A Workflow for the Functional Characterization of Noncoding RNAs in Legume Symbiotic Bacteria.

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.

A double-negative feedback loop between NtrBC and a small RNA rewires nitrogen metabolism in legume symbionts.

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 N2 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.