Fig. 5: AI-2 induces the activity of KinD from B. subtilis and of DGC rpHK1S-Z16 from R. palustris.
From: Sensing of autoinducer-2 by functionally distinct receptors in prokaryotes
a The LBD of KinD from B. subtilis and rpHK1S-Z16 from R. palustris is capable of retaining AI-2. Bioluminescence in V. harveyi MM32 was induced by addition of ligands released from purified proteins expressed in a luxS+ (white bars) or luxS- (purple bars) E. coli strain. Results are shown as fold induction relative to the light production induced by a buffer control (mean ± s.e.m.; n = 5 independent experiments). b, c AI-2 specifically binds to the LBDs of KinD and rpHK1S-Z16 with high affinity. The binding affinity was evaluated using ITC analysis. The upper panels show the sequential heat pