The roles of the polytopic membrane proteins NarK, NarU and NirC in Escherichia coli K‐12: two nitrate and three nitrite transporters

Abstract

The roles of the polytopic membrane proteins NarK, NarU and NirC in Escherichia coli K-12: two nitrate and three nitrite transporters bic product of the third gene of the nir operon, which is predicted to be a polytopic membrane protein with six membrane-spanning helices. Deletion of both NarK and NirC decreased nitrite uptake and reduction to a basal rate that was fully restored by a single chromosomal copy of either narK or nirC. A multicopy plasmid encoding NarU complemented a narK mutation for nitrite excretion, but not for nitrite uptake. We conclude that, in contrast to NirC, which transports only nitrite, NarK and NarU provide alternative mechanisms for both nitrate and nitrite transport. However, NarU might selectively promote nitrite excretion, not nitrite uptake.

Role of the Escherichia coli nitrate transport protein, NarU, in survival during severe nutrient starvation and slow growth

“…As the catalytic site of NarG is located on the cytoplasmic side of the membrane, nitrate reduction by this strain and its derivatives is totally dependent on active nitrate transport by NarK or NarU. Strains JCB4014, 4016 and 4018 are derivatives of JCB4011 with deletions in narK, narU, or both narU and narK, respectively (Clegg et al, 2002). Note that the fusaric acid method was used to cure the tetracycline resistance determinant from Tn10 in strain JCB4014, which is therefore a stable narK deletion mutant.…”

“…Note that the fusaric acid method was used to cure the tetracycline resistance determinant from Tn10 in strain JCB4014, which is therefore a stable narK deletion mutant. This strain was checked by PCR for loss of the narK + gene, and by biochemical assays for retention of the adjacent narXL and narG genes required for induction of nitrate reductase A activity during anaerobic growth in the presence of nitrate (see Clegg et al, 2002, for further details). Strain JCB4016 carries a deletion-insertion mutation in narU: it was constructed in strain RK4353 using the method of Datsenko & Wanner (2000), and transferred into strain JCB4011 by bacteriophage P1-mediated transduction.…”

“…1991; Rowe et al, 1994;Clegg et al, 2002). This is consistent with a nitrate : nitrite antiport role for NarK, but does not exclude other transport mechanisms, for example, nitrate : H + symport, or nitrite : H + export.…”

“…This is consistent with a nitrate : nitrite antiport role for NarK, but does not exclude other transport mechanisms, for example, nitrate : H + symport, or nitrite : H + export. Although less nitrite accumulates during anaerobic exponential growth of a narK mutant than the parental strain in the presence of nitrate, NarU expressed from a multicopy plasmid fully complements the complex phenotypes of the narK mutant, including nitrite accumulation during exponential growth (Bonnefoy et al, 1997;Clegg et al, 2002). This suggests that NarU duplicates the functions of NarK, except that it is expressed at a much lower level that limits the rate of nitrate uptake and reduction by a narK mutant.…”

“…There are two nitrate transport proteins in E. coli, NarK and NarU (Clegg et al, 2002). Both NarK and NarU, as well as a third protein, NirC, were all shown to catalyse nitrite transport, but the different physiological roles of these three proteins in nitrite transport were not defined.…”