phrR-like gene praR of Azorhizobium caulinodans ORS571 is essential for symbiosis with Sesbania rostrata and is involved in expression of reb genes

Abstract

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.

FIG. 1.

Phenotypes of A. caulinodans praR mutant. (A) Growth of ORS571 (wild type) and Anx7 (ΔpraR) in the free-living states. Each strain was grown in NH₄⁺-sufficient (+; 10 mM) or -deficient (−; 0 mM) L3 medium under aerobic (21% O₂) or microaerobic (3% O₂) conditions, and the OD₆₀₀ of the culture was monitored. The values are means ± standard deviations from three replicate cultures. (B) Nitrogen fixation activities of ORS571 and Anx7 in the free-living state. Each strain was grown under NH₄⁺-deficient and microaerobic conditions to an OD₆₀₀ of approximately 0.3, and ARA was measured. The values are means ± standard deviations from five replicate cultures. (C) Stem nodules formed by ORS571, Anx7, and Anx13 (ΔpraR+praR). Each strain was inoculated onto the stems of S. rostrata, and the stem nodules were observed at 7 and 12 dpi. (D) Nitrogen fixation activities of the stem nodules formed by ORS571, Anx7, and Anx13. The stem nodules formed by each strain were harvested at 5 to 15 dpi, and ARA was measured. The values are means ± standard deviations from five replicate plants. Different letters above each data point indicate significant differences (P < 0.05; Tukey-Kramer). (E) Survival abilities of ORS571 and Anx7 in stem nodules. The stem nodules formed by each strain were harvested at 5 to 15 dpi, and bacteria were isolated from the crushed stem nodules. The survival ability was monitored by measuring CFU of the isolated bacteria. The values are means ± standard deviations from five replicate plants. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; Student's t test). FW, fresh weight.

FIG. 2.

Optical microscope images of the stem nodules of strains ORS571 and Anx7. Stem nodules were longitudinally sectioned and stained with toluidine blue O. (A to C) Stem nodules formed by ORS571 at 7 (A) and 12 (B and C) dpi. (D to I) Stem nodules formed by Anx7 at 7 (D), 9 (E, G, and H), and 12 (F and I) dpi. Abbreviations: oc, oval or elongated host cell; sc, shrunken host cell; uc, unstained host cells; v, vacuole.

FIG. 3.

Quantitative RT-PCR analyses. (A) Expression of praR in the free-living and symbiotic states. Total RNAs were isolated from the free-living bacteria of ORS571 grown in NH₄⁺-sufficient (+N) L3 medium at various pH values (pH 6.0, 6.2, and 7.0) under aerobic conditions, from bacteria grown in NH₄⁺-deficient (−N) L3 medium at pH 7.0 under microaerobic (3% O₂) conditions, and from the stem nodules formed by ORS571 at 12 dpi. The amounts of transcripts of praR and 16S rRNA in the total RNA from each sample were estimated by quantitative RT-PCR, and expression levels of praR were evaluated by normalizing to the 16S rRNA level. The values are means ± standard deviations from three replicate cultures or plants and are presented relative to the results for the free-living bacteria grown in NH₄⁺-sufficient L3 medium at pH 7.0 under aerobic conditions. Different letters indicate significant differences (P < 0.05; Tukey-Kramer). (B) Expressions of reb genes (AZC_3781 and AZC_3782), an unknown gene (AZC_1189), and an Omp25/Omp31 gene (AZC_3726) in the free-living and symbiotic states. Total RNAs were isolated from the free-living bacteria of ORS571 (wild type) and Anx7 (ΔpraR) grown under NH₄⁺-sufficient and aerobic conditions and from the stem nodules formed by ORS571 and Anx7 at 12 dpi. The amounts of transcripts of each gene and 16S rRNA in the total RNA from each sample were estimated by quantitative RT-PCR, and the expression levels of each gene were evaluated by normalizing to the 16S rRNA level. The values are means ± standard deviations from three replicate cultures or plants and are presented relative to the results for the free-living bacteria of ORS571.

FIG. 4.

WAD value for each gene plotted against its position within the genome in a comparison between free-living Anx7 and ORS571. Three replicate cultures for each strain were grown under NH₄⁺-sufficient and aerobic conditions to an OD₆₀₀ of approximately 0.8, and microarray analyses were carried out. When the WAD value of a gene is positive, the expression level of this gene is higher in Anx7 than in ORS571.

FIG. 5.

Phenotypes of the A. caulinodans praR and/or reb mutants. (A) Stem nodules formed by ORS571 (wild type), Anx7 (ΔpraR), Anx156 (Δreb), and Anx157 (ΔpraR Δreb). Each strain was inoculated onto the stems of S. rostrata, and the stem nodules were observed at 12 dpi. (B) Nitrogen fixation activities of the stem nodules formed by ORS571, Anx7, Anx156, and Anx157. The stem nodules formed by each strain were harvested at 12 dpi, and ARA was measured. The values are means ± standard deviations from five replicate plants. Different letters indicate significant differences (P < 0.05; Tukey-Kramer). FW, fresh weight. (C) Optical microscope images of the stem nodules formed by Anx156 and Anx157. The stem nodules were longitudinally sectioned and stained with toluidine blue O at 12 dpi.