José Ignacio Jiménez-Zurdo

Bacterial ribonucleases (RNases) are central components of post-transcriptional networks underlying environmental adaptation. However, their contribution to the ecological specialization of bacteria with complex lifestyles, such as nitrogen-fixing legume symbionts, remains poorly understood. Here, we investigated the role of the double-stranded RNase III ortholog ( Sm RNase III) in Sinorhizobium meliloti , the symbiotic partner of alfalfa ( Medicago sativa L.). Loss of Sm RNase III function affected the expression of nearly 30% of protein-coding genes and 12% of annotated non-coding RNAs (sRNAs). Remarkably, more than 70% of these changes occurred under the microaerobic conditions typical of symbiotic root nodules. Many Sm RNase III-dependent transcripts encode pathways supporting microaerobic metabolism and nitrogen fixation in endosymbiotic bacteroids. Analysis of sequencing read coverage revealed putative consensus cleavage signatures biased toward mRNA 5′ untranslated regions, suggesting preferential processing at these sites. Altered expression of sRNAs and/or their predicted mRNA targets further supports a role for Sm RNase III in sRNA-mediated silencing. Consistently, in vivo and in vitro assays demonstrated that Sm RNase III is required for the repression of nifK (encoding the β-subunit of the nitrogenase MoFe protein) and dctA (encoding a major dicarboxylate transporter) by the antisense sRNA asNifK1 and the trans -sRNA AbcR1, respectively. Our findings reveal a major impact of Sm RNase III on shaping the symbiotic transcriptome of S. meliloti and provide a foundation for deeper investigation into the mechanisms and regulatory roles of RNase III activity in rhizobia.

Bacterial small non-coding RNAs (sRNAs) remain understudied in the ecologically crucial nitrogen (N ₂ )-fixing root-nodule Rhizobium -legume symbiosis. The only known rhizobial RNA regulator with broad symbiotic influence is the N-responsive trans -acting sRNA NfeR1, identified in the alfalfa symbiont Sinorhizobium meliloti . To pinpoint NfeR1 function, we profiled its RNA targets using MS2 affinity purification coupled with RNA sequencing (MAPS) in N stressed bacteria, a condition that drives nodulation. NfeR1 targets distinct regions of numerous mRNAs and sRNAs via three redundant anti-Shine-Dalgarno motifs, with silencing as major regulatory outcome. Target mRNAs span pathways differentially regulated throughout symbiosis, including N metabolism, motility, stress adaptation, and cell cycle control. Notably, NfeR1 modulates cell morphology and DNA replication by pervasive regulation of cell cycle mRNAs. It also silences gdhA , repressing glutamine dehydrogenase-dependent N assimilation and enhancing nodulation gene expression, further fine-tuned by a novel RNA feedback loop between NfeR1 and the dual-function sRNA SmelC549. Our findings position NfeR1 as a central hub within a structurally and functionally complex RNA network that coordinates N signaling and symbiotic performance in S. meliloti .

Computational comparative genomics and, later, high-throughput transcriptome profiling (RNAseq) have uncovered a plethora of small noncoding RNA species (sRNAs) with potential regulatory roles in bacteria. A large fraction of sRNAs are differentially regulated in response to different biotic and abiotic stimuli and have the ability to fine-tune posttranscriptional reprogramming of gene expression through protein-assisted antisense interactions with trans-encoded target mRNAs. However, this level of gene regulation is still understudied in most non-model bacteria. Here, we compile experimental methods to detect expression, determine 5′/3′-ends, assess transcriptional regulation, generate mutants, and validate candidate target mRNAs of trans-acting sRNAs (trans-sRNAs) identified in the nitrogen-fixing α-rhizobium Sinorhizobium meliloti. The workflow, molecular tools, and methods are suited to investigate the function of newly identified base-pairing trans-sRNAs in phylogenetically related α–rhizobia. Key words Sinorhizobium meliloti Rhizobia α-Proteobacteria trans-sRNA Riboregulation

The activity mechanism and function of bacterial base-pairing small non-coding RNA regulators (sRNAs) are largely shaped by their main interacting cellular partners, i.e., proteins and mRNAs. We describe here an MS2 affinity chromatography–based procedure adapted to unravel the sRNA interactome in nitrogen-fixing legume endosymbiotic bacteria. The method consists of tagging of the bait sRNA at its 5′-end with the MS2 aptamer followed by pulse overexpression and immobilization of the chimeric transcript from cell lysates by an MS2-MBP fusion protein conjugated to an amylose resin. The sRNA-binding proteins and target mRNAs are further profiled by mass spectrometry and RNAseq, respectively. Key words Plant symbionts Sinorhizobium meliloti trans-sRNA MS2 RNA-binding proteins Riboregulation

