Characterisation of oxygen regulation mechanisms in Rhizobium leguminosarum for repurposing as tools in the engineering of nitrogen fixation

Rhizobia are alpha- and beta -proteobacteria that form a symbiotic partnership with legumes, fixing atmospheric dinitrogen to ammonia and providing it to the plant. Fixation is performed by an oxygen-intolerant nitrogenase enzyme but requires respiration to meet its high energy demands. To overcome this paradox, regulation by oxygen (O2) in rhizobia is essential for symbiosis and involves multiple O2 sensing proteins. Interactions between O2 regulatory systems are common, but their importance was not well understood.
We studied the pea microsymbiont Rhizobium leguminosarum biovar viciae 3841 (Rlv3841), which employs three systems: hFixL-FxkR-FixK, FnrN and NifA. We found that both the hFixL-FxkR-FixK and FnrN systems are functional, but act at different O2 concentrations. hFixL-FxkR-FixK is active at a relatively high O2 concentration (1%). The system induces key symbiosis targets including the high-affinity cbb3-type terminal oxidase fixNOQP and the O2 sensor fnrN. FnrN is largely inactive at 1% O2 but becomes active inside nodules, where it autoregulates fnrN and is critical for full fixNOQP expression. Both hFixL-FxkR-FixK and FnrN are required to attain wild-type nitrogen fixation activity. With confocal microscopy, we observed that the two systems act in a hierarchical manner, with hFixL-FxkR-FixK activating at the tip of nodules (in zones I and II), followed by FnrN closer to the nodule core (at the II-III interzone).
The NifA regulator is also O2 sensitive and of particular interest to engineering efforts because of its central role in the control of nitrogen fixation. Little is known about how rhizobial NifA proteins are controlled at the protein level. Most rhizobial NifA proteins have a GAF domain, but the function of the domain remains unknown. We found that very weak activity could be observed from Rlv3841 NifA when native transcriptional regulation was bypassed. Deleting the GAF domain of Rlv3841 NifA critically impaired its activity. Finally, we engineered NifA and NifV activity in Rlv3841 and were able to detect nitrogen fixation activity in free living conditions. This confirmed the potential of NifA engineering as an avenue to modify native biological nitrogen fixation regulation, albeit substantial work will be needed to improve activity.
The hierarchical arrangement of oxygen regulation in Rlv3841 provides a framework which explains both the multiplicity of oxygen sensors in other rhizobia and past findings of partial redundancy between them. Our findings demonstrate the complexity of oxygen regulation in nitrogen fixation, and how one of these systems, NifA, could be repurposed to engineer this regulation. A better understanding of oxygen regulation in biological nitrogen fixation could eventually reduce our need for nitrogen fertilizers, substantially improving the carbon footprint and sustainability of modern agriculture.

Keywords: FnrN
GAF
legume
oxygen regulation
FixK
nitrogen fixation
FxkR
proteobacteria
NifA
synthetic biology
biological nitrogen fixation
rhizobia
Advisor: Poole, P
Sweetlove, L