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.

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