A single small RNA shapes multiple symbiotic traits 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 .