An <i>Azorhizobium caulinodans</i> ORS571 mutant with deletion of a gene encoding a TIGR02302 family protein overproduces exopolysaccharides and is defective in infection into plant host cells

<p><i>Azorhizobium caulinodans</i> is a microsymbiont of <i>Sesbania rostrata</i> Bremek. & Oberm., and is able to fix nitrogen in both the free-living and symbiotic states. In this study, we focused on the <i>ggm</i> 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 alpha-proteobacteria including rhizobia, the functions of this protein are still unknown. To investigate the functions of this protein in <i>A. caulinodans</i>, we made a <i>ggm</i> mutant, and analyzed its phenotypes. The <i>ggm</i> 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 <i>ggm</i> mutant overproduced exopolysaccharides (EPSs). The amounts of EPSs produced by the <i>ggm</i> mutant on L3 + N plates were about 1.3-fold higher than those by the wild-type strain, and expression levels of EPS production-related genes in the <i>ggm</i> 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 <i>ggm</i> mutant on the stems of <i>S. rostrata</i> 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 <i>ggm</i> mutant, suggesting that the <i>ggm</i> 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 <i>A. caulinodans</i> to invade into host cells.</p

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

<p><i>Azorhizobium caulinodans</i> ORS571 is a microsymbiont of the legume <i>Sesbania rostrata</i>, which forms nitrogen-fixing nodules on stems and roots. This bacterium harbors a <i>reb</i> operon, which is associated with R-body production. R-bodies are large proteinaceous ribbons and were first observed in <i>Caedibacter</i> species, which are obligate bacterial endosymbionts in paramecia. R-body-producing <i>Caedibacter</i> species released from their host paramecia are toxic to the symbiont-free paramecia. R-body-producing cells of <i>A. caulinodans</i> mutants are also toxic to the plant host cells. To maintain harmonic symbiosis with <i>S. rostrata, A. caulinodans</i> has to repress the expression of the <i>reb</i> operon. To date, it has been revealed that the PraR transcription factor and Lon protease repress <i>reb</i> 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 <i>reb</i> operon expression. The AZC_3265 gene deletion mutant showed high levels of <i>reb</i> 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 <i>reb</i> operon promoter region. Additionally, an electrophoretic mobility shift assay (EMSA) also revealed that AZC_3265 could not bind to the <i>reb</i> operon promoter region. These results suggested that AZC_3265 repressed the expression of the <i>reb</i> operon in an indirect manner. In conclusion, the present data demonstrated that multiple regulators participate in the regulation of expression of the <i>reb</i> operon. The presence of multiple mechanisms for regulating the expression of the <i>reb</i> operon suggested that its expression was controlled in response to multiple biological and environmental factors.</p