Jimenez-Zurdo, J. I. | Search bioRxiv

bioRxiv2026

RNase III influences microaerobic symbiotic pathways and RNA regulation in Sinorhizobium meliloti

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 (SmRNase III) in Sinorhizobium meliloti, the symbiotic partner of alfalfa (Medicago sativa L.). Loss of SmRNase 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 SmRNase 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 SmRNase III in sRNA-mediated silencing. Consistently, in vivo and in vitro assays demonstrated that SmRNase III is required for the repression of nifK (encoding the {beta}-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 SmRNase 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.

microbiology↗

bioRxiv2025

A single small RNA shapes multiple symbiotic traits in rhizobia

Bacterial small non-coding RNAs (sRNAs) remain understudied in the ecologically crucial nitrogen (N2)-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.

microbiology↗