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

• Published in: Environmental microbiology Vol. 23; no. 10; pp. 6194 - 6209
• Main Authors: Cabrera, Juan J., Jiménez‐Leiva, Andrea, Tomás‐Gallardo, Laura, Parejo, Sergio, Casado, Sara, Torres, María J., Bedmar, Eulogio J., Delgado, María J., Mesa, Socorro
• Format: Journal Article
• Language: English
• Published: Hoboken, USA John Wiley & Sons, Inc 01.10.2021; Wiley Subscription Services, Inc
• Subjects: Bradyrhizobium diazoefficiens; Deoxyribonucleic acid; DNA; Genes; Mutation; Nitric oxide; Nitric-oxide reductase; Nitrogen; Nucleotides; Promoters; Proteins; Reductases; Specificity; Stability; Symbionts; Symbiosis; Transcription; Transcription activation
• ISSN: 1462-2912; 1462-2920
• DOI: 10.1111/1462-2920.15661

Summary 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.