Azorhizobium caulinodans ORS571 contains an 87.6 kb integrative and conjugative element (ICE^Ac) that conjugatively transfers symbiosis genes to other rhizobia. Many hypothetical redundant gene fragments (rgfs) are abundant in ICE^Ac, but their potential function in horizontal gene transfer (HGT) is unknown. Molecular biological methods were employed to delete hypothetical rgfs, expecting to acquire a minimal ICE^Ac and consider non-functional rgfs as editable regions for inserting genes related to new symbiotic functions. We determined the significance of rgf4 in HGT and identified the physiological function of genes designated rihF1a (AZC_3879), rihF1b (AZC_RS26200), and rihR (AZC_3881). In-frame deletion and complementation assays revealed that rihF1a and rihF1b work as a unit (rihF1) that positively affects HGT frequency. The EMSA assay and lacZ-based reporter system showed that the XRE-family protein RihR is not a regulator of rihF1 but promotes the expression of the integrase (intC) that has been reported to be upregulated by the LysR-family protein, AhaR, through sensing host’s flavonoid. Overall, a conservative module containing rihF1 and rihR was characterized, eliminating the size of ICE^Ac by 18.5%. We propose the feasibility of constructing a minimal ICE^Ac element to facilitate the exchange of new genetic components essential for symbiosis or other metabolic functions between soil bacteria.

Many rhizobia use quorum sensing (QS) systems to detect their population density and modify their symbiotic behavior with the legume host. There are three LuxRI-type QS systems in Rhizobium etli CFN42, and CinR plays a key role in symbiotic performance. However, the details of how CinR regulates the symbiotic process remain unknown. In this study, we employed the RNA-Seq method to screen differentially expressed genes between the wild-type strain and the ΔcinR mutant of R. etli CFN42. We found that most of the genes related to reactive oxygen species (ROS) were expressed at lower levels in the ΔcinR mutant than in CFN42. We also found that the ΔcinR mutant was more sensitive to H₂O₂ than to CFN42. We then showed that CinR positively regulated katG expression and possessed an affinity to bind the katG promoter in the absence of the AHL ligand. The addition of AHLs promoted CinR binding to the katG promoter and enhanced katG expression. Accumulation of H₂O₂ and O₂^•− was observed in root nodules formed by the ΔcinR mutant. Crucially, katG overexpression rescued the H₂O₂-sensitive phenotype in vitro and partially restored defective symbiotic performance in nodules formed by the ΔcinR mutant on the common bean. These results suggest that CinR globally regulates ROS scavenging gene expression in order to balance oxidative stress within root nodules, promoting nitrogenase activity of R. etli CFN42.