# Riboregulation in Nitrogen-Fixing Endosymbiotic Bacteria
## 4 Assigning Functions to Rhizbial _Trans_-sRNAs
### Cell Cycle  
Cell cycle progression has been widely studied in \(\alpha\)-proteobacteria, in which there is a single chromosomal replication event per cycle (Figure 3). Mechanisms to control chromosome replication and cell division in fluctuating environments are of critical relevance for bacterial survival [83]. Accordingly, rhizobia completely rewire cell cycle during the establishment of symbiosis with leguminous plants [34; 84]. Control of \(\alpha\)-proteobacterial cell cycle relies on several conserved transcription factors regulating the main stages [10]. Among them, DnaA mediates replication initiation of _S. meliloti_ chromosome and GcrA has been reported to control expression of several genes involved in the segregation machinery during S-phase in _Caulobacter_[85; 86]. Interestingly, _gcrA_-depleted cells resemble bacteroid genome content and morphology in _S. meliloti_[87], suggesting a similar role of GcrA in cell cycle and also in bacteroid differentiation. Finally, the master cell cycle regulator CtrA controls genes involved in cell division and inhibits replication reinitiation [88]. This essential protein is tightly controlled and negatively regulated, together with GcrA, by the widely-studied plant peptide NCR247, which suggests its involvement in differentiation of N-fixing bacteroids [84]. To date, two _S. meliloti trans_-sRNAs have been related to the adjustment of cell cycle progression under adverse conditions by different mechanisms (Figure 3); EcpR1 represses _dnaA_ and _gcrA_ expression [87] whereas GspR contributes to _ctrA_ silencing [89]. Target predictions for conserved sRNA families in related \(\alpha\)-proteobacteria showed an enrichment of cell cycle-related mRNAs for EcpR1 (formerly SmelC291; Table 1), whereas reverse searches using the 5'-UTR of the _ctrA_ mRNA as a bait ended up with the identification of GspR (formerly SmelC775) as possible CtrA riboregulator. EcpR1 homologs are broadly distributed among Rhizoles, whilst GspR occurrence is likely restricted to the genus _Sinorhizobium_[43]. The name of both _trans_-sRNAs stand for the main phenotypes associated to their gain-of-function. EcpR1 overexpression results into an elongated cell phenotype, along with cell filamentation and genome endoduplication, mimicking the phenotypes of bacteroid and GcrA-depleted cells [87]. Over accumulation of GspR hampers cell growth (growth stop phenotype) in accordance with cell cycle progression defects [89]. Interestingly, these effects are also visible in related species upon overproduction of the respective sRNA homologs. Deletion mutants do not show clear growth phenotypes, however, EcpR1- and GspR- _S. meliloti_ derivatives are outcompeted by their parent strains when co-cultured, suggesting that both sRNAs confer a fitness advantage to  
Figure 3: Activity mechanisms of _S. meliloti_ cell-cycle _trans_-sRNAs. EcpR1 (left) is transcriptionally induced by the alarmone ppGpp and represses the early cell cycle transcriptional regulators DnaA and GcrA, restraining transition to G2 phase. GspR (right) regulates the expression of the gene encoding the essential cell division protein CtrA, preventing cytokinesis, and the cold-shock protein homolog CspA5, which is a CtrA activator. bacteria. Expression of both EcpR1 and GspR is induced in growth-limiting stationary phase in rich media and is not detected in mature nodules [87; 89]. EcpR1 is preceded by a \(\sigma^{70}\)-dependent promoter and this sRNAs is also activated by other stresses like cold and heat shocks or nutrient deprivation, stimulated by the stringent response signal ppGpp. GspR strongly accumulates in bacteria grown in minimal medium. In agreement with in silico predictions and profiling of their dependent proteomes, EcpR1- and GspR-mediated regulation of cell cycle-related genes was experimentally validated by an eGFP-based genetic reporter system [47]. However, EcpR1 and GspR use different targeting mechanisms for mRNA regulation.