Two domains of a dual-function NarK protein are required for nitrate uptake, the first step of denitrification in Paracoccus pantotrophus

Uptake of nitrate into the cytoplasm is the first but least well understood step of denitrification; no gene has previously been identified to be necessary for this process. Upstream from the structural genes of the membrane-bound nitrate reductase (narGHJI) in Paracoccus pantotrophus there is a fusion of two genes, each homologous to members of the narK family. The single open reading frame is predicted to encode 24 transmembrane helices, comprising two domains, NarK1 and NarK2. Analysis of both the accumulation of intracellular nitrite and electron transport through the nitrate reductase enzyme in narK mutants reveals that NarK1 and NarK2 are both involved in nitrate uptake. Maximal rate of nitrate transport via NarK2 was dependent upon nitrite, indicating that NarK2 encodes a nitrate/nitrite antiporter. The uncouplers S13 and dinitrophenol showed that NarK2 was not dependent on the proton motive force for maximal nitrate transport activity. Nitrate transport via NarK1 was dependent on proton motive force, indicating that it is likely to be a nitrate/proton symporter. Low expression of membrane-bound nitrate reductase in narK mutants was counteracted by azide, which induced nitrate reductase expression only if the transcriptional activator NarR was present.