Control of N₂ fixation and NH₃ excretion in Azorhizobium caulinodans ORS571

Abstract

Due to the costly energy demands of N₂ fixation, diazotrophic bacteria have evolved complex regulatory networks that permit expression of the N₂-fixing catalyst nitrogenase only under conditions of N starvation, whereas the same condition stimulates upregulation of high-affinity NH₃ assimilation by glutamine synthetase (GS), preventing excess release of excess NH₃ for plants. Diazotrophic bacteria can be engineered to excrete NH₃ by interference with GS, however control is required to minimise growth penalties and prevent unintended provision of NH₃ to non-target plants. Here, we attempted two strategies to control GS regulation and NH₃ 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 adenylyltransferases (uATs) in a ΔglnE mutant of AcLP which permitted strong GS shutdown and excretion of NH₃ derived from N₂ 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 NH₃ excretion specifically under microaerobic conditions, the same cue that initiates N₂ fixation, then deleted nifA and transferred a rhizopine-inducible nifA_L94Q/D95Q-rpoN controller plasmid into this strain, permitting coupled rhizopine-dependent activation of N₂ fixation with NH₃ excretion. In future, this highly sophisticated and multi-layered control circuitry could be used to activate N₂ fixation and NH₃ excretion specifically by AcLP colonising transgenic rhizopine producing cereals, targeting delivery of fixed N to the crop, and preventing interaction with non-target plants.