Figure 7.
Assays for the effects of c-di-GMP on the kinase activity of DevS and their co-regulation on mycobacterial tolerance to oxidative stress. A, effect of c-di-GMP on the autophosphorylation activity of DevS. DevSC was co-incubated with [γ-32P]ATP, and 10-μl aliquots were removed from the mixture at 10, 15, and 20 min (lanes 1–3), and reaction was quenched immediately. Activity was also detected in the presence of 500 μm c-di-GMP (lanes 4–6). The phosphorylation signal intensity of DevSC was quantified and is shown in the lower panel. B, effect of c-di-GMP on the phosphorylation of DevR by DevS. A total of 15 μm DevSC was fully autophosphorylated for 60 min and mixed with 15 μm DevR for further phosphotransfer reactions in the presence of 100–400 μm c-di-GMP. The phosphorylation signal intensity of DevR was quantified and is shown in the lower panel.