Abstract
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 14bp-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.
Highlights
SummaryThe 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
The FixK2 protein is able to interact with its DNA recognition site, the so-called FixK2 box, within a series of amino acids but only three of them, L195, E196, and R200, located in helix F of the HTH motif of the protein make specific contacts with nucleotides of the FixK2 binding site (Bonnet et al, 2013a)
To further study the contribution of these amino acids on FixK2–DNA interaction, we analysed the effects of replacing the FixK2 residues L195, E196, or R200 with alanine
Summary
SummaryThe 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. 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 Biological nitrogen fixation (BNF) and denitrification constitute two processes that play a key role in the balance between biologically accessible and non-reactive nitrogen species within the biogeochemical nitrogen cycle. BNF is the most important process of nitrogen contribution to ecosystems and is mainly carried out by nitrogen-fixing organisms, the diazotrophs. These organisms, through the nitrogenase enzyme encoded by the fix and nif genes, catalyse the reduction of N2 to NH3, whose activity takes place under oxygen-limiting conditions (microoxia) and requires high amounts of energy (Newton, 2007). The anthropogenic use of nitrogenous fertilizers in agriculture triggers an input of nitrogen in excess, which causes an imbalance in the cycle (Galloway, 1998; Canfield et al, 2010; Martínez-Espinosa et al, 2011; Shibata et al, 2015)
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