Disrupting hierarchical control of nitrogen fixation enables carbon-dependent regulation of ammonia excretion in soil diazotrophs

• Published in: PLoS genetics 2021-06, Vol.17 (6), p.e1009617
• Main Authors: Bueno Batista, Marcelo, Brett, Paul, Appia-Ayme, Corinne, Wang, Yi-Ping, Dixon, Ray
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Ammonia; Ammonia - metabolism; Analysis; Assimilation; Azotobacter vinelandii - enzymology; Azotobacter vinelandii - genetics; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biology and Life Sciences; Biosynthesis; Carbon; Carbon - metabolism; Carbon sources; Cereals; E coli; Enzymes; Excretion; Fixation; Gene expression; Gene Expression Regulation, Bacterial; Genetic Complementation Test; Genetic transcription; Glutamate-ammonia ligase; Glutamate-Ammonia Ligase - genetics; Glutamate-Ammonia Ligase - metabolism; Glutamine; Metabolism; Molecular modelling; Mutation; Nitrogen; Nitrogen - metabolism; Nitrogen Fixation; Nitrogen-fixing microorganisms; Nitrogenase; Nitrogenase - genetics; Nitrogenase - metabolism; Oxygen - metabolism; Physical Sciences; Physiological aspects; Protein engineering; Proteins; Proteobacteria; Research and analysis methods; Signal transduction; Soil - chemistry; Soil Microbiology; Transcription; Transcription Factors - genetics; Transcription Factors - metabolism; Transcription, Genetic
• ISSN: 1553-7404; 1553-7390; 1553-7404
• DOI: 10.1371/journal.pgen.1009617

The energetic requirements for biological nitrogen fixation necessitate stringent regulation of this process in response to diverse environmental constraints. To ensure that the nitrogen fixation machinery is expressed only under appropriate physiological conditions, the dedicated NifL-NifA regulatory system, prevalent in Proteobacteria, plays a crucial role in integrating signals of the oxygen, carbon and nitrogen status to control transcription of nitrogen fixation (nif) genes. Greater understanding of the intricate molecular mechanisms driving transcriptional control of nif genes may provide a blueprint for engineering diazotrophs that associate with cereals. In this study, we investigated the properties of a single amino acid substitution in NifA, (NifA-E356K) which disrupts the hierarchy of nif regulation in response to carbon and nitrogen status in Azotobacter vinelandii. The NifA-E356K substitution enabled overexpression of nitrogenase in the presence of excess fixed nitrogen and release of ammonia outside the cell. However, both of these properties were conditional upon the nature of the carbon source. Our studies reveal that the uncoupling of nitrogen fixation from its assimilation is likely to result from feedback regulation of glutamine synthetase, allowing surplus fixed nitrogen to be excreted. Reciprocal substitutions in NifA from other Proteobacteria yielded similar properties to the A. vinelandii counterpart, suggesting that this variant protein may facilitate engineering of carbon source-dependent ammonia excretion amongst diverse members of this family.