of the surrounding medium. The following experiments were designed to examine whether ammonia oxidation and MA uptake are coupled. At first, the energy status of ‘fresh’ and ‘starved cells’, was determined. The ATP and NADH concentration in ‘fresh cells’ was 6.9±0.5 µmol ATP (g protein)−1 and 8.8±1.3 µmol NADH (g protein)−1. In ‘starved cells’, the ATP and NADH concentrations were as low as 0.1 µmol ATP (g protein)−1 and 0.3 µmol NADH (g protein)−1, respectively.

The [14C]MA uptake rate of the ‘acetylene cells’ (AMO inhibited) was not significantly different from ‘fresh cells’ (Fig. 5). In ‘acetylene cells’ treated with MSF (AMO and GS inhibited), the MA uptake was not reduced (data not shown) and equaled the uptake activity of ‘fresh cells’ (Fig. 5). The ‘starved cells’ of N. europaea showed a low MA uptake within the first minute, and at the same time the pH value in the periplasmic space declined below pH 6, followed by an increased MA uptake (2–4 min). In contrast, in ‘starved acetylene cells’, the pH value in the periplasmic space did not decrease, and the [14C]MA uptake rate remained low throughout the whole experiment (Fig. 5).

Figure 5

[14C]MA uptake by Nitrosomonas europaea cells with either active or acetylene inhibited AMO. The MA concentration during the assay was adjusted to 10 mM and the cell number was 5 × 109 cells mL−1 [0.8 mg (protein mL−1)]. The pH value in the assay buffer was 7.25. (▪) MA uptake of ‘fresh cells’, (Δ) MA uptake of ‘acetylene cells’, (•) MA uptake of ‘starved cells’, (□) MA uptake of ‘starved acetylene cells’. Results are mean values±SD (n=5).

Ammonia oxidation and N-assimilation by N. europaea

To evaluate the importance of the ammonium transport for energy conservation and assimilation in N. europaea, the maximum ammonia oxidation activity and the ratio of oxidized to assimilated ammonium was determined. At 5 mM ammonium, the maximum ammonia oxidation activity of 82.3±5.6 nmol (mg protein)−1 min−1 was detectable. It remained unchanged when the ammonium concentration was further increased up to 50 mM. During the lag and log phase on average 99.3±2.5% of the consumed ammonium were oxidized and about 1.2±0.9% were converted into biomass (20 replicated experiments). In the stationary phase, hardly any cell growth or N-assimilation was detectable, although the ammonia oxidation activity remained at about 48.7±12.3 nmol (mg protein)−1 min−1.


Ammonium transport in the nitrifying bacterium N. europaea was supposed to occur via diffusion over the cytoplasmic membrane (Kleiner, 1981). This suggestion came into conflict with the observation that N. europaea accumulates ammonium during ammonia oxidation (Schmidt et al., 2004a). In natural environments, N. europaea competes with many bacteria/organisms possessing highly efficient ammonium transport proteins (Marini et al., 2000; Huang & Peng, 2005). AMT ammonium transporters are responsible for the membrane potential-dependent acquisition and accumulation of ammonium (Ludewig et al., 2002, 2003; Mayer et al., 2006). In contrast, the Rh-type transporters equilibrate ammonia across membranes independent of the membrane potential (Westhoff et al., 2002; Ludewig, 2004; Mak et al., 2006; Mayer et al., 2006).

The current study provides evidence that the Rh1 protein of N. europaea is a functional Rhesus-type ammonia transporter. First, in transport assays, the MA uptake rate of nitrifying N. europaea cells (high transcription level of rh1) was about 10 times higher than in denitrifying cells (low transcription level of rh1) (Fig. 2). In denitrifying N. europaea cells, ammonium is only required for assimilation. As a consequence, the ammonium demand is about 99% lower than in nitrifying cells, and matching with this finding, the transcription rate of the rh1 gene was about 95% reduced (Fig. 1). Second, the MA uptake kinetics (Fig. 3a) of nitrifying cells suggested a transporter mediated MA uptake, while the MA uptake of denitrifying cells is obviously dominated by passive diffusion. The MA uptake capacity of Rh1 seems to be saturated at about 20 mM MA (Fig. 3a). The further increase of the MA uptake rate (50 mM MA) is most likely mediated by passive diffusion, because a similar increase was observed for denitrifying cells (Fig. 3a). Third, a competition in the ammonium and MA uptake was detectable (Fig. 3c), also indicating that both substances were taken up via a transport protein. A transport via passive diffusion would not lead to an interaction of the ammonium and the MA uptake. The Km values calculated for uncharged MA as a substrate were constant independent of the pH value of the medium, pointing to NH3 as the transported compound. Fourth, all properties of Rh1 in yeast (Fig. 4) are similar to other Rh-type proteins, which have been shown to transport ammonia (MA) in an electroneutral manner (Marini et al., 2000; Mayer et al., 2006). The expression of the rh1 gene in a triple mep-deficient mutant of S. cerevisiae resulted in an increased growth of the mutant. Consistent with this result, S. cerevisiae growth was enhanced by Rh1 at higher pH values, hinting at NH3 as being the transported compound (or NH4+/H+ exchange). In ammonia-oxidizing cells of N. europaea (pH value of the medium 6.8–8.0), the intracellular pH varies between pH 7 and 8 (Kumar & Nicholas, 1983) and the pH value of the periplasmic space is below 6.0 (this study). The pH optimum for the ammonia oxidation of the AMO is above pH 7 (7.0–7.8) (Suzuki et al., 1974; Schmidt & Bock, 1998), but the hydroxylamine oxidoreductase (HAO) is still active at pH values as low as 5.0 (Frijlink et al., 1992). The HAO is a periplasmatic enzyme adapted to the low pH values in the periplasmic space. The high pH optimum of the ammonia oxidation by the AMO provides strong evidence that the ammonia oxidation is located in the cytoplasm (cytoplasmic side of the cytoplasmic membrane), although a location on the periplasmic side was suggested earlier (Hooper et al., 1997).

