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

The FixK 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 FixK-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. FixK is a CRP/FNR-type transcription factor, which recognizes a 14 bp-palindrome (FixK 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 FixK-mediated regulation. While R200 was essential for DNA binding and activity of FixK, L195 was involved in protein–DNA complex stability. Mutation at positions 1, 3, or 11 in the genuine FixK box at the fixNOQP promoter impaired transcription activation by FixK, 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 FixK-mediated activation in response to microoxia. Thus, position 11 within the FixK/NnrR boxes constitutes a key element that changes FixK targets specificity, and consequently, it might modulate B. diazoefficiens lifestyle as nitrogen fixer or as denitrifier.This work was funded by Fondo Europeo de Desarrollo Regional (FEDER)-co-financed grants AGL2011-23383 and AGL2015-63651-P to S.M., and AGL2013-45087-R and AGL2017-85676-R to M.J.D. [Ministerio de Economía y Competitividad, presently Ministerio de Ciencia e Innovación (MICINN), Spain]. Grants P12-AGR-1968 to E.J.B., and P18-RT-1401 to S.M. and M.J.D., and support from the Junta de Andalucía, Spain, to Group BIO-275 are also acknowledged. J.J.C., A.J.-L., and M.J.T. were supported by contracts funded by grants AGL2015-63651-P, AGL2017-85676-R, and AGL2013-45087-R, respectively. J.J.C. also acknowledged to Alfonso Martín Escudero Foundation the support by its abroad postdoctoral grants program. A.J.-L. was also financed by grant P12-AGR-1968 during her PhD thesis' period. S.P. acknowledged the FPU Program (Ministerio de Educación, Cultura y Deporte, presently Ministerio de Universidades, Spain; grant FPU2015/04716) for financial support. Juan Sanjuán (Estación Experimental de Zaidín, CSIC, Granada, Spain) is acknowledged for his critical comments on the manuscript. We are grateful to the financial support of the publication fee by the CSIC Open Access Publication Support Program through its Unit of Scientific Information Resources for Research (URICI). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript