Activation of theKlebsiella pneumoniae nifUpromoter: identification of multiple and overlapping upstream NifA binding sites

Abstract

The Klebsiella pneumoniae nifU promoter is positively controlled by the NifA protein and requires a form of RNA polymerase holoenzyme containing the rpoN encoded sigma factor, σ⁵⁴ Occupancy of the K. pneumoniae nifU promoter by NifA was examined using in vivo dimethyl sulphate footprinting. Three binding sites for NifA (Upstream Activator Sequences, UASs 1,2 and 3) located at - 125, - 116 and - 72 were identified which conform to the UAS consensus sequence TGTN₁₀-ACA. An additional NifA binding site was identified at position - 90. The UASs located at - 125 (UAS1) and - 116 (UAS2) overlap and do not appear to bind NifA as independent sites. They may represent a NifA binding site interacting with two NifA dlmers. UAS3 is located at - 72, and abuts a binding site for integration host factor (IHF) and is not normally highty occupied by NifA. In the absence of IHF UAS3 showed increased occupancy by NifA. Mutational and footprinting analysis of the three UASs indicates (1) IHF and NifA can compete for binding and that this competition influences the level of expression from the nifU promoter (2) that UAS2 is a principle sequence of the UAS 1,2 region required for acttivation and (3) that none of the NifA binding sites interacts with NifA independently. In vivo KMnO₄, footprinting demonstrated that NifA catalyses open complex formation at the nifU promoter. IHF was required for maximal expression from the nifU and nifH promoters in Escherichia coli, and for the establishment of a Nif⁺ phenotype in E. coli from the nif piasmid pRD1.