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 antiactivator that tightly controls the activity of the nitrogen fixation-specific transcriptional activator NifA in response to redox, nitrogen, and carbon status. While several studies reported that mutations in A. vinelandii nifL resulted in the deregulation of nitrogenase expression and the release of large quantities of ammonium, knowledge about the specific determinants for this ammonium-excreting phenotype is lacking. In this work, we report that only specific disruptions of nifL lead to large quantities of ammonium accumulated in liquid culture (∼12 mM). The ammonium excretion phenotype is associated solely with deletions of NifL domains combined with the insertion of a promoter sequence in the orientation opposite that of nifLA transcription. We further demonstrated that the strength of the inserted promoter could influence the amounts of ammonium excreted by affecting rnf1 gene expression as an additional requirement for ammonium excretion. These ammonium-excreting nifL mutants significantly stimulate the transfer of fixed nitrogen to rice. This work defines discrete determinants that bring about A. vinelandii ammonium excretion and demonstrates that strains can be generated through simple gene editing to provide promising biofertilizers capable of transferring nitrogen to crops. IMPORTANCE There is considerable interest in the engineering of ammonium-excreting bacteria for use in agriculture to promote the growth of plants under fixed-nitrogen-limiting conditions. This work defines discrete determinants that bring about A. vinelandii ammonium excretion and demonstrates that strains can be generated through simple gene editing to provide promising biofertilizers capable of transferring nitrogen to crops.
Original language | English (US) |
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Article number | e01876-21 |
Journal | Applied and environmental microbiology |
Volume | 88 |
Issue number | 6 |
DOIs | |
State | Published - Mar 2022 |
Externally published | Yes |
Bibliographical note
Funding Information:This work was supported by the National Science Foundation (grant number NSF-1331098). Partial salary support for J.W.P. was provided by the U.S. Department of Agriculture National Institute of Food and Agriculture, Hatch umbrella project number 1015621.
Publisher Copyright:
© 2022 American Society for Microbiology.
Keywords
- A. vinelandii
- ammonium excretion
- biofertilizer
- nifL
- nitrogen fixation
- regulation
- rice
- transfer of fixed nitrogen
- Bacterial Proteins/genetics
- Nitrogenase/genetics
- Nitrogen Fixation/genetics
- Azotobacter vinelandii
- Gene Expression Regulation, Bacterial
- Ammonium Compounds/metabolism
PubMed: MeSH publication types
- Research Support, U.S. Gov't, Non-P.H.S.
- Journal Article