Multiple sensors provide spatiotemporal oxygen regulation of gene expression in a Rhizobium-legume symbiosis

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.

Why are rhizobial symbiosis genes mobile?

“…Modularity of sym elements within the genome-and potentially symbiosis genes within their mobile repliconsdemonstrates how such complex traits are able to maintain mobility in rhizobial populations. One counterpoint to this, however, is the existence of direct regulatory control between replicons in several known cases related to fixNOQP and fix-GHIS genes [94][95][96]. In pRetCFN42d, expression of fix genes is regulated by genes on another, less mobile, plasmid, pRetCFN42f [95].…”

Genome-scale metabolic modelling of lifestyle changes in Rhizobium leguminosarum

“…The model for nodule bacteria was obtained using published dRNA-Seq data for RNA extracted from nodule tips (65), as well as a list of genes that were predicted to be specifically essential for nodule bacteria (13). Nutrient availability was defined based on a study using biosensors to detect metabolites inside nodules (12) and our direct measurement of metabolites in pea root exudate, in pea bacteroids and in the nodule cytosol as described previously (40) (Table S4 and S5, Supplementary Data 7).…”

“…To extract a model specific for nitrogen-fixing bacteroids, we used dRNA-Seq data derived from the middle of nodules (65), which contains fully differentiated bacteroids performing nitrogen fixation (64). In addition, a list of 38 genes that were present in the model and encoded proteins significantly upregulated in bacteroids compared to free-living bacteria (23) and the dct genes (73) were specified to ensure inclusion of those genes in the bacteroid model.…”

“…The dRNA-Seq data used for creation of the nodule bacteria and the bacteroid model were described previously (65) and are available on the NCBI SRA database, BioProject number PRJNA667846.…”

Distinct within-host bacterial populations ensure function, colonization and transmission in leaf symbiosis

“…In addition to controlling bacterial proliferation, specific signals may also control expression of bacterial symbiotic functions in target tissue (60, 61). The smp and opk genes of O. dioscoreae encode putative enzymes of the secondary metabolism, which we hypothesized to play a central role in the leaf symbiosis (29, 30).…”