Two polytopic membrane proteins, NarK and NarU, are involved in nitrate and nitrite uptake and nitrite extrusion by Escherichia coli. A third polytopic membrane protein, NirC, functions only in nitrite transport. During exponential growth, the quantity of NarU in membrane fractions was <0.01% of the quantity of NarK. During the stationary phase of growth, the ratio of NarU to NarK increased to 0.1%. However, in the exponential phase of growth, the strain expressing only NarK transports and reduces nitrate and nitrite at a rate only slightly higher than that of the strain expressing only NarU, indicating that, in a NarK+ strain, the rate of nitrate reduction is not limited by the rate of nitrate transport. By measuring nitrate and nitrite transport abilities of strains expressing only narK or expressing both narK and nirC, we hypothesized that NarK might function as a primary nitrate–nitrite antiporter. After nitrate is imported by NarK and reduced to nitrite, some nitrite is expelled from the cell and then reimported for reduction to ammonia. Two highly conserved positively charged residues, Arg-87 and Arg-303 of NarU, were shown by site-directed mutagenesis to play a key role in anion transport. This result indicates that NarU might form a single channel for nitrate and nitrite transport.
- Arg-87 in NarU
- Arg-303 in NarU
- nitrate transport
- nitrite transport
- polytopic transporters
Under different growth conditions, Escherichia coli expresses three different nitrate reductases, nitrate reductases A and Z with active sites located in the cytoplasm, and a periplasmic nitrate reductase Nap. There are also two Nir (nitrite reductases), the NADH-dependent Nir located in the cytoplasm and the periplasmic Nrf (for nitrite reduction by formate). Nitrate transport across the cytoplasmic membrane is essential for nitrate-dependent anaerobic growth of strains that express only the cytoplasmic nitrate reductase. Clegg et al.  showed that at least three polytopic membrane proteins, NarK, NarU and NirC, are involved in nitrate and nitrite transport. NarK functions in nitrate uptake, nitrite extrusion and nitrite uptake [1–3]. NarU is capable of importing nitrate and expelling nitrite [1,4], but it was not clear whether NarU is involved in nitrite uptake. The first aim of this work was to establish a complete profile of the transport activities for each of the three proteins. The second aim was to reveal the relative quantities of NarK and NarU expressed under different conditions. Primary roles of NarK and NirC in nitrate and nitrite transport and the transport mechanism of NarU have also been studied.
Materials and methods
Assays to estimate the rate of nitrate or nitrite uptake and reduction
Bacteria were grown anaerobically in 1 litre of minimal salts medium  supplemented with Luria broth, glucose and nitrate. They were harvested in the middle of the exponential phase of growth and rates of nitrate reduction were measured using the nitrate electrode with formate as the electron donor. To measure the rates of nitrite reduction, samples were taken at intervals from the reaction mixture composed of Mops buffer (pH 8.5), cells, glucose and nitrite. The samples were then mixed with sulphanilamide and N-1-naphthylethylenediamine dihydrochloride. The rate of nitrite reduction or accumulation was determined by the change in A540.
To determine the quantity of NarU, a chromosomal narU gene with its own promoter region was fused with a C-terminal myc tag, and inserted into a pGEM-TEasy vector. This plasmid was designated JCB901. Plasmid JCB902 encodes NarK with a myc tag. The plasmids were transformed into a triple mutant strain, narKnarUnirC (JCB4520 ), and were grown anaerobically in the 1 litre medium stated above. Bacteria were harvested at either exponential phase or stationary phase. Cells were broken in the French pressure cell and fractionated by centrifugation. Membrane proteins were separated by SDS/PAGE and probed by a commercial anti-myc antibody.
Measurement of the effects of a mutation on NarU
On the basis of pSJC901, a plasmid encoding narU gene, the codon for residue Arg-87 or Arg-303 was mutated randomly by site-directed mutagenesis (Stratagene). These plasmids were transformed into the narKnarUnirC triple mutant strain JCB4520 . The transformants were grown in 100 ml of minimal salts medium supplemented with 10% (v/v) Luria broth, glycerol, nitrate, thiamine and ampicillin. Samples were taken at intervals to measure the density of bacteria and the concentrations of extracellular nitrite.
