Diazotrophy overcomes the deleterious growth phenotype of glnE deletion in Azotobacter vinelandii
Florence Mus, Alex Tseng, Ray Dixon, John W. Peters
Applied and Environmental Microbiology
Overcoming the inhibitory effects of excess environmental ammonium on nitrogenase synthesis or activity, or preventing ammonium assimilation, have both been considered as strategies to increase the amount of fixed nitrogen transferred from bacterial to plant partners in associative or symbiotic plant-diazotroph relationships. The GlnE adenylyltransferase/adenylyl-removing enzyme catalyzes reversible adenylylation of glutamine synthetase (GS), thereby affecting the post translational regulation of ammonium assimilation critical for appropriate coordination of carbon and nitrogen assimilation. Since GS is key to Azotobacter vinelandii's sole ammonium-assimilation pathway, attempts to obtain deletion mutants in the gene encoding GS (glnA) have been unsuccessful. We have generated a glnE deletion strain thus preventing post-translational regulation of GS. The resultant strain containing constitutively active GS is unable to grow well on ammonium-containing medium as previously observed in other organisms and can be cultured only at low ammonium concentrations. This phenotype is caused by the lack of down regulation of GS activity resulting in high intracellular glutamine levels and severe perturbation of the glutamine:2-oxoglutarate ratio under nitrogen excess conditions. Interestingly, the mutant can grow diazotropically with rates comparable to wild-type. This observation suggests that the control of nitrogen fixation specific gene expression at the transcriptional level in response to 2-oxoglutarate via NifA is sufficiently tight to alone regulate ammonium production at appropriate levels for optimal carbon and nitrogen balance. Importance: In this study, the characterization of the glnE knock out mutant of the model diazotroph Azotobacter vinelandii provides significant insights into the integration of the regulatory mechanisms of ammonium production and ammonium assimilation during nitrogen fixation. The work reveals the profound fidelity of nitrogen fixation regulation in providing ammonium sufficient for maximal growth but constraining energetically costly excess production. A detailed fundamental understanding of the interplay between the regulation of ammonium production and assimilation are of paramount importance in exploiting existing and potentially engineering new plant-diazotroph relationships for improved agriculture.
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