Elevated nitrate levels inhibit nitrogen fixation in Azotobacter chroococcum via an ammonium-independent pathway

Nitrate impairs both symbiotic and free-living biological nitrogen fixation (BNF). While nitrate-induced phosphorylation signalling has been implicated in the inhibition of symbiotic BNF, the suppression of free-living BNF has generally been attributed to ammonium generated during nitrate assimilation. However, whether nitrate can inhibit free-living BNF independently of ammonium feedback regulation remains unclear. Here, an ammonium-deregulated mutant of Azotobacter chroococcum (A4) was used to investigate whether nitrate inhibits nitrogen fixation independently of ammonium regulation. Despite the loss of ammonium-mediated inhibition, nitrate significantly suppressed nitrogen fixation. Nitrate at concentrations above 2 mM reduced extracellular ammonium accumulation, with 10 mM nitrate decreasing ammonium production to 61% of that observed under nitrogen-free conditions. Integrated multi-omics analyses revealed that nitrate triggered extensive regulatory reprogramming across multiple molecular layers, with both coordinated and layer-specific responses across transcriptomic, proteomic and phosphoproteomic levels. These responses differed from the typical ammonium-mediated feedback regulation characterized by substantial repression of nitrogen fixation-related genes or proteins, but nevertheless resulted in reduced ammonium excretion, accompanied by enhanced biomass accumulation and extracellular polymeric substance (EPS) production in A4. Together, these findings indicate that, rather than directly repressing the nitrogen fixation machinery, prolonged nitrate exposure suppresses nitrogen fixation output through global regulatory reprogramming that redirects cellular metabolism and resource allocation away from nitrogen fixation. This study provides new insights into nitrate-mediated regulation of free-living diazotrophs and has implications for optimising nitrogen management and improving the application of nitrogen-fixing microorganisms.