In vivo genomic footprinting analysis reveals that the complex Bradyrhizobium japonicum fixRnifA promoter region is differently occupied by two distinct RNA polymerase holoenzymes

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Abstract
The Bradyrhizobium japonicum fixRnifA operon is transcribed from two promoters: fixR p1, a −24/−12 promoter recognized by the σ 54 -holoenzyme form of the RNA polymerase, and fixR p2, a −35/−10 promoter that is transcribed by a second, unidentified, form of RNA polymerase holoenzyme. The fixR p1 promoter is autoregulated during microaerobiosis by NifA, whereas fixR p2 is also activated, but by a different regulatory protein. The main transcription start sites for these promoters are just two nucleotides apart, such that the conserved −12 and −10 regions of fixR p1 and fixR p2, respectively, must overlap each other, whereas the −24 and −35 regions lie one DNA helical turn apart. Using in vivo genomic dimethyl sulfate and KMnO 4 footprinting, we showed that the promoter region is differentially protected, depending upon which holoenzyme is bound. Mutagenesis analyses indicated that positions from −12 to −14 are critical for the activity of both promoters, whereas mutations at −10 and −11 affected mainly fixR p2 expression. When the sequence of the putative −35 region of fixR p2 was modified to match the putative consensus, expression from this promoter was increased 3-fold and the reactivity toward KMnO 4 , but not the transcriptional start site, moved two nucleotides further upstream, indicating that the altered promoter forms a different open complex. Additionally, we detected NifA-dependent methylation protection of two atypical NifA binding sites and protection of guanine −75. The latter residue is located in a region critical for fixR p2 promoter activation. The results present direct physical evidence of the complexity of the organization, regulation, and function of the fixRnifA promoter region.