FIG 7.

The ΔatpD strain shows respiratory slowdown and is hypersensitive to peroxide and oxidative stresses. (A) Change in dissolved oxygen (i.e., percent oxygen saturation) that occurs in each case with time. At the start of the experiment, this value was considered 100%. Oxygen consumption in all three bacteria, viz., the WT, ΔatpD, and ΔatpD^C strains, is shown as the reduction in oxygen saturation, which is observed as a downward slope. The mutant bacterium showed reduced oxygen consumption compared to the wild-type and the complemented strains. (B) Cellular ROS levels, as determined by use of the DCFH-DA dye. Higher fluorescence is suggestive of higher ROS levels, which was observed in the case of the knockout strain. (C and D) Gene expression analyses of several genes involved in the antioxidant defense system, carried out by RT-qPCR. The expression levels of katG, sodA, and sodC (C) and ahpC and ahpD (D) in the atpD-knockout strain with respect to those in the wild-type (control), which was considered 1, are shown. (E) MB7H9 agar plate images of the results of the spot assays carried out with the ΔatpD strain in the presence of H₂O₂ are shown. The knockout strain was found to be hypersensitive to peroxide stress. Bacteria were treated for 6 h with either 0 mM (top row) or 20 mM (subsequent three rows) of H₂O₂ and in the presence of 4-HT or thiourea (bottom two rows). Dilutions of the spotted cultures are marked as triangles above the panels. The ΔatpD strain was found to be susceptible to 20 mM H₂O₂ and could be rescued by antioxidants, such as 4-HT and thiourea. For the experiments whose results are presented in both panels A and E, the experiments were repeated at least thrice, and only representative data are shown. In panels B, C, and D, the data represent averages from at least three independent experiments with standard deviations. *, P ≤ 0.05; **, P ≤ 0.01.