Abstract
SummaryNitrate and nitrite transport across biological membranes is often facilitated by protein transporters that are members of the major facilitator superfamily. Paracoccus denitrificans contains an unusual arrangement whereby two of these transporters, NarK1 and NarK2, are fused into a single protein, NarK, which delivers nitrate to the respiratory nitrate reductase and transfers the product, nitrite, to the periplasm. Our complementation studies, using a mutant lacking the nitrate/proton symporter NasA from the assimilatory nitrate reductase pathway, support that NarK1 functions as a nitrate/proton symporter while NarK2 is a nitrate/nitrite antiporter. Through the same experimental system, we find that Escherichia coli NarK and NarU can complement deletions in both narK and nasA in P. denitrificans, suggesting that, while these proteins are most likely nitrate/nitrite antiporters, they can also act in the net uptake of nitrate. Finally, we argue that primary sequence analysis and structural modelling do not readily explain why NasA, NarK1 and NarK2, as well as other transporters from this protein family, have such different functions, ranging from net nitrate uptake to nitrate/nitrite exchange.
Highlights
IntroductionThe transport of nitrate into bacterial cells is important for two processes, assimilation (where nitrate is transported to the cytoplasm and is reduced to nitrite and to ammonium) and respiration (where nitrate can act as a terminal electron acceptor)
The transport of nitrate into bacterial cells is important for two processes, assimilation and respiration
In the case of assimilation, there is evidence that in some bacteria nitrate uptake is driven at the expense of ATP hydrolysis by an ABC-type transport protein (Ohashi et al, 2011) while in other organisms, this process involves a member of the major facilitator superfamily (MFS) (Ogawa et al, 1995; Gates et al, 2011)
Summary
The transport of nitrate into bacterial cells is important for two processes, assimilation (where nitrate is transported to the cytoplasm and is reduced to nitrite and to ammonium) and respiration (where nitrate can act as a terminal electron acceptor). The negatively charged nitrate has to enter the cytoplasm against a membrane potential of 180 mV (negative in the cytoplasm) which, if transport was relying on a passive nitrate entry pore, would restrict cytoplasmic nitrate concentrations to a mere 0.001% of the external concentration. Both assimilation and respiration occur at extracellular nitrate concentrations in the low micromolar range (Parsonage et al, 1985).
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