Disparate response to microoxia and nitrogen oxides of the Bradyrhizobium japonicum napEDABC, nirK and norCBQD denitrification genes.

Expression of the Bradyrhizobium japonicum napEDABC, nirK and norCBQD denitrification genes requires low oxygen (O) tension and nitrate (NO), through a regulatory network comprised of two coordinated cascades, FixLJ-FixK-NnrR and RegSR-NifA. To precisely understand how these signals are integrated in the FixLJ-FixK-NnrR circuit, we analyzed β-Galactosidase activities from napE-lacZ, nirK-lacZ and norC-lacZ fusions, and performed analyses of NapC and NorC levels as well as periplasmic nitrate reductase (Nap) activity, in B. japonicum wildtype and fixK and nnrR mutant backgrounds. While microoxic conditions (2% O at headspace) were sufficient to induce expression of napEDABC and nirK genes and this control depends on FixK, norCBQD expression requires, in addition to microoxia, nitric oxide gas (NO) and both FixK and NnrR transcription factors. Purified FixK protein directly interacted and activated transcription in collaboration with B. japonicum RNA polymerase (RNAP) from the napEDABC and nirK promoters, but not from the norCBQD promoter. Further, recombinant NnrR protein bound exclusively to the norCBQD promoter in an O-sensitive manner. Our work suggest a disparate regulation of B. japonicum denitrifying genes expression with regard to their dependency to microoxia, nitrogen oxides (NOx), and the regulatory proteins FixK and NnrR. In this control, expression of napEDABC and nirK genes requires microoxic conditions and directly depends on FixK, while expression of norCBQD genes relies on NO, being NnrR the candidate which directly interacts with the norCBQD promoter.