Abstract
Regulation by oxygen (O2) in rhizobia is essential for their symbioses with plants and involves multiple O2 sensing proteins. Three sensors exist in the pea microsymbiont Rhizobium leguminosarum Rlv3841: hFixL, FnrN and NifA. At low O2 concentrations (1%) hFixL signals via FxkR to induce expression of the FixK transcription factor, which activates transcription of downstream genes. These include fixNOQP, encoding the high-affinity cbb3-type terminal oxidase used in symbiosis. In free-living Rlv3841, the hFixL-FxkR-FixK pathway was active at 1% O2, and confocal microscopy showed hFixL-FxkR-FixK activity in the earliest stages of Rlv3841 differentiation in nodules (zones I and II). Work on Rlv3841 inside and outside nodules showed that the hFixL-FxkR-FixK pathway also induces transcription of fnrN at 1% O2 and in the earliest stages of Rlv3841 differentiation in nodules. We confirmed past findings suggesting a role for FnrN in fixNOQP expression. However, unlike hFixL-FxkR-FixK, Rlv3841 FnrN was only active in the near-anaerobic zones III and IV of pea nodules. Quantification of fixNOQP 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 fixNOQP. Without FnrN, nitrogen fixation was reduced by 85% in Rlv3841, while eliminating hFixL only reduced fixation by 25%. The hFixL-FxkR-FixK pathway effectively primes the O2 response by increasing fnrN expression in early differentiation (zones I-II). In zone III of mature nodules, near-anaerobic conditions activate FnrN, which induces fixNOQP transcription to the level required for wild-type nitrogen fixation activity. Modelling and transcriptional analysis indicates that the different O2 sensitivities of hFixL and FnrN lead to a nuanced spatiotemporal pattern of gene regulation in different nodule zones in response to changing O2 concentration. Multi-sensor O2 regulation is prevalent in rhizobia, suggesting the fine-tuned control this enables is common and maximizes the effectiveness of the symbioses.
Highlights
Rhizobia are alpha-proteobacteria that engage in symbiosis with legume plants [1]
Rhizobia are soil bacteria that form a symbiosis with legume plants
Fixation is catalysed by the nitrogenase enzyme, which is inactivated by oxygen
Summary
Rhizobia are alpha-proteobacteria that engage in symbiosis with legume plants [1]. The bacteria convert inert atmospheric N2 into biologically accessible ammonia and provide it to their plant host in a process called nitrogen fixation [2,3]. Rhizobia are obligate aerobes and must respire to meet the high energy demands of nitrogen fixation [7,8] These competing requirements create an ‘oxygen paradox’ in symbiotic nitrogen fixation [9,10]. Nodules create a near-anoxic internal environment suitable for nitrogenase activity [15,16,17] To produce this environment, oxygen (O2) is captured and shuttled to bacteroids by plant leghaemoglobins [18,19,20,21]. Rhizobia undergo a radical lifestyle change after nodule entry to survive and fix nitrogen in these conditions (reviewed in [24,25]) In indeterminate nodules, such as those produced by Pisum sativum (pea), rhizobia are initially free-living upon entry [26,27]. Rhizobial regulatory mechanisms sensitive to O2 tension are essential for successful differentiation into bacteroids and the establishment of a productive symbiosis [33,34,35]
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