AUTHOR=Jiménez-Leiva Andrea , Cabrera Juan J. , Bueno Emilio , Torres María J. , Salazar Sergio , Bedmar Eulogio J. , Delgado María J. , Mesa Socorro 

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

JOURNAL=Frontiers in Microbiology

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2019.01926

DOI=10.3389/fmicb.2019.01926

ISSN=1664-302X

ABSTRACT=<p>Denitrification in the soybean endosymbiont <italic>Bradyrhizobium diazoefficiens</italic> is controlled by a complex regulatory network composed of two hierarchical cascades, FixLJ-FixK<sub>2</sub>-NnrR and RegSR-NifA. In the former cascade, the CRP/FNR-type transcription factors FixK<sub>2</sub> and NnrR exert disparate control on expression of core denitrifying systems encoded by <italic>napEDABC</italic>, <italic>nirK</italic>, <italic>norCBQD</italic>, and <italic>nosRZDFYLX</italic> genes in response to microoxia and nitrogen oxides, respectively. To identify additional genes controlled by NnrR and involved in the denitrification process in <italic>B. diazoefficiens</italic>, we compared the transcriptional profile of an <italic>nnrR</italic> 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 <italic>cycA</italic> gene which codes for the <italic>c</italic><sub>550</sub> soluble cytochrome (CycA), previously identified as an intermediate electron donor between the <italic>bc</italic><sub>1</sub> complex and the denitrifying nitrite reductase NirK. Here, we demonstrated that CycA is also required for nitrous oxide reductase activity. However, mutation in <italic>cycA</italic> neither affected <italic>nosZ</italic> gene expression nor NosZ protein steady-state levels. Furthermore, <italic>cycA</italic>, <italic>nnrR</italic> and its proximal divergently oriented <italic>nnrS</italic> gene, are direct targets for FixK<sub>2</sub> as determined by <italic>in vitro</italic> transcription activation assays. The dependence of <italic>cycA</italic> expression on FixK<sub>2</sub> 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 <italic>c</italic>-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.</p>