Figure 6: Nature Communications

Fig. 6

From: Non-canonical activation of OmpR drives acid and osmotic stress responses in single bacterial cells

Fig. 6

Spurious results from using pHluorin, MC4100, and clamping agent sodium benzoate. a S. Typhimurium and E. coli strains harboring a plasmid containing pH-sensitive gfp (pHluorin) exhibit heterogeneity in pHi. Representative epifluorescence (R405/488) ratio images of emission channel 525 nm upon 405 nm excitation and 488 nm excitation were obtained for wild-type cultures at either acidic pHe 5.6 or neutral pHe 7.2. Using ImageJ software, the ratio images were color coded with blue (ratio = 0.1) to white (ratio = 1). Scale bar, 3 µm. The pH of individual cells after 30 min is indicated in the panel. b Representative epifluorescence ratio images (R488/440) were obtained for wild-type cultures of E. coli MC4100 incubated at pHe 5.6, pHe 7.2, and pHe 7.2 with 15% (w/v) sucrose at the indicated times. Scale bar, 3 µm. c A plot of the intracellular pH of 50 cells incubated under acid and osmotic stress conditions at each indicated time. Error bars represent mean ± s.e.m. (n = 3). d Cells were clamped by either 40 μM nigericin or 30 mM sodium benzoate and the R488/440 ratio intensities were plotted as a function of pH and overlaid on their in vitro calibration curve. Intracellular pH values determined from BCECF flluorescence of e E. coli MG1655 and f MC4100 clamped with sodium benzoate at pHe 5.6, pHe 6.1, and pHe 7.2 were plotted