FIG 1.
Gallein inhibits P. aeruginosa PPK1 and PPK2 enzymes in vitro and in vivo. (A) IC50 curves for gallein inhibition of purified PPK1 (polyP synthesis from ATP), PPK2A (ADP phosphorylation by polyP), PPK2B (polyP synthesis from ATP), and PPK2C (AMP phosphorylation by polyP) in vitro. (B) Dose-dependent effects of gallein on WT PA14 polyP accumulation following phosphate starvation. (C) Effect of gallein on starvation-induced polyP accumulation in PA14 strains. (D) Bacterial polyP extracts analyzed via electrophoresis on Tris-borate-EDTA (TBE)–urea gel negatively stained with DAPI. PolyP standards (14, 60, and 130 Pi residues) from RegenTiss are presented for chain length comparison. Image is representative of n = 3 gels. (E and F) Michaelis-Menten kinetics of purified PPK1 (E) and PPK2A (F) in the presence of gallein. Units represent specific activity (μmol per minute per mg PPK protein). Curves were fit with the substrate inhibition equation {Y = Vmax × X/[Km + X × (1 + X/Ki)]}. In panels B and C, symbols are as follows: ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001 (one-way analysis of variance [ANOVA] for panel B, two-way ANOVA for panel C, Tukey’s multiple-comparison test, n = 3). All data points are the average from triplicates; error bars are ±standard deviation.