An imperfect inverted repeat is critical for DNA binding of the response regulator RegR of Bradyrhizobium japonicum

RegR is the response regulator of the RegSR two-component regulatory system in Bradyrhizobium japonicum. The only target known so far is the fixR-nifA operon, encoding the redox-responsive transcription factor NifA, which act ivates many genes required for symbiotic nitrogen fixation in soybean nodul es. In previous in vivo studies, we identified a 32 bp upstream activating sequence located around position -68, which is essential for RegR-dependent expression of the fixR-nifA operon. Here, we used an in vitro binding-site selection assay (SELEX) to more precisely define the DNA-binding specifici ty of RegR. The selected sequences comprised an imperfect inverted repeat ( GCGGC-N-5-GTCGC) which is highly similar-to an imperfect inverted repeat in the fixR UAS (GCGAC-N-5-GACGC). In a parallel approach, band-shift experim ents were performed with oligonucleotides comprising defined point or delet ion mutations in the fixR UAS. This led to the identification of 11 critica l nucleotides within a 17 bp minimal RegR binding site centered at position -64 upstream of the fixR-nifA transcription start site. Notably, all 11 cr itical nucleotides were located either within the half sites of the inverte d repeat (four nucleotides in each half site) or in the 5 bp spacer that se parates the half sites (three nucleotides). Based on these results, we defi ned a DNA motif comprising those nucleotides that are critical for RegR bin ding (RegR box; 5'-GNG(G)(A)C(G)(A)TTNNGNCGC-3'). A comparison of the RegR box with functional binding sites of the RegR-like regulator RegA of Rhodob acter capsulatus revealed considerable similarities. Thus, the RegR box may assist in the identification of new RegR target genes not only in B.japoni cum but also in other alpha -proteobacteria possessing RegR-like response r egulators.