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 nodules. Many SmRNase III-dependent transcripts encode pathways supporting microaerobic metabolism and nitrogen fixation in endosymbiotic bacteroids. Analysis of sequencing read coverage identified putative consensus cleavage signatures enriched in 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 vitro assays showed that base‑pairing between nifK (encoding the β‑subunit of the nitrogenase MoFe protein) and the antisense RNA asNifK promotes SmRNase III-mediated cleavage. In vivo assays further supported that silencing of nifK and dctA (encoding a major dicarboxylate transporter) requires SmRNase III, with dctA regulation involving a base‑pairing interaction between the trans‑sRNA AbcR1 and a predicted SmRNase III cleavage site within the mRNA. 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 RNase III-mediated regulation in rhizobia.