Expanding the Regulon of the Bradyrhizobium diazoefficiens NnrR Transcription Factor: New Insights Into the Denitrification Pathway

Abstract

Denitrification in the soybean endosymbiont Bradyrhizobium diazoefficiens is controlled by a complex regulatory network composed of two hierarchical cascades, FixLJ-FixK 2 -NnrR and RegSR-NifA. In the former cascade, the CRP/FNR-type transcription factors FixK 2 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 c 550 soluble cytochrome (CycA), previously identified as an intermediate electron donor between the bc 1 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 FixK 2 as determined by in vitro transcription activation assays. The dependence of cycA expression on FixK 2 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.

Regulation of the Emissions of the Greenhouse Gas Nitrous Oxide by the Soybean Endosymbiont Bradyrhizobium diazoefficiens

“…Environmental cues (oxygen tensions and nitrogen oxides) and regulatory proteins (FixK 2 and NnrR) governing denitrification in B. diazoefficiens are well-known [19,20,25] (Figure 1). In this work, we have validated, under physiological conditions, the importance of the FixK 2 and NnrR transcription factors in real-time N 2 O dynamics using a robotized incubation system.…”

Competition for electrons favours N2O reduction in denitrifying Bradyrhizobium isolates

“…Our results from the denitrification analyses support other studies showing that complete denitrification pathways are more common in the B. japonicum supergroup than in other phylogenetic branches of Bradyrhizobium Although this suggests that some taxonomic groups of bradyrhizobia have higher frequency of complete denitrifiers than others, taxonomic position cannot be used as an indicator of the denitrification end-product since even very closely related strains may differ in this regard (Mania et al, 2020). Recent research has revealed several transcriptional and post-transcriptional mechanisms that influence the amounts of intermediate products released from denitrifying organisms (Liu et al, 2013;Torres et al, 2016;Lycus et al, 2018;Gaimster et al, 2019;Jiménez-Leiva et al, 2019). Although of value by itself, such information may be anecdotal.…”

“…Recent research has revealed several transcriptional and post‐transcriptional mechanisms that influence the amounts of intermediate products released from denitrifying organisms (Liu et al ., 2013; Torres et al ., 2016; Lycus et al ., 2018; Gaimster et al ., 2019; Jiménez‐Leiva et al ., 2019). Although of value by itself, such information may be anecdotal.…”

Competition for electrons favors N2O reduction in denitrifyingBradyrhizobium isolates

“…Recent research has revealed several transcriptional and post-transcriptional mechanisms that influence the amounts of intermediate products released from denitrifying organisms [56][57][58][59][60]. Although of value by itself, such information may be anecdotal.…”