Figure 4. Oxygen consumption, ROS accumulation, and protein oxidation during growth.

(A) Oxygen consumption rate throughout cell growth. The wild type (JH43) and Δphx1 (ESX5) mutant cells were inoculated to an initial OD600 of 0.02 in EMM, and monitored for growth at 30°C by measuring OD₆₀₀ (diamonds). Aliquots were taken at different growth phases, and measured for oxygen consumption rates (circles). Average values with standard deviations were obtained from three independent experiments, and presented as %O₂ consumed per ml of cell culture per min per OD600 to normalize the amount of cells in each sample. (B) Relative levels of intracellular ROS. Cells were grown in the same way as in (A). At the indicated time points, aliquots were taken and mixed with 2′,7′-dichlorofluorescin diacetate (DCFH-DA). Fluorescence from 10,000 cells for each sample was monitored by flow cytometry. Values relative to the fluorescence from wild type and Δphx1 cells at 12 h were presented for the rest of the samples. Average values with standard deviations were obtained from three independent culture samples. (C) Relative levels of protein carbonylation. Cells were grown in the same way as in (A). Carbonylated proteins were detected with anti-DNP antibodies. The carbonylation level of wild type cells at 48 h was taken as 1, and relative values were presented for other samples. Average values with standard deviations were obtained from three independent culture samples. * and ** represent p-values of <0.1, and <0.05, respectively, in Student t-test.