Rates of nitrate and nitrite transport and reduction by NarK, NarU or NirC
To investigate the transport activity of each protein, strains that express only a cytoplasmic nitrate reductase and an Nir were constructed. In this genetic background, strains expressing only NarK, only NarU or only NirC were established. The strains were grown anaerobically in 1 litre medium and harvested during exponential growth. The rate of nitrate uptake and reduction by the strain expressing only NarK [80 nmol·min−1·(mg of dry bacteria)−1] was higher than that by the strain expressing only NarU [49 nmol·min−1·(mg of dry bacteria)−1]. The same trend was also observed in nitrite uptake and reduction. The NirC+ strain is unable to transport nitrate; however, the activity of nitrite uptake and reduction by this strain was 5-fold higher than that of the NarK+ strain.
The relative quantity of NarK and NarU
Western-blot analysis was used to detect both NarK and NarU and to provide an indication of the relative quantities of the two proteins expressed under different conditions. Plasmids pJCB901 and pJCB902 fully restored nitrate-dependent anaerobic growth to the narKnarUnirC triple mutant strain JCB4520. Membrane proteins from strains expressing either NarK-myc or NarU-myc were then probed with the anti-myc antibody. During exponential growth, the quantity of NarU in membrane fractions was <0.01% of the quantity of NarK. During the stationary phase of growth, the concentration of NarU increased considerably, whereas the concentration of NarK decreased. However, the ratio of NarK to NarU in stationary phase bacteria was still more than 1000:1.
The primary roles of NarK and NirC
Glucose is a source of both formate and NADH. Formate is an electron donor for the cytoplasmic nitrate reductase A, and NADH is an electron donor for Nir. When the bacteria expressing only NarK were incubated with nitrate and formate, as expected, extracellular nitrite accumulated rapidly. When the same culture was incubated with nitrate and glucose, less extracellular nitrite was accumulated and the rate of accumulation was slower, suggesting that some of the nitrite produced from nitrate had been reduced further to ammonia (Figure 1A). In contrast, when the bacteria expressing both NarK and NirC were incubated with nitrate and glucose, no nitrite accumulation was observed, which suggests that nitrite extruded by NarK had been reimported through NirC and then reduced by Nir (Figure 1B).
The essential roles of two arginine residues in NarU
Moir and Wood  proposed that NarK and NarU are composed of 12 transmembrane residues. Two positively charged residues, Arg-87 in helix 2 and Arg-303 in helix 8, are highly conserved in NarK and NarU homologues across the different genera . The conservation of charged residues within membrane-spanning helices made them obvious candidates for functional significance and hence targets for mutagenesis studies. Site-directed mutagenesis of the Arg-87 codon of narU to a leucine codon abolished nitrate-dependent anaerobic growth, nitrate reduction by nitrate reductase A and nitrite reduction by Nir (Figure 2). Similarly, substitution of NarU Arg-303 by leucine resulted in total loss of both nitrate and nitrite transport and reduction, indicating that both of these conserved arginine residues are essential for both nitrate and nitrite transport. It was confirmed by Western analysis that the substituted proteins were accumulated at normal levels in the cytoplasmic membrane.
Transcription of narZ is RpoS-dependent [6,7]. Therefore the significant increase in NarU expression from exponential phase to stationary phase is compatible with previous studies and suggests that narU might be the first gene of a five-gene operon narUZWYV .
Rates of nitrite accumulation by the NarK+ strain and the NarK+NirC+ strain suggest that the primary role of NarK is to import nitrate and export nitrite, and that NirC is most active in nitrite import. The result also indicates that some of the nitrate-N is expelled as nitrite by NarK before it can be reimported for reduction to ammonia. Therefore NarK is primarily a nitrate–nitrite antiporter. As the homologue of NarK, NarU might also function as a nitrate–nitrite antiporter.
Although the first and second six helices of NarU have significant sequence similarity, we have shown by site-directed mutagenesis that residues Arg-87 and Arg-303 are both essential for nitrate and nitrite transport (Figure 2 and unpublished work). This indicates that NarU might form a single channel for nitrate and nitrite transport. Similar to the glycerol 3-phosphate/Pi antiporter (GlpT) , NarU might function as a nitrate–nitrite antiporter using an alternating-access mechanism, with the two highly conserved residues forming a ‘rocker-switch’.
The 10th Nitrogen Cycle Meeting 2004: Focused Meeting held at the University of East Anglia, Norwich, U.K., 2–4 September 2004. Edited by C.S. Butler (Newcastle upon Tyne, U.K.) and D.J. Richardson (Norwich, U.K.). Sponsored by the COST (European Cooperation in the field of Scientific and Technical Research) Office and the ESF (European Science Foundation).
Abbreviations: Nir, nitrite reductase
- © 2005 The Biochemical Society