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 PII 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 {Delta}glnE 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 nifAL94Q/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. Author SummaryInoculation of cereal crops with associative "diazotrophic" bacteria that convert atmospheric N2 to NH3 could be used to sustainably improve delivery of nitrogen in agriculture. However, due to the costly energy demands of N2 fixation, natural diazotrophic bacteria have evolved to conserve energy by preventing excess production of NH3 and release to the plants. Diazotrophs can be engineered for excess NH3 production and release, however genetic control is required to minimise growth penalties and prevent unintended provision of NH3 to non-target weed species. Here, we engineer control of N2 fixation and NH3 excretion in response to the signalling molecule rhizopine which is produced by transgenic barley. This control could be used to establish plant host-specific activation of N2 fixation and NH3 release following root colonisation in the field, minimising bacterial energy requirements in the bulk soil and preventing provision of NH3 to non-target plants.