The powerful greenhouse gas, nitrous oxide (N₂O) has a strong potential to drive climate change. Soils are the major source of N₂O and microbial nitrification and denitrification the main processes involved. The soybean endosymbiont Bradyrhizobium diazoefficiens is considered a model to study rhizobial denitrification, which depends on the napEDABC, nirK, norCBQD, and nosRZDYFLX genes. In this bacterium, the role of the regulatory cascade FixLJ-FixK₂-NnrR in the expression of napEDABC, nirK, and norCBQD genes involved in N₂O synthesis has been previously unraveled. However, much remains to be discovered regarding the regulation of the respiratory N₂O reductase (N₂OR), the key enzyme that mitigates N₂O emissions. In this work, we have demonstrated that nosRZDYFLX genes constitute an operon which is transcribed from a major promoter located upstream of the nosR gene. Low oxygen was shown to be the main inducer of expression of nosRZDYFLX genes and N₂OR activity, FixK₂ being the regulatory protein involved in such control. Further, by using an in vitro transcription assay with purified FixK₂ protein and B. diazoefficiens RNA polymerase we were able to show that the nosRZDYFLX genes are direct targets of FixK₂.

Denitrification in the soybean endosymbiont Bradyrhizobium diazoefficiens is controlled by a complex regulatory network composed of two hierarchical cascades, FixLJ-FixK₂-NnrR and RegSR-NifA. In the former cascade, the CRP/FNR-type transcription factors FixK₂ and NnrR exert disparate control on expression of core denitrifying systems encoded by napEDABC, nirK, norCBQD, and nosRZDFYLX genes in response to microoxia and nitrogen oxides, respectively. To identify additional genes controlled by NnrR and involved in the denitrification process in B. diazoefficiens, we compared the transcriptional profile of an nnrR mutant with that of the wild type, both grown under anoxic denitrifying conditions. This approach revealed more than 170 genes were simultaneously induced in the wild type and under the positive control of NnrR. Among them, we found the cycA gene which codes for the c₅₅₀ soluble cytochrome (CycA), previously identified as an intermediate electron donor between the bc₁ complex and the denitrifying nitrite reductase NirK. Here, we demonstrated that CycA is also required for nitrous oxide reductase activity. However, mutation in cycA neither affected nosZ gene expression nor NosZ protein steady-state levels. Furthermore, cycA, nnrR and its proximal divergently oriented nnrS gene, are direct targets for FixK₂ as determined by in vitro transcription activation assays. The dependence of cycA expression on FixK₂ and NnrR in anoxic denitrifying conditions was validated at transcriptional level, determined by quantitative reverse transcription PCR, and at the level of protein by performing heme c-staining of soluble cytochromes. Thus, this study expands the regulon of NnrR and demonstrates the role of CycA in the activity of the nitrous oxide reductase, the key enzyme for nitrous oxide mitigation.

Nitrous oxide (N₂O) is a powerful greenhouse gas that contributes to climate change. Denitrification is one of the largest sources of N₂O in soils. The soybean endosymbiont Bradyrhizobium diazoefficiens is a model for rhizobial denitrification studies since, in addition to fixing N₂, it has the ability to grow anaerobically under free-living conditions by reducing nitrate from the medium through the complete denitrification pathway. This bacterium contains a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a Cu-dependent nitrous oxide reductase (Nos) encoded by the napEDABC, nirK, norCBQD and nosRZDFYLX genes, respectively. In this work, an integrated study of the role of Cu in B. diazoefficiens denitrification has been performed. A notable reduction in nirK, nor, and nos gene expression observed under Cu limitation was correlated with a significant decrease in NirK, NorC and NosZ protein levels and activities. Meanwhile, nap expression was not affected by Cu, but a remarkable depletion in Nap activity was found, presumably due to an inhibitory effect of nitrite accumulated under Cu-limiting conditions. Interestingly, a post-transcriptional regulation by increasing Nap and NirK activities, as well as NorC and NosZ protein levels, was observed in response to high Cu. Our results demonstrate, for the first time, the role of Cu in transcriptional and post-transcriptional control of B. diazoefficiens denitrification. Thus, this study will contribute by proposing useful strategies for reducing N₂O emissions from agricultural soils.

Summary: The FixK ₂ 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 ₂ ‐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 ₂ is a CRP/FNR‐type transcription factor, which recognizes a 14 bp‐palindrome (FixK ₂ 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 ₂ ‐mediated regulation. While R200 was essential for DNA binding and activity of FixK ₂ , L195 was involved in protein–DNA complex stability. Mutation at positions 1, 3, or 11 in the genuine FixK ₂ box at the fixNOQP promoter impaired transcription activation by FixK ₂ , 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 ₂ ‐mediated activation in response to microoxia. Thus, position 11 within the FixK ₂ /NnrR boxes constitutes a key element that changes FixK ₂ targets specificity, and consequently, it might modulate B. diazoefficiens lifestyle as nitrogen fixer or as denitrifier.

FixK₂ is a CRP/FNR-type transcription factor that plays a central role in a sophisticated regulatory network for the anoxic, microoxic and symbiotic lifestyles of the soybean endosymbiont Bradyrhizobium diazoefficiens. Aside from the balanced expression of the fixK₂ gene under microoxic conditions (induced by the two-component regulatory system FixLJ and negatively auto-repressed), FixK₂ activity is posttranslationally controlled by proteolysis, and by the oxidation of a singular cysteine residue (C183) near its DNA-binding domain. To simulate the permanent oxidation of FixK₂, we replaced C183 for aspartic acid. Purified C183D FixK₂ protein showed both low DNA binding and in vitro transcriptional activation from the promoter of the fixNOQP operon, required for respiration under symbiosis. However, in a B. diazoefficiens strain coding for C183D FixK₂, expression of a fixNOQP’-‘lacZ fusion was similar to that in the wild type, when both strains were grown microoxically. The C183D FixK₂ encoding strain also showed a wild-type phenotype in symbiosis with soybeans, and increased fixK₂ gene expression levels and FixK₂ protein abundance in cells. These two latter observations, together with the global transcriptional profile of the microoxically cultured C183D FixK₂ encoding strain, suggest the existence of a finely tuned regulatory strategy to counterbalance the oxidation-mediated inactivation of FixK₂ in vivo.