A multi-sensor system provides spatiotemporal oxygen regulation of gene expression in a Rhizobium-legume symbiosis

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 vitro, the Rlv3841 hFixL-FxkR-FixK cascade was active at 1% O2, and confocal microscopy showed the cascade is active in the earliest stages of Rlv3841 differentiation in nodules (zones I-II). In vitro and in vivo work showed that the hFixL-FxkR-FixK cascade 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-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 system effectively primes the O2 response by increasing fnrN expression in early differentiation (zones I-II). In Zone III of mature nodules, the near-anaerobic conditions activate FnrN, which induces fixNOQP transcription to the level required to achieve 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 systems are prevalent in rhizobia, suggesting the fine-tuned control they enable is common and maximizes the effectiveness of the symbioses. Author Summary 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 Rhizobium 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.


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The second common O 2 regulation system is based on a variant of FixL called hybrid FixL

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(hFixL) [35,36]. This forms an alternative TCS with FxkR acting as the receiver protein. FxkR   87 is not a FixJ homolog but similarly induces expression of fixK, by binding to an upstream 88 'K-box' motif (GTTACA-N 4 -GTTACA) [37]. The third O 2 sensing system is based on the

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The significance of these multi-sensor arrangements, and the relationship between 8 132 Oxygen is shown in red diamonds. Proteins are shown as ovals, operator sites as squares and genes as pointed rectangles. Promoters are shown as right-angled arrows. Line endings indicate activation (arrows), inhibition (blunt end) and translation (circle). (A) The single pathway formed by the two systems acts in two stages. Stage I starts under microaerobic conditions and can function outside the nodule. In this stage, hFixL is active but FnrN is not. hFixL activates FxkR, which binds to the K-box operator (orange squares) to induce expression of fixK. FixK binds to anaerobox operators (blue squares) to induce expression, including of fnrN and fixNOQP (dashed line). Once oxygen reaches near-anaerobic conditions inside the nodule, FnrN becomes active and stage II begins. Like FixK, FnrN binds anaeroboxes. It auto-regulates fnrN both positively and negatively and induces fixNOQP expression. (B) Rlv3841 has multiple copies of many oxygen regulation genes and many are arranged in clusters. On plasmid pRL9, fixK 9a forms an operon with hfixL 9 , both regulated by a K-box. This operon is adjacent to fixNOQP 9 , regulated by an anaerobox. (C) fixK 9b and fxkR 9 are adjacent, with an anaerobox and a K-box in their intergenic region. Both operators are downstream of the fxkR 9 promoter and likely repress it. The K-box also likely serves to induce fixK 9b expression. (D) The Rlv3841 chromosome also has a cluster, containing fxkR c , fixK c and hfixL c .
Unlike the similar clusters on pRL9, the intergenic region of this cluster contains no anaerobox or Kbox operators. 154 fxkR 9 forms an O 2 regulation cluster with fixK 9b ( Figure 1C) and fxkR c forms a cluster with 155 fixK c -hfixL c ( Figure 1D). The first cluster contains an anaerobox and a K-box, but the 156 second cluster contains neither ( Figure 1D). Thus, FxkR 9 is likely the main FxkR protein 157 and fxkR 9 was deleted to produce strain OPS1808 (∆fxkR 9 ). This mutant reproduced the 158 reduced induction of fnrN under free-living microaerobic conditions observed in the 159 double hfixL mutant ( Figure 3). This finding supports the role of FxkR 9 as the mediator of 160 hFixL O 2 regulation in Rlv3841, in agreement with studies in other rhizobia [35,36].

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Studying the role of the hFixL-FxkR-FixK system in fnrN expression in planta, we observed 162 that the double hfixL mutant reduced fnrN expression to 28% of WT levels ( Figure 4A).

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This indicates the system also plays an important role in inducing fnrN during symbiosis. Individual values (Fluo/OD 600 ) are normalised such that the WT average is 100% for each reporter.
Activity from all three promoters was critically reduced or nearly abolished in the double hfixL and fxkR mutant backgrounds. The hfixL 9 homolog had a far more pronounced effect on expression of all three genes than did the hfixL c homolog. Little or no reduction in expression was observed when fnrN was mutated. Data are averages (±SEM) from at least four biological replicates.

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of fixK 9a was abolished (Supplementary 1, Figure S1), suggesting minimal FixK production 217 in the absence of hFixL-FxkR TCS activity. Taken together, our results indicate that FnrN is 218 critical for fixNOQP expression during symbiosis but the hFixL-FxkR system also plays a 219 significant role.

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To assess the impact of FnrN and hFixL on symbiotic nitrogen fixation, acetylene zone I was greatly reduced in nodules infected with the double hfixL mutant ( Figure 6B).

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This suggests the O 2 concentration in the relatively aerobic environment of zone I is 258 sufficiently low to activate the hFixL-FxkR-FixK system. In the absence of this system, 259 some fnrN expression was retained in zone II and interzone II-III, but this was weaker 260 than WT. Minimal fnrN expression was observed in zone III in the hfixL double mutant.

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In the fnrN mutant, expression of fnrN appeared to be localized primarily in infection 262 threads, around the entire periphery of the nodule ( Figure 6B). Nodules infected by this 263 mutant were severely impaired in their development, failed to elongate and contained 264 little to no leghaemoglobin ( Figure 5D). Free O 2 concentration is unlikely to drop as much 265 in these nodules as it does in fully developed nodules. It is therefore noteworthy that the 266 hFixL-FxkR-FixK system is nevertheless active, suggesting even poorly developed nodules 267 produce a sufficiently low O 2 concentration to activate it.

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In WT Rlv3841, expression of both started abruptly in the II-III interzone of nodules, in 270 agreement with past studies [46,47,87,88]. This abrupt start was absent in nodules 271 infected with the double hfixL mutant, indicating it requires the hFixL-FxkR-FixK system.

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In the fnrN mutant, we observed minimal expression of fixNOQP 9 . This confirms that the