A multi-sensor system provides spatiotemporal oxygen regulation of gene expression in a Rhizobium-legume symbiosis
Rutten PJ., Steel H., Hood GA., RAMACHANDRAN V., McMurtry L., Geddes B., Papachristodoulou A., Poole PS.
<jats:title>Abstract</jats:title><jats:p>Regulation by oxygen (O<jats:sub>2</jats:sub>) in rhizobia is essential for their symbioses with plants and involves multiple O<jats:sub>2</jats:sub> sensing proteins. Three sensors exist in the pea microsymbiont <jats:italic>Rhizobium leguminosarum</jats:italic> Rlv3841: hFixL, FnrN and NifA. At low O<jats:sub>2</jats:sub> concentrations (1%) hFixL signals via FxkR to induce expression of the FixK transcription factor, which activates transcription of downstream genes. These include <jats:italic>fixNOQP</jats:italic>, encoding the high-affinity cbb<jats:sub>3</jats:sub>-type terminal oxidase used in symbiosis. <jats:italic>In vitro</jats:italic>, the Rlv3841 hFixL-FxkR-FixK cascade was active at 1% O<jats:sub>2</jats:sub>, and confocal microscopy showed the cascade is active in the earliest stages of Rlv3841 differentiation in nodules (zones I-II). <jats:italic>In vitro</jats:italic> and <jats:italic>in vivo</jats:italic> work showed that the hFixL-FxkR-FixK cascade also induces transcription of <jats:italic>fnrN</jats:italic> at 1% O<jats:sub>2</jats:sub> and in the earliest stages of Rlv3841 differentiation in nodules. We confirmed past findings suggesting a role for FnrN in <jats:italic>fixNOQP</jats:italic> expression. However, unlike hFixL-FxkR-FixK, Rlv3841 FnrN was only active in the near-anaerobic zones III-IV of pea nodules. Quantification of <jats:italic>fixNOQP</jats:italic> expression in nodules showed this was driven primarily by FnrN, with minimal direct hFixL-FxkR-FixK induction. Thus, FnrN is key for full symbiotic expression of <jats:italic>fixNOQP</jats:italic>. Without FnrN, nitrogen fixation was reduced by 85% in Rlv3841, while eliminating hFixL only reduced fixation by 25%. The hFixL-FxkR-FixK system effectively primes the O<jats:sub>2</jats:sub> response by increasing <jats:italic>fnrN</jats:italic> expression in early differentiation (zones I-II). In Zone III of mature nodules, the near-anaerobic conditions activate FnrN, which induces <jats:italic>fixNOQP</jats:italic> transcription to the level required to achieve wild-type nitrogen fixation activity. Modelling and transcriptional analysis indicates that the different O<jats:sub>2</jats:sub> sensitivities of hFixL and FnrN lead to a nuanced spatiotemporal pattern of gene regulation in different nodule zones in response to changing O<jats:sub>2</jats:sub> concentration. Multi-sensor O<jats:sub>2</jats:sub> regulation systems are prevalent in rhizobia, suggesting the fine-tuned control they enable is common and maximizes the effectiveness of the symbioses.</jats:p><jats:sec><jats:title>Author Summary</jats:title><jats:p>Rhizobia are soil bacteria that form a symbiosis with legume plants. In exchange for shelter from the plant, rhizobia provide nitrogen fertilizer, produced by nitrogen fixation. Fixation is catalysed by the nitrogenase enzyme, which is inactivated by oxygen. To prevent this, plants house rhizobia in root nodules, which create a low oxygen environment. However, rhizobia need oxygen, and must adapt to survive low oxygen in the nodule. Key to this is regulating their genes based on oxygen concentration. We studied one <jats:italic>Rhizobium</jats:italic> species which uses three different protein sensors of oxygen, each turning on at a different oxygen concentration. As the bacteria get deeper inside the plant nodule and the oxygen concentration drops, each sensor switches on in turn. Our results also show that the first sensor to turn on, hFixL, primes the second sensor, FnrN. This prepares the rhizobia for the core region of the nodule where oxygen concentration is lowest and most nitrogen fixation takes place. If both sensors are removed, the bacteria cannot fix nitrogen. Many rhizobia have several oxygen sensing proteins, so using multiple sensors is likely a common strategy that makes it possible for rhizobia to adapt to low oxygen gradually in stages during symbiosis.</jats:p></jats:sec>