c-di-GMP hydrolytic activity of PA4108.

<p>(A) Representative time-courses of pGpG and GMP formation obtained after incubation of 0.64 µM PA4108 with 150 µM c-di-GMP (in the presence of 10 mM MgCl₂ and 2.5 mM MnCl₂). The nucleotide content of the reaction mixture was analyzed by RP-HPLC at different times (30, 60, 100, 180 min, at 30°C; solid lines in the figure) and compared with a calibration curve of standard solutions of GMP and pGpG (the chromatogram of a 5 µM solution of both nucleotides is reported as reference, dashed lines). (B) Plot of the initial rate of c-di-GMP hydrolysis (µM of c-di-GMP consumed/min) measured at different c-di-GMP concentrations (black circles). Data were fitted with the Michaelis-Menten equation (continuous line) in order to extrapolate the K_M and V_max parameters (20 µM and 5.8x10^-3 µM/min, respectively). Data are the average of at least two independent experiments.