The nitrogen (N) status transduced via the NtrBC two-component system is a major signaling cue in the root nodule endosymbiosis of diazotrophic rhizobia with legumes. NtrBC is upregulated in the N-limiting rhizosphere environment at the onset of nodulation but silenced in nodules to favor the assimilation of the fixed N into plant biomass. We reported that the trans -acting sRNA NfeR1 (Nodule Formation Efficiency RNA) broadly influences the symbiotic performance of the α-rhizobium Sinorhizobium meliloti . Here, we show that NfeR1 is indeed an N-responsive sRNA that fine-tunes NtrBC output during the symbiotic transition. Biochemical and genetic approaches unveiled that NtrC and the LysR-type symbiotic regulator LsrB bind at distinct nearby sites in the NfeR1 promoter, acting antagonistically as repressor and activator of transcription, respectively. This complex transcriptional control specifies peak NfeR1 steady-state levels in N-starved and endosymbiotic bacteria. Furthermore, NfeR1 base pairs the translation initiation region of the histidine kinase coding mRNA ntrB , causing a decrease in both NtrB and NtrC abundance as assessed by double-plasmid genetic assays. In the context of endogenous regulation, NfeR1-mediated ntrBC silencing most likely amends the effective strength of the known operon autorepression exerted by NtrC. Accordingly, a lack of NfeR1 shifts the wild-type NtrBC output, restraining the fitness of free-living rhizobia under N stress and plant growth upon nodulation. The mixed NtrBC-NfeR1 double-negative feedback loop is thus an unprecedented adaptive network motif that helps α-rhizobia adjust N metabolism to the demands of an efficient symbiosis with legume plants. IMPORTANCE: Root nodule endosymbioses between diazotrophic rhizobia and legumes provide the largest input of combined N to the biosphere, thus representing an alternative to harmful chemical fertilizers for sustainable crop production. Rhizobia have evolved intricate strategies to coordinate N assimilation for their own benefit with N ₂ fixation to sustain plant growth. The rhizobial N status is transduced by the NtrBC two-component system, the seemingly ubiquitous form of N signal transduction in Proteobacteria. Here, we show that the regulatory sRNA NfeR1 (nodule formation efficiency RNA) of the alfalfa symbiont Sinorhizobium meliloti is transcribed from a complex promoter repressed by NtrC in a N-dependent manner and feedback silences ntrBC by complementary base-pairing. These findings unveil a more prominent role of NtrC as a transcriptional repressor than hitherto anticipated and a novel RNA-based mechanism for NtrBC regulation. The NtrBC-NfeR1 double-negative feedback loop accurately rewires symbiotic S. meliloti N metabolism and is likely conserved in α-rhizobia.

Nitrogen-fixing root nodule symbioses between rhizobia and legume plants provide more than half of the combined nitrogen incorporated annually into terrestrial ecosystems, rendering plant growth independent of environmentally unfriendly chemical fertilizers. The success of symbiosis depends primarily on the capacity of rhizobia to establish competitive populations in soil and rhizosphere environments.

Function of bacterial small non-coding RNAs (sRNAs) and overall RNA metabolism is largely shaped by a vast diversity of RNA-protein interactions. However, in non-model bacteria with defined non-coding transcriptomes the sRNA interactome remains almost unexplored. We used affinity chromatography to capture proteins associated in vivo with MS2-tagged trans-sRNAs that regulate nutrient uptake (AbcR2 and NfeR1) and cell cycle (EcpR1) mRNAs by antisense-based translational inhibition in the nitrogen-fixing α-rhizobia Sinorhizobium meliloti. The three proteomes were rather distinct, with that of EcpR1 particularly enriched in cell cycle-related enzymes, whilst sharing several transcription/translation-related proteins recurrently identified associated with sRNAs. Strikingly, MetK, the synthetase of the major methyl donor S-adenosylmethionine, was reliably recovered as a binding partner of the three sRNAs, which reciprocally co-immunoprecipitated with a FLAG-tagged MetK variant. Induced (over)expression of the trans-sRNAs and MetK depletion did not influence canonical riboregulatory traits, `for example, protein titration or sRNA stability, respectively. An in vitro filter assay confirmed binding of AbcR2, NfeR1 and EcpR1 to MetK and further revealed interaction of the protein with other non-coding and coding transcripts but not with the 5S rRNA. These findings uncover a broad specificity for RNA binding as an unprecedented feature of this housekeeping prokaryotic enzyme.

Small non-coding RNAs (sRNAs) are ubiquitous components of bacterial adaptive regulatory networks underlying stress responses and chronic intracellular infection of eukaryotic hosts. Thus, sRNA-mediated regulation of gene expression is expected to play a major role in the establishment of mutualistic root nodule endosymbiosis between nitrogen-fixing rhizobia and legume plants. However, knowledge about this level of genetic regulation in this group of plant-interacting bacteria is still rather scarce. Here, we review insights into the rhizobial non-coding transcriptome and sRNA-mediated post-transcriptional regulation of symbiotic relevant traits such as nutrient uptake, cell cycle, quorum sensing, or nodule development. We provide details about the transcriptional control and protein-assisted activity mechanisms of the functionally characterized sRNAs involved in these processes. Finally, we discuss the forthcoming research on riboregulation in legume symbionts.