1 Department of Microbiology, University of Bayreuth, Universitaetsstrasse 30, 95447 Bayreuth, Germany
2 Department of Microbiology, University of Hamburg, Ohnhorststrasse 18, 22609 Hamburg, Germany
3 Department of Microbiology, University of Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands
Correspondence
Ingo Schmidt
ingo.schmidt1{at}uni-bayreuth.de
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ABSTRACT |
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INTRODUCTION |
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The kinetic coefficient for ammonia oxidation was derived from experiments with nitrifying activated sludge, pure cultures, and cell-free extracts of ammonia oxidizers. The KS value (half saturation constant) for ammonia of ammonia oxidizers like Nsm. europaea was found to be fairly constant at about 20 µM between pH 6·5 and 8·5 (Suzuki et al., 1974; Wood, 1986
). The current assumption that free ammonia (NH3) rather than ammonium (
) is the substrate for ammonia oxidation in Nitrosomonas is based on numerous publications (Suzuki et al., 1974
; Drozd, 1976
; Hooper & Dispirito, 1985
; Painter, 1988
; Wiesmann, 1994
). Cell membranes are highly permeable for ammonia, in contrast to ammonium, and passive ammonia diffusion over cell membranes to enter the cytoplasm has been discussed as a possible ammonia uptake mechanism (Kleiner, 1981
, 1985
). Other micro-organisms, like Corynebacterium glutamicum, use a membrane-potential-driven transport system for active ammonium uptake (Krämer & Lambert, 1990
; Meier-Wagner et al., 2001
). Since the ratio between ammonia and ammonium concentration only depends on the pH value, an increase in ammonia in the cytoplasm results in a concomitant increase in ammonium. Therefore, ammonia oxidizers have to tackle several difficulties. The pKa value (negative logarithm of the dissociation constant for acids) of ammonia is 9·25. As a consequence, at pH values between 7·0 and 8·0 (optimal for most ammonia oxidizers), even at high ammonium concentrations, the ammonia concentration is low. The pH value of the cytoplasm in active Nsm. europaea cells is about 6·87·2 (Hollocher et al., 1982
; Kumar & Nicholas, 1983
; C. Look, unpublished data). To establish an ammonia concentration that only meets the KS value for ammonia oxidation (20 µM, Nsm. europaea), the cytoplasmic ammonium concentration should range between 2000 and 5000 µM. If the ammonia uptake over the membrane is limited by diffusion or passive transport, an even higher environmental ammonium concentration would be necessary to establish an internal ammonia concentration of 20 µM. However, the actual ammonium concentration in ecosystems is usually very low (100 µM would result in a maximal cytoplasmic ammonia concentration of 1 µM). It is difficult to envision how ammonia oxidizers can establish a high ammonia oxidation activity that necessitates internal ammonia concentrations in the range of the KS value (20 µM, Nsm. europaea). The ammonium/ammonia concentration was shown to be an important factor for the niche differentiation of ammonia oxidizers (Bollmann et al., 2002
), and ammonia oxidizers in niches with low ammonia concentrations will depend on an efficient ammonium/ammonia transport system. Though the genome of Nsm. europaea encodes many transporters for inorganic ions, only one ammonium transporter is present, supplementing the passive uptake of ammonium at low pH values (Chain et al., 2003
); an active transporter has not yet been described.
The present study shows that starving Nitrosomonas cells rapidly take up and accumulate ammonium/ammonia or hydroxylamine without simultaneous nitrite formation. In short-term kinetic analyses, the uptake capacity of ammonium and hydroxylamine of cells and the accumulation capacity of cell-free extracts were determined. 15N-labelling experiments were performed to document that ammonium and hydroxylamine were accumulated, but not immediately oxidized to nitrite. The internal ammonium concentrations of both active and starving cells were determined to provide evidence for an active ammonia/ammonium accumulation.
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METHODS |
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Medium and growth conditions.
The mineral medium consisted of the following components (l1 distilled water): NH4Cl (535 mg), NaCl (585 mg), CaCl2.2H2O (147 mg), KCl (74 mg), KH2PO4 (54 mg), MgSO4.7H2O (49 mg), HEPES (12 g) and 1 ml trace element solution containing 0·02 M HCl (1 l), FeSO4.7H2O (973 mg), H3BO3 (49 mg), ZnSO4.7H2O (43 mg), (NH4)6Mo7O24.4H2O (37 mg), MnSO4.H2O (34 mg), CuSO4 (16 mg). The medium was adjusted to pH 7·2.
Experimental design.
All experiments were carried out in Clark-type Oxygen Electrode Units (volume 10 ml) flushed with synthetic air. The oxygen concentration was kept constant at 3 mg l1. Cells of Nitrosomonas and Nitrosolobus were harvested from the precultures by centrifugation. The experiments were performed with starved cells' or active cells. Before the ammonium/ammonia uptake experiments were started, starved cells' were incubated between 20 min and 48 h in the absence of ammonium and hydroxylamine. In contrast, active cells' were directly transferred into Clark-type Oxygen Electrode Units to start the experiments (these cells were never incubated without ammonium or hydroxylamine). Experiments were performed at 28 °C with suspensions of 1x1061x109 cells ml1. Medium and biomass samples were transferred into an Eppendorf tube and immediately frozen in liquid nitrogen.
