Phylogenetic analysis and symbiotic functional characterization of opa22-homologous genes in three rhizobial strains

• Opa22 homologs exist in multiple Rhizobiales bacteria with symbiotic-life cycle.
• Opa22 homolog is essential for nodulation but exhibits functional divergence.
• Opa22 homolog may function via a consensus mechanism in bacteria–host interaction.
• Opa22-homologous gene is absent in some rhizobia but independent of taxonomy.

In a previous study, we identified the novel opa22 (DQ199621) gene in Mesorhizobium huakuii 7653R, which is required for nodulation. In this study, we determined that opa22-homologous genes exist in the multiple species of bacteria in Rhizobiales. A conserved domain analysis revealed that the Opa22 homologues are a group of β-barrel outer membrane proteins, belonging to the surface antigen superfamily. A phylogenetic tree was constructed based on the Opa22 homologues and showed that Opa22 supports the validity of the 16S rRNA gene for phylogenic classification. Isogenic mutants were made for each homologue of Sinorhizobium medicae USDA1037, Rhizobium leguminosarum bv. trifolii LPR5045, and Bradyrhizobium japonicum USDA2110 via double-cross replacement. Plant experiments showed that the mutants had lost nodulation ability with their original host plants, and that this symbiotic phenotype could be restored by functional complementation. Interestingly, opa22 from M. huakuii 7653R partially restored nodulation ability of S. medicae isogenic mutant, but not that of B. japonicum isogenic mutant. Taken together, these results indicate that Opa22 is phylogenetically conserved in rhizobial families and is essential for root nodule formation in some species.