# Riboregulation in Nitrogen-Fixing Endosymbiotic Bacteria
## 3 Conservation of Rhizbial sRNAs: \(\alpha\)-Proteobacterial sRNA Families  
Reverse comparative RNomics contributes to unravel sRNA function by identifying either functionally characterized homologs to the query transcripts or conservation patterns potentially related to the particular lifestyle of phylogenetically close bacterial species. Homology predictions typically rely on stochastic covariance models (CMs) capturing both sequence and secondary structure (folding) conservation from a multiple alignment to the query sRNA. CMs can be automatically generated by INFERNAL, which builds RNA families collected by the Rfam database ([[https://rfam.xfam.org/](https://rfam.xfam.org/)]([https://rfam.xfam.org/](https://rfam.xfam.org/))) [35]. Regardless of their assignment to specific family models, a handful of chromosomally encoded rhizbial sRNAs that occur ubiquitously and exert housekeeping functions in bacteria can be unequivocally identified by the sole comparison of the primary nucleotide sequence. This set includes:  
- RNase P (Rfam family model RF00010), which is the ribozyme that cleaves off 5'-extra sequences on tRNA precursors [36]. - tmRNA or SsrA (RF00023), which has a dual function as transfer and mRNA, operating in _trans_-translation for stalled ribosome recycling [37]. Of note, tmRNA has been the only rhizbial sRNA identified experimentally by a random mutagenesis screen for symbiotic genes in _B. japonicum_[38]. The _S. meliloti_ tmRNA homolog is expressed in a growth- and stress-dependent manner as an unstable precursor, further processed into two readily detectable 214 and 82 nt-long RNA species likely derived from the mRNA and tRNA domains of the primary transcript [39]. - The 4.5S RNA (RF00169), which associates with the multidomain Ffh protein, in the bacterial SRP (small signal recognition particle) ribonucleoprotein complex, universally required for cotranslational protein targeting [40]. - The 6S RNA or SsrS (RF00013) RNA, which relies on an open promoter-like structure to counteract the activity of the \(\sigma^{70}\) RNA polymerase holoenzyme by a target mimicry mechanism, thus contributing to the transcriptional reprogramming during transition from exponential to stationary growth [41]. Interestingly, the 6S RNA is also required for the proper multiplication of the pathogen _Legionella pneumophila_ within macrophages [42], which could anticipate novel yet unexplored wider roles of this riboregulator in both virulence and endosymbiosis. CM-based phylogenetic distribution of other 57 _S. meliloti trans_-sRNAs without associated functional evidence has been also addressed by several independent studies [43; 44; 45; 46]. In two particular cases where high secondary structure but low sequence conservation hindered construction of the CMs, RNA families were modeled using a complementary thermodynamic matchers (TDMs) based approach [43; 44]. TDMs are RNA predictors that ignore sequence conservation in some parts, rather scoring the folding energy of conserved motifs elsewhere in the structure. Altogether, these analyses collected the 57 _trans_-sRNA input sequences into 43 family models, indicating that several of these sRNA _loci_ occur with different levels of paralogy in the Rm1021 genome. Distribution of the majority of these families was restricted to members of the family Rhizobiaceae within \(\alpha\)-proteobacteria, including _Agrobacteriumum_ species. Only a few chromosomally encoded sRNAs are conserved beyond Rhizobiaceae, occurring in representatives of Brucellaceae or even Bartonellaceae, Xanthobacteriaceae, Beijerinckaceae, or Bradyrhizobiaceae. In contrast, sRNA loci mapping to _S. meliloti_ pSymB or pSymA are almost unique to _Sinorhizobium_ spp. and _S. meliloti_, respectively, further supporting that these two megapalsmids mostly encode accessory functions, with pSymA being the most recently acquired replicon in genus _Sinorhizobium_.