RT Journal Article SR Electronic(1) A1 Nilavongse, Arjaree A1 Brondijk, T. Harma C. A1 Overton, Tim W. A1 Richardson, David J. A1 Leach, Emily R. A1 Cole, Jeffrey A.YR 2006 T1 The NapF protein of the Escherichia coli periplasmic nitrate reductase system: demonstration of a cytoplasmic location and interaction with the catalytic subunit, NapA JF Microbiology, VO 152 IS 11 SP 3227 OP 3237 DO https://doi.org/10.1099/mic.0.29157-0 PB Microbiology Society, SN 1465-2080, AB The periplasmic nitrate reductase of Escherichia coli is important during anaerobic growth in low-nitrate environments. The nap operon encoding this nitrate reductase comprises seven genes including a gene, napF, that encodes a putative cytoplasmic iron–sulphur protein of uncertain subcellular location and function. In this study, N-terminal sequence analysis, cell fractionation coupled with immunoblotting and construction of LacZ and PhoA fusion proteins were used together to establish that NapF is located in the E. coli cytoplasm. A bacterial two-hybrid protein–protein interaction system was used to demonstrate that NapF interacted in the cytoplasm with the terminal oxidoreductase NapA, but that it did not self-associate or interact with other electron-transport components of the Nap system, NapC, NapG or NapH, or with another cytoplasmic component, NapD. NapF, purified as a His6-tagged protein, exhibited spectral properties characteristic of an iron–sulphur protein. This protein was able to pull down NapA from soluble extracts of E. coli. A growth-based assay for NapF function in intact cell cultures was developed and applied to assess the effect of mutation of a number of conserved amino acids. It emerged that neither a highly conserved N-terminal double-arginine motif, nor a conserved proline motif, is essential for NapF-dependent growth. The combined data indicate that NapF plays one or more currently unidentified roles in the post-translational modification of NapA prior to the export of folded NapA via the twin-arginine translocation pathway into the periplasm., UL https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.29157-0