An integrated biochemical system for nitrate assimilation and nitric oxide detoxification in Bradyrhizobium japonicum

Rhizobia are recognized to establish N-2-fixing symbiotic interactions with legume plants. Bradyrhizobium japonicum, the symbiont of soybeans, can denitrify and grow under free-living conditions with nitrate (NO3-) or nitrite (NO2-) as sole nitrogen source. Unlike related bacteria that assimilate NO3-, genes encoding the assimilatory NO3- reductase (nasC) and NO2- reductase (nirA) in B. japonicum are located at distinct chromosomal loci. The nasC gene is located with genes encoding an ABC-type NO3- transporter, a major facilitator family NO3-/NO2- transporter (NarK), flavoprotein (Flp) and single-domain haemoglobin (termed Bjgb). However, nirA clusters with genes for a NO3-/NO2--responsive regulator (NasS-NasT). In the present study, we demonstrate NasC and NirA are both key for NO3- assimilation and that growth with NO3-, but not NO2- requires flp, implying Flp may function as electron donor to NasC. In addition, bjgb and flp encode a nitric oxide (NO) detoxification system that functions to mitigate cytotoxic NO formed as a by-product of NO3- assimilation. Additional experiments reveal NasT is required for NO3--responsive expression of the narK-bjgb-flp-nasC transcriptional unit and the nirA gene and that NasS is also involved in the regulatory control of this novel bipartite assimilatory NO3-/NO2- reductase pathway.