Analytical procedures.
Measurements were carried out according to Schmidt & Bock (1997) (ammonium), according to van de Graaf et al. (1996)
(nitrite, nitrate) and by a modified method according to Verstraete & Alexander (1972)
(NH2OH). The protein concentration was determined according to Bradford (1976)
. The intracellular pool of ATP was determined by a method according to Strehler & Trotter (1952)
. Cell numbers were determined by light microscopy with the aid of a Helber chamber. Cell dimensions were evaluated by microscopy (multi-dimensional imaging; Universal Imaging Corporation). Cells were fixed in glutaraldehyde and the cell dimensions were determined with the aid of a distance marker placed on the video screen. Starved and active cells had on average the same volume of 0·2385 fl (0·93x0·53x0·53 µm).
To measure the ammonium, hydroxylamine and nitrite concentration in the medium, cells were sedimented (5 min at 8000 g) and the supernatant was used for the analytical procedures. To determine the internal (cytoplasmic plus periplasmic) ammonium concentration, the cells were washed (three times) and concentrated in a 0·9 % NaCl solution before homogenization by passing the cell suspension (1x1010 cells ml1) through a French pressure cell at 140 MPa. Samples were taken directly to determine the concentration of free ammonium and hydroxylamine in the cells. The bound ammonium in the cell-free extracts was solubilized by adding an HCl stock solution to a final concentration of 0·1 M, whereas hydroxylamine was solublized by adding detergents (SDS) to a concentration of 10 %. The cell-free extracts were centrifuged (5 min at 8000 g) and the ammonium or hydroxylamine concentrations in the supernatant were measured. The measured concentrations and the cell volume were used to calculate the internal ammonium and hydroxylamine concentrations. The 15N-analysis was performed by isotope ratio MS (Robo-Prep-G and Tracer Mass; Europa Scientific). The 15N-labelled ammonium was analysed after conversion to N2 with hypobromite (Risgaard-Petersen et al., 1995). The detection limit for all 15N-compounds was 10 nM.
Enzyme purification.
Preparation of cell-free extracts and protein purification were performed at 4 °C. Nitrosomonas cells were washed three times with 10 mM Tris/HCl (pH 7·5), homogenized by passing the cell suspension (1x1010 cells ml1) through a French pressure cell at 140 MPa, and the crude extract was supplemented with DNase I (Sigma) and centrifuged for 30 min at 48 000 g. The supernatant was loaded onto the HiPrep 16/10 DEAE column (Amersham Pharmacia) and a linear gradient of 01 M NaCl was applied at 1 ml min1. Twenty-five fractions were sampled and their ammonium and hydroxylamine binding capacities were determined. The three fractions with the highest binding capacity were pooled and further purified by anion-exchange chromatography on a UNO Q6 column (Bio-Rad) and eluted by a 01 M NaCl gradient. The hydroxylamine-binding fractions were transferred onto an SDS-PAGE gel (Mini-Protean II Cell vertical gel electrophoresis chamber; Bio-Rad) (Schmidt et al., 2001c) and an enzyme assay for hydroxylamine oxidoredutase (HAO) was performed according to Schalk et al. (2000)
.
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RESULTS |
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For a first evaluation, crude cell-free extracts of Nsm. europaea were separated into extracellular polymer substances (EPS), soluble protein and a membrane fraction. The fractions were supplemented with ammonium or hydroxylamine and the binding capacities were determined (Table 3). The ammonium-binding capacity of all fractions was extremely low. Only in the crude cell-free extracts and in the membrane fraction were small amounts of ammonium bound. The hydroxylamine-binding capacity was high in both the crude cell-free extract and in the soluble protein fraction. To elucidate which proteins were responsible for hydroxylamine binding, the soluble protein fraction was further separated. After each separation step the fractions with the highest hydroxylamine-binding capacity were pooled. After purification, the hydroxylamine-binding protein was identified as HAO by SDS-PAGE and an enzyme assay. On SDS gels, proteins of 125 and 65 kDa were detectable, which resemble the masses of the HAO (125140 kDa) and its subunits (63 kDa) (Masson et al., 1990
; Nejidat et al., 1997
; Arciero & Hooper, 1993
). In the enzyme assay, a hydroxylamine oxidation activity of 58 µmol (mg protein)1 min1 was measured. The binding of hydroxylamine to HAO was a reversible process. Following addition of SDS (10 %), the bound hydroxylamine was released from the protein and was detectable in the medium. Hence, hydroxylamine was not covalently bound to HAO. Heat-treated HAO (3 min at 90 °C) was unable to bind hydroxylamine, indicating that a native protein is necessary for the binding process.
