Fig 1: Ammonia liberation by GlsA/B raises intracellular pH. (A) Cellular ammonia release from the unfed and manganese-fed E. coli strains was estimated using ab83360 ammonia assay kit (Abcam) and plotted. ****, P < 0.0001. (B) Intracellular pH was measured and plotted for the E. coli strains grown in the presence or absence of manganese using GFPmut3* reporter. ****, P < 0.0001. (C) Relative activity of glsA promoter in the different E. coli strains grown in the presence or absence of manganese was plotted (using a lacZ reporter). The promoter activity in the WT strain was 37.59 ± 2.12 Miller units, which was normalized to 1 for fold change calculation. ****, P < 0.0001 against untreated WT control. (D) Absorption spectrums of bromocresol green pH indicator dye was recorded after allowing the cells to liberate ammonia. Note that the absorption at 620 nm sharply increased for the ΔmntP strain treated with manganese at the 3-min time point (also see Fig. S5A in the supplemental material for all time points). (E) A620/A420 of bromocresol green dye was obtained and plotted to show the kinetics of ammonia release from different strains grown in the presence or absence of manganese.