Fig. 6: Phosphorylation of WspR decreases its binding affinity to RpfG and blocks the capacity of RpfG to function as an adaptor protein. | npj Biofilms and Microbiomes

Fig. 6: Phosphorylation of WspR decreases its binding affinity to RpfG and blocks the capacity of RpfG to function as an adaptor protein.

From: The Wsp chemosensory system modulates c-di-GMP-dependent biofilm formation by integrating DSF quorum sensing through the WspR-RpfG complex in Lysobacter

Fig. 6

a MST showing that phosphorylated WspR (WspR-His~P) interacts with MBP-RpfG with moderate affinity (Kd, 0.93 μM). b Phosphorylation of WspR (WspR~P) reduces c-di-GMP synthesis in the presence of MBP-RpfG using GTP as a substrate but not MBP-RpfGH190A. The peak area of the HPLC chromatogram is expressed as the relative yield of c-di-GMP (y-axis). MBP-RpfGH190A represents an enzymatically inactive variant of MBP-RpfG. c HPLC chromatogram corresponding to b. The c-di-GMP and GTP standard are represented in black and red, respectively. d MST showing that WspR-His~P interacts with MBP-RpfGH190A with moderate affinity (Kd, 0.56 μM). e Western blotting showing that the reaction system in b did not affect the stability of the tested protein. Anti-MBP and anti-His antibodies were used to detect WspR~P (39.96 kDa), RpfG, or RpfGH190A (83.92 kDa). All blots derive from the same experiment and that they were processed in parallel. Statistical comparisons were performed using one-way ANOVA of GraphPad software (GraphPad, La Jolla, CA). In panels b, mean data ± SD from three experiments were shown, **P < 0.01. NS means not significant.

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