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.
Azorhizobium caulinodans is a microsymbiont of Sesbania rostrata Bremek. & Oberm., and is able to fix nitrogen in both the free-living and symbiotic states. In this study, we focused on the ggm gene (locus tag, AZC_4606) that encodes a putative membrane protein belonging to the TIGR02302 family. Although the genes encoding TIGR02302 family protein are distributed in a wide range of alphaproteobacteria including rhizobia, the functions of this protein are still unknown. To investigate the functions of this protein in A. caulinodans, we made a ggm mutant, and analyzed its phenotypes. The ggm mutant produced more bubbles than the wild-type strain in L3 + N medium liquid cultures, and formed mucoid colonies on L3 + N medium agar plates, suggesting that the ggm mutant overproduced exopolysaccharides (EPSs). The amounts of EPSs produced by the ggm mutant on L3 + N plates were about 1.3-fold higher than those by the wild-type strain, and expression levels of EPS productionrelated genes in the ggm mutant grown in L3 + N liquid medium were about 2-to 4-fold higher than those of the wild-type strain. In addition, the stem nodules formed by the ggm mutant on the stems of S. rostrata showed little or no nitrogen-fixing activity. By microscopic analyses, large infection pockets and a few infected cells were observed in the stem nodules formed by ggm mutant, suggesting that the ggm mutant is defective in invasion into plant cells. Taken together, our results suggest that Ggm is involved in EPS production and that adequate levels of EPS production are required for A. caulinodans to invade into host cells.
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