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DISCUSSION |
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The major results of this study are as follows. First, all ammonia oxidizers tested (Nsm. europaea, Nsm. eutropha, Nsl. multiformis) accumulated ammonium to a final internal concentration of about 1 M (about 6x108 molecules per cell). Second, this internal ammonium concentration remained constant during ammonia oxidation, but decreased rapidly when the external ammonium was consumed. Third, starved cells accumulated 1 M ammonium before the first catabolic products (nitrite) were detected, and the specific ammonium uptake rate was about 40 times higher than the highest specific ammonia oxidation activity observed. Fourth, inactive cells and cell components were unable to bind significant amounts of ammonium/ammonia. Fifth, the results provide evidence for a membrane-potential-driven ammonium uptake mechanism.
According to the results of 15N-labelling studies the processes of ammonium uptake/accumulation and catabolic ammonia oxidation forming nitrite have to be distinguished. Starved cells accumulate 15N-labelled ammonium and increase their internal ammonium concentration to about 1 M. Throughout this time period ammonia is not oxidized and 15N-labelled nitrite is not formed. Cells might need several minutes to recover their ammonia oxidation activity (Hooper, 1969b) or they have to establish a sufficient internal ammonia concentration before oxidation starts. In contrast, in experiments with active cells (internal ammonium concentration already 1 M) ammonia was oxidized immediately and stochiometric amounts of nitrite were produced.
Ammonia oxidizers have to tackle substrate limitations in many environments. At an ammonium concentration of 100 µM, the ammonia concentration is about 0·8 µM at pH 7. Passive transport (diffusion) over the membrane would be a slow process and even an internal ammonia concentration of about 0·8 µM would be significantly below the KS value of ammonia monooxygenase (20 µM, Nsm. europaea), resulting in a low specific ammonia oxidation activity. This ammonia limitation can be compensated by internal ammonium accumulation. At an internal ammonium concentration of 1 M, as measured in this study, an ammonia concentration of about 80 mM would be available, which is 500 times higher than the KS value of the ammonia-oxidizing system. Ammonium accumulation might be a strategy adopted by ammonia oxidizers in environmental niches with low ammonium concentrations to maintain high ammonia oxidation activities. This accumulation strategy would also protect the ammonia-oxidizing enzyme system from an evolutionary pressure to increase its affinity for ammonia, but instead this pressure is placed on the effectiveness of the ammonium/ammonia uptake (transport) system, since ammonia oxidizers have to compete with other organisms (heterotrophic micro-organisms, fungi, algae, plants) for ammonium (Laanbroek & Gerards, 1993; Bodelier & Frenzel, 1999
). Although ammonia oxidizers are able to establish high internal ammonium concentrations, this pool has obviously no function as an ammonium (substrate) stock. When the ammonium in the medium was depleted, the internal ammonium concentration decreased rapidly. Possibly, the internal ammonium pool is used to prepare the ammonia oxidizers for starvation, for example by synthesizing compatible solutes (Prosser, 1989
).
The results presented indicate that ammonium uptake (transport) is a membrane-potential-driven mechanism [proton motive force (pmf)-dependent]. First, the instant uptake inhibition after addition of CCCP or DNP favours pmf-driven transport (Krämer & Lambert, 1990; Meier-Wagner et al., 2001
). Second, the ATP pool of the cells and subsequently primary ATP-driven transport systems remained unaffected for several minutes. ATP would have been accessible if necessary for ammonium transport. Third, supplementation of ATP synthase inhibitors had no effect on the uptake rates for several minutes. Also, hydroxylamine uptake is most likely to be based on an active transport mechanism. At 40 mM the internal free hydroxylamine concentration was at least 40 times higher than the external hydroxylamine concentration in the medium. Together with the high uptake rate, these results provide evidence for active hydroxylamine transport.
Experiments investigating the binding capacity of ammonium/ammonia (Nsm. europaea) showed only low binding capacity in the membrane fraction and the crude cell-free extract. It seems possible that the binding of ammonium is dependent on an energized membrane and that the positively charged ammonium molecules are electrostatically bound to the negatively charged side of cytoplasmic membrane. Hydroxylamine was shown to be bound to HAO (Table 3) and the addition of SDS displaced it. The process of hydroxylamine binding was dependent on a native protein. A model of the surface charge of native HAO shows extensive negatively or positively charged areas (Igarashi et al., 1997
). This allows an electrostatic interaction between the enzyme and the (partially) charged hydroxylamine molecule, leading to its immobilization.
The results presented above strongly indicate an active ammonium transport system in ammonia oxidizers. Analysis of the Nsm. europaea genome provides evidence for an ammonium transporter that might support ammonium uptake at low pH values (Chain et al., 2003). Database analysis of the protein (ORF2457) revealed a high level of homology to a family of active ammonium transporters in, for example, E. coli, B. subtilis and Saccharomyces cerevisiae, containing domains for an Na+/H+ antiporter and cation transport. Furthermore, the genome encodes 13 putative active transporters (ABC), including one nitrate transport system (Chain et al., 2003
). It can be speculated that a modified ABC-type transporter might be responsible for the active ammonium uptake by ammonia oxidizers.
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Received 18 August 2003;
revised 5 November 2003;
accepted 2 February 2004.
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