Abstract
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.
Highlights
Biological nitrogen fixation requires diversion of reducing equivalents and ATP derived from carbon metabolism to support the high energetic demands of the enzyme nitrogenase, which converts dinitrogen to ammonia
In this study we demonstrate that both expression and activity of nitrogenase are insensitive to the nitrogen status when the nifA-E356K mutation is introduced into A. vinelandii, resulting in excretion of ammonia at millimolar levels during exponential growth, a phenomenon correlated with feedback regulation of glutamine synthetase when nitrogenase is constitutively active
The activity of NifA-E356K is not regulated in response to the nitrogen status in A. vinelandii, resulting in ammonia excretion
Summary
Biological nitrogen fixation requires diversion of reducing equivalents and ATP derived from carbon metabolism to support the high energetic demands of the enzyme nitrogenase, which converts dinitrogen to ammonia. A second PAS domain (PAS2) appears to play a structural role in relaying the redox changes perceived by the PAS1 domain to the central (H) and C-terminal (GHKL) domains of NifL [8,9]. The latter is responsible for ADP binding [10,11] and is probably the site of interaction for the GlnK signal transduction protein, allowing integration of the nitrogen input into NifLNifA regulation [12,13]. NifA can only escape inhibition by NifL, when the GAF domain is saturated with 2-oxoglutarate, potentially providing a mechanism for the NifL-NifA system to respond to the carbon status
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