| Summary: | <p>Due to the costly energy demands of nitrogen (N) fixation, diazotrophic bacteria have evolved complex regulatory networks that permit expression of the catalyst nitrogenase only under conditions of N starvation, whereas the same condition stimulates upregulation of high-affinity ammonia (NH<sub>3</sub>) assimilation by glutamine synthetase (GS), preventing excess release of excess NH<sub>3</sub> for plants. Diazotrophic bacteria can be engineered to excrete NH<sub>3</sub> by interference with GS, however control is required to minimise growth penalties and prevent unintended provision of NH<sub>3</sub> to non-target plants. Here, we tested two strategies to control GS regulation and NH<sub>3</sub> excretion in our model cereal symbiont <em>Azorhizobium caulinodans Ac</em>LP, a derivative of ORS571. We first attempted to recapitulate previous work where mutation of both P<sub>II</sub> homologues <em>glnB</em> and <em>glnK</em> stimulated GS shutdown but found that one of these genes was essential for growth. Secondly, we expressed unidirectional adenylyl transferases (uATs) in a Δ<em>glnE</em> mutant of <em>Ac</em>LP which permitted strong GS shutdown and excretion of NH<sub>3</sub> derived from N<sub>2</sub> fixation and completely alleviated negative feedback regulation on nitrogenase expression. We placed a <em>uAT</em> allele under control of the NifA-dependent promoter P<em>nifH</em>, permitting GS shutdown and NH<sub>3</sub> excretion specifically under microaerobic conditions, the same cue that initiates N<sub>2</sub> fixation, then deleted <em>nifA</em> and transferred a rhizopine <em>nifA</em><sub><em>L94Q/D95Q</em></sub><em>-rpoN</em> controller plasmid into this strain, permitting coupled rhizopine-dependent activation of N<sub>2</sub> fixation and NH<sub>3</sub> excretion. This highly sophisticated and multi-layered control circuitry brings us a step closer to the development of a "synthetic symbioses” where N<sub>2</sub> fixation and NH<sub>3</sub> excretion could be specifically activated in diazotrophic bacteria colonising transgenic rhizopine producing cereals, targeting delivery of fixed N to the crop while preventing interaction with non-target plants.</p>
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