Genetic determinants of ammonia excretion in nifL mutants of Azotobacter vinelandii

Abstract The ubiquitous diazotrophic soil bacterium Azotobacter vinelandii has been extensively studied as a model organism for biological nitrogen fixation (BNF). In A. vinelandii, BNF is regulated by the NifL-NifA two-component system, where NifL acts as an anti-activator that tightly controls that activity of the nitrogen fixation specific transcriptional activator, NifA, in response to redox, nitrogen, and carbon status. While several studies reported mutations in A. vinelandii nifL resulted in the deregulation of nitrogenase expression and the release of large quantities of ammonia, knowledge about the specific determinants for this ammonia-excreting phenotype is lacking. In this work, we report that only specific disruptions of nifL lead to large quantities of ammonia accumulated in liquid culture (~ 12 mM). The ammonia excretion phenotype is solely associated with deletions of NifL domains combined with the insertion of a promoter sequence in the opposite orientation to nifLA transcription. We further demonstrated that the strength of the inserted promoter could influence the amounts of ammonia excreted by affecting rnf1 gene expression as an additional requirement for ammonia excretion. These ammonia-excreting nifL mutants significantly stimulate the transfer of fixed nitrogen to rice. This work defines the discreet determinants that bring about A. vinelandii ammonia excretion and demonstrates that strains can be generated through simple gene editing to provide promising biofertilizers capable of transferring nitrogen to crops.