Abstract
Rhizobia are recognized to establish N2-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.
Highlights
Fixation of atmospheric dinitrogen (N2) by plant-associated symbiotic soil bacteria, collectively termed rhizobia, is a significant agricultural process that reduces dependence on synthetic nitrogen (N) containing fertilizers in crop production
In order to test the involvement of the NasT regulatory protein in NO3 − -dependent induction of the narK-bjgb-flp-nasC operon and nirA gene, we examined expression of narK-lacZ and nirA-lacZ transcriptional fusion constructs in WT and nasT mutant cells following aerobic culture in the presence or absence of the inducer NO3 − (Table 2)
A series of molecular genetics studies have established that genes encoded at two distinct loci, blr2806–09 and bll4571–73 of the B. japonicum genome, encode structural and regulatory components of a combined assimilatory NO3 − reductase and nitric oxide (NO) detoxification system (Figure 1)
Summary
Fixation of atmospheric dinitrogen (N2) by plant-associated symbiotic soil bacteria, collectively termed rhizobia, is a significant agricultural process that reduces dependence on synthetic nitrogen (N) containing fertilizers in crop production. Growth curves for WT (᭹), narK (×), bjgb (ᮀ), flp (᭞), nasC (᭛) and nirA (᭝) strains were measured under aerobic (A) and anaerobic (B) conditions in BN3 minimal medium with NO3 − as sole N-source.
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