Essential roles for the products of the napABCD genes, but not napFGH, in periplasmic nitrate reduction by Escherichia coli K-12

Abstract

The seven nap genes at minute 47 on the Escherichia coli K-12 chromosome encode a functional nitrate reductase located in the periplasm. The molybdoprotein, NapA, is known to be essential for nitrate reduction. We now demonstrate that the two c-type cytochromes, the periplasmic NapB and the membrane-associated NapC, as well as a fourth polypeptide, NapD, are also essential for nitrate reduction in the periplasm by physiological substrates such as glycerol, formate and glucose. None of the three iron-sulphur proteins, NapF, NapG or NapH, are essential, irrespective of whether the bacteria are grown anaerobically in the presence of nitrate or fumarate as a terminal electron acceptor, or by glucose fermentation. Mutation of napD resulted in the total loss of Methyl Viologen-dependent nitrate reductase activity of the molybdoprotein, NapA, consistent with an earlier suggestion by others that NapD might be required for post-translational modification of NapA.

Structural diversity in twin-arginine signal peptide-binding proteins

“…1 A). In the absence of the napD gene, E. coli lacks all periplasmic nitrate reductase activity (23). Western blot analysis demonstrated the NapA polypeptide was completely destabilized and degraded in a napD Ϫ background (Fig.…”

“…The other proteins encoded by the nap operon have roles either in electron transfer to NapA (20) or in the biosynthesis of NapA (21,22). NapD has been shown to be essential for NapA-dependent periplasmic nitrate reductase activity in E. coli (23). The similar cofactor requirements of NapA and TorA (24) suggest that NapA should also be subject to Tat proofreading during assembly.…”

Purification and Spectropotentiometric Characterization of Escherichia coli NrfB, a Decaheme Homodimer That Transfers Electrons to the Decaheme Periplasmic Nitrite Reductase Complex

“…Purification of Native E. coli NrfB and NrfA-Periplasmic proteins were extracted from the harvested cells as described previously (14). The enzyme was precipitated from the periplasm using 65% saturated ammonium sulfate and resuspended in 50 mM Tris-HCl, pH 7.…”

“…In this paper we present the first purification and spectropotentiometric characterization of NrfB and demonstrate its competence as an electron donor to NrfA, with which it must transiently form a 20-heme [NrfB] 2 [NrfA] 2 electron transfer complex. levels during anaerobic growth in the presence of nitrate (14). Cultures of strain LCB2048 were grown without aeration overnight at 37°C in minimal salts medium (14) supplemented with 0.4% glycerol, 20 mM nitrate, kanamycin (25 g/ml), and spectinomycin (25 g/ml).…”

“…levels during anaerobic growth in the presence of nitrate (14). Cultures of strain LCB2048 were grown without aeration overnight at 37°C in minimal salts medium (14) supplemented with 0.4% glycerol, 20 mM nitrate, kanamycin (25 g/ml), and spectinomycin (25 g/ml). Initial cultures (250 ml) were inoculated with 0.5 ml of an overnight culture grown aerobically on Luria-Bertani broth medium.…”