A putative TetR-type transcription factor AZC_3265 from the legume symbiont Azorhizobium caulinodans represses the production of R-bodies that are toxic to eukaryotic cells

Azorhizobium caulinodans ORS571 is a microsymbiont of the legume Sesbania rostrata, which forms nitrogen-fixing nodules on stems and roots. This bacterium harbors a reb operon, which is associated with R-body production. R-bodies are large proteinaceous ribbons and were first observed in Caedibacter species, which are obligate bacterial endosymbionts in paramecia. R-body-producing Caedibacter species released from their host paramecia are toxic to the symbiont-free paramecia. R-body-producing cells of A. caulinodans mutants are also toxic to the plant host cells. To maintain harmonic symbiosis with S. rostrata, A. caulinodans has to repress the expression of the reb operon. To date, it has been revealed that the PraR transcription factor and Lon protease repress reb operon expression, in direct and indirect manners, respectively. In this study, we carried out transposon-based mutagenesis screening, and found that the AZC_3265 (locus tag on the genome) gene encoding a putative TetR-type transcription factor was involved in the repression of reb operon expression. The AZC_3265 gene deletion mutant showed high levels of reb operon expression and R-body formation, and this strain formed stem nodules defective in nitrogen-fixing activity. Systematic evolution of ligands by exponential enrichment (SELEX) experiment revealed that AZC_3265 protein could bind to the consensus palindromic sequence TTGC-N6-GCAA. However, this consensus sequence was not found in the reb operon promoter region. Additionally, an electrophoretic mobility shift assay (EMSA) also revealed that AZC_3265 could not bind to the reb operon promoter region. These results suggested that AZC_3265 repressed the expression of the reb operon in an indirect manner. In conclusion, the present data demonstrated that multiple regulators participate in the regulation of expression of the reb operon. The presence of multiple mechanisms for regulating the expression of the reb operon suggested that its expression was controlled in response to multiple biological and environmental factors.