Dissection of FixK₂ protein-DNA interaction unveils new insights into Bradyrhizobium diazoefficiens lifestyles control

The FixK₂ protein plays a pivotal role in a complex regulatory network, which controls genes for microoxic, denitrifying, and symbiotic nitrogen-fixing lifestyles in Bradyrhizobium diazoefficiens. Among the microoxic-responsive FixK₂ -activated genes are the fixNOQP operon, indispensable for respiration in symbiosis, and the nnrR regulatory gene needed for the nitric-oxide dependent induction of the norCBQD genes encoding the denitrifying nitric oxide reductase. FixK₂ is a CRP/FNR-type transcription factor, which recognizes a 14 bp-palindrome (FixK₂ box) at the regulated promoters through three residues (L195, E196, and R200) within a C-terminal helix-turn-helix motif. Here, we mapped the determinants for discriminatory FixK₂ -mediated regulation. While R200 was essential for DNA binding and activity of FixK₂ , L195 was involved in protein-DNA complex stability. Mutation at positions 1, 3, or 11 in the genuine FixK₂ box at the fixNOQP promoter impaired transcription activation by FixK₂ , which was residual when a second mutation affecting the box palindromy was introduced. The substitution of nucleotide 11 within the NnrR box at the norCBQD promoter allowed FixK₂ -mediated activation in response to microoxia. Thus, position 11 within the FixK₂ /NnrR boxes constitutes a key element that changes FixK₂ targets specificity, and consequently, it might modulate B. diazoefficiens lifestyle as nitrogen fixer or as denitrifier.