Figure 4.

Complementation of ubiTUV-KO strains restores bacterial growth over the entire O₂ range in a UQ-dependent manner. A, Photographs of culture tubes after overnight growth under anaerobic conditions in denitrification medium of ubiTUV-KO strains transformed with the empty vector pSW196 or the same vector carrying the corresponding WT allele (ubiT_Pa, ubiU_Pa, and ubiV_Pa). The parental strain PAO1 was used as a control (Ct), and the UQ₉ content of WT and ubiTUV-KO strains cultured anaerobically was assayed (n = 3). B, ubiTUV-KO strains were cultured in denitrification medium supplemented with methanol-solubilized UQ₄ at 5 or 50 μm final concentration. After 24 h of incubation, the numbers of CFU per ml (CFU/ml) of each KO strain were estimated and compared with those of the same strain grown without UQ₄. As a control (P > 0.05 by unpaired Student's t test), the toxicity of methanol was tested on the parental strain grown in the same medium but supplemented with 4.5% (v/v) methanol (Ct), which is the final concentration of methanol corresponding to the adding of 50 μm UQ₄. Data are representative of three independent experiments (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001; by unpaired Student's t test compared with condition without addition of UQ₄; ns, not significant). C, As described for panel A, but in soft-agar tubes after overnight culture. All the strains studied were inoculated into anaerobic tubes and then exposed to ambient air to create an oxygen gradient. The controls correspond to soft-agar tubes supplemented with 2.5 µg/ml resazurin and then incubated with (1) or without (2) air. Oxic and anoxic parts of the agar are indicated. For all strains containing pSW196 vectors, denitrification medium was also supplemented with a 0.1% (w/v) final concentration of arabinose to induce the P_BAD promoter.