This study focuses on the function of the gene praR that encodes a putative transcription factor in Azorhizobium caulinodans ORS571, a microsymbiont of Sesbania rostrata. The praR gene is a homolog of the phrR gene of Sinorhizobium medicae WSM419, and the praR and phrR homologs are distributed throughout the class Alphaproteobacteria. The growth and nitrogen fixation activity of an A. caulinodans praR deletion mutant in the free-living state were not significantly different from those of the wild-type strain. However, the stem nodules formed by the praR mutant showed lower nitrogen fixation activity than the wild-type stem nodules. Microscopy revealed that infected host cells with an oval or elongated shape were observed at early stages in the nodules formed by the praR mutant, but these infected cells gradually fell into two types. One maintained an oval or elongated shape, but the vacuoles in these cells gradually enlarged and the bacteria gradually disappeared. The other cells were shrunken with bacteria remaining inside. Microarrays revealed that genes homologous to the reb genes of Caedibacter taeniospiralis were highly expressed in the praR mutant. Furthermore, the stem nodules formed by an A. caulinodans mutant with a deletion of praR and reb-homologous genes showed high nitrogen fixation activity, comparable to that of the wild-type stem nodules, and were filled with oval or elongated host cells. These results suggest that PraR controls the expression of the reb-homologous genes and that high expression of reb-homologous genes causes aberrance in A. caulinodans-S. rostrata symbiosis.
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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