Control of nitrogen fixation and ammonia excretion in Azorhizobium caulinodans

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(3)) assimilation by glutamine synthetase (GS), preventing excess release of excess NH(3) for plants. Diazotrophic bacteria can be engineered to excrete NH(3) by interference with GS, however control is required to minimise growth penalties and prevent unintended provision of NH(3) to non-target plants. Here, we tested two strategies to control GS regulation and NH(3) excretion in our model cereal symbiont Azorhizobium caulinodans AcLP, a derivative of ORS571. We first attempted to recapitulate previous work where mutation of both P(II) homologues glnB and glnK stimulated GS shutdown but found that one of these genes was essential for growth. Secondly, we expressed unidirectional adenylyl transferases (uATs) in a ΔglnE mutant of AcLP which permitted strong GS shutdown and excretion of NH(3) derived from N(2) fixation and completely alleviated negative feedback regulation on nitrogenase expression. We placed a uAT allele under control of the NifA-dependent promoter PnifH, permitting GS shutdown and NH(3) excretion specifically under microaerobic conditions, the same cue that initiates N(2) fixation, then deleted nifA and transferred a rhizopine nifA(L94Q/D95Q)-rpoN controller plasmid into this strain, permitting coupled rhizopine-dependent activation of N(2) fixation and NH(3) excretion. This highly sophisticated and multi-layered control circuitry brings us a step closer to the development of a "synthetic symbioses” where N(2) fixation and NH(3) 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.