Fig 2. WhiB3 regulates ATP levels, NADH/NAD⁺ ratios and bioenergetic homeostasis in Mtb.

(A) Growth curves/rates for wt Mtb H37Rv, MtbΔwhiB3, and the complemented strain (comp) are matching as indicated by CFU over 5 days. Inset: growth rate in logarithmic format. (B) ATP levels in MtbΔwhiB3 were significantly lower than those in wt Mtb and the complemented strain on Day 5 as measured by luminescence (RLU) normalized to CFU. Error bars of (A) and (B) represent SEM of the mean of two independent experiments performed in duplicate. *p<0.01 (Unpaired Student’s t test). (C) NADH/NAD⁺ ratios of Mtb strains grown in 50 μM of the indicated carbon sources until an OD₆₀₀ of 0.8. Error bars represent SD of the mean of at least three independent cultures, *p<0.05, **p<0.001 (Unpaired Student’s t test). (D) Schematic representation of Mtb extracellular flux measured by the XF96. (E-H) OCR, ECAR and a phenogram of wt Mtb (blue), MtbΔwhiB3 (red) and the complemented strain (green) after the addition of one of the following carbon sources (CS): (E) no carbon sources, (F) 50 μM glucose, (G) 50 μM acetate or (H) 50 μM propionate before and after addition of 1, 8 and 20 μM CCCP. Error bars represent SD from the mean of triplicate wells. Data are representative of three independent experiments.