Fig. 3. Carbon source modulates the energetics of bacterial cells undergoing nitrosative stress.
Effect of NO on respiration (A) and PMF (B) of E. coli grown in MOPS-GLC or MOPS-CAA. O2 consumption was measured polarographically, whereas the PMF was estimated fluorometrically by the accumulation of 3,3′-dipropylthiadicarbocyanine iodide [DiSC3(5)]. Selected samples were treated for 5 min with 250 μM pNO, 750 μM sNO, or 50 μM CCCP. ****P < 0.0001 versus untreated controls, as calculated by one-way analysis of variance (ANOVA). A.U., arbitrary units. (C) Thin-layer chromatography (TLC) analysis of nucleoside triphosphates (NTPs) extracted from E. coli cells treated with sNO, pNO, or CCCP for 5 min. UTP, uridine 5′-triphosphate; CTP, cytidine 5′-triphosphate; PPi, inorganic pyrophosphate; ppGpp, guanosine 5′-diphosphate 3′-diphosphate. (D) ATP was measured in cytoplasmic extracts by luciferase-dependent chemiluminescence. ****P < 0.0001 versus untreated controls, as calculated by one-way ANOVA. (E) Localization of FtsZ-GFP was evaluated by fluorescence microscopy. **P < 0.001 versus wild-type (WT) control (Ctrl). The data in (A) are representative of three independent experiments, whereas the data in (B), (D), and (E) are means ± SD from four independent observations. Strain EC448 was used for (A), (C), and (D). Strain MC4100 was used for (B). Strains EC3749 and EC3751 were used for (E). ns, not significant.