Different studies have shown that Rh-type transporters equilibrate ammonia across membranes (Westhoff et al., 2002; Ludewig, 2004; Mak et al., 2006; Mayer et al., 2006). The present results would clearly seem to suggest the uncharged MA/ammonia as the transported substrate in N. europaea, although the bacterium is known to accumulate ammonium (Schmidt et al., 2004a). While the membrane potential does not primarily influence the equilibration of NH3 across a membrane, the pH value in the cell and/or the periplasmic space is crucial to the total MA/ammonium uptake and accumulation. The data of this study show that a low pH value in the periplasmic space is required for a rapid MA (ammonium) uptake (Fig. 5). The inhibitory effect of the MA on the ammonia oxidation obstructs the acidification of the periplasmic space and consequently impedes the accumulation of MA. Data from earlier studies showed that the protonophor carbonyl cyanide m-chlorophenylhydrazone (CCCP), which destroys the pH gradient over the cytoplasmic membrane, inhibits the ammonium uptake of N. europaea (Kumar & Nicholas, 1983; Schmidt et al., 2004a). However, the energy status of the cell (NADH, ATP concentration) did not directly correlate with the MA uptake. This study suggests that the pH difference between medium and periplasmic space (the pH value of the periplasmic space was two to three pH units below the pH value of the medium) allows an accumulation of ammonium by an acid NH4+ trap. Such an acid-trap mechanism would allow an accumulation of ammonium by a factor of 102–103 (factor of 10 per pH unit), but is obviously not sufficient to explain the accumulation by a factor of about 104 reported earlier (Schmidt et al., 2004a). According to the current data, an explanation for this discrepancy cannot be given. Subsequent studies should aim at investigating additional mechanisms that would make possible a further accumulation of ammonium. The increased ammonium concentration in the periplasmic space might initiate or support the Rh1-dependent ammonia uptake of N. europaea.

The ammonia uptake capacity seems to be regulated by the cells according to their requirements to generate energy, but not according to the requirements of nitrogen assimilation. First, this is supported by the observation that the ammonium requirement of denitrifying cells is low and the rh1 gene is only expressed at a low rate (Fig. 1). Second, the inhibition of the glutamine synthetase, a key element controlling the ammonium uptake of E. coli (Javelle et al., 2005), has no effect on the MA/ammonium uptake of N. europaea. Third, a PII-like protein, regulating the ammonium uptake in other organisms in relation to the requirements for assimilation (Hsieh et al., 1998; Arcondeguy et al., 2001), is not encoded in the genome of N. europaea (Chain et al., 2003). Interestingly, the maximum MA/ammonium uptake rate of N. europaea was about five times higher than the highest ammonia oxidation activity measured. In natural ecosystems, N. europaea has to compete with other (heterotrophic) bacteria for the resource ammonium. The ammonium demand of heterotrophic bacteria for assimilation calculated per cell and time interval is about the same as the ammonium demand of an N. europaea cell for assimilation plus energy conservation. Taking into account that many heterotrophic bacteria (for example strains of Escherichia, Corynebacterium, Bacillus) have shorter generation times and efficient ammonium uptake systems (Siewe et al., 1996; Javelle et al., 2005), one should assume that N. europaea loses the competition for ammonium — but as is well known, it does not. It might be speculated that the ability to rapidly take up ammonium and accumulate it already during the lag phase is part of the strategy of N. europaea to compete successfully for the resource ammonium. The overcapacity of the ammonium/MA uptake might explain the high uptake capacity of N. europaea compared with other bacteria (Javelle 2005). A rhesus-type protein is also coded in the genome of Nitrosospira multiformis, but is obviously missing in the genome of Nitrosococcus oceani and Nitrosomonas eutropha. Nitrosomonas eutropha was often found in ammonium-rich environments (Koops et al., 1991), where an ammonium transporter might not be required. Whether the existence of rh/amtB in the genome of ammonia oxidizers reflects the concentration of ammonium in the preferred habitat is at the moment speculative. More genomes of ammonia oxidizers have to be checked for the occurrence of these genes to evaluate the importance of ammonium transporters for different ammonia oxidizers.


  • Editor: Sergio Casella


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