FIG 9 .

Effects of 2OG on the expression of the reb operon and R-body formation in bacteria under free-living conditions. (A) β-Galactosidase activities of the reb-lacZ strain in the presence of various 2OG concentrations. The reb-lacZ strain was grown in medium with the indicated concentrations of 2OG (0 to 50 mM) at 26°C for 24 h to an OD₆₀₀ of approximately 1.0. Data are presented as means ± standard deviations from three replicate cultures. Different letters indicate significant differences (P < 0.05, Tukey-Kramer). (B) Quantitative RT-PCR analysis to investigate the effects of 2OG and temperature on the reb operon expression in the WT strain. Cultures of the WT strain were supplemented with 10 mM 2OG (+2OG) or no 2OG (−2OG) and were grown at 26 or 38°C. The data are expressed relative to those from the cultures under –2OG conditions at 26°C and are presented means ± standard deviations from three replicate cultures. Asterisks indicate significant difference (**, P < 0.01, Student’s t test). (C) TEM observations of the WT cells cultured with 2OG at 26 and 38°C. Bacterial cells were collected from the cultures that were used for the quantitative RT-PCR conducted in panel B. Arrowheads indicate R bodies. (D) Quantitative RT-PCR analysis of the reb operon in the WT, ΔpraR, ΔrebR, and ΔpraR ΔrebR strains. These strains were grown under +2OG or –2OG conditions at 26°C. The data are expressed relative to corresponding values in the WT strain under –2OG conditions at 26°C and are presented as means ± standard deviations from three replicate cultures. Asterisks indicate significant difference (**, P < 0.01, Student’s t test). (E) Western blotting with an anti-His₅ antibody to investigate the effects of 2OG on RGS-His₆-PraR expression. Whole-cell lysates from the rgs-his₆-praR strain grown under +2OG or –2OG conditions and from the WT strain grown under –2OG conditions were electrophoresed. (F) EMSA analysis to investigate the effects of 2OG on binding activities of His₆-PraR to the reb promoter. FITC-labeled dsDNA probe corresponding to the reb promoter was incubated with the purified His₆-PraR in the presence of 2OG (0.01 to 10 mM) or succinate (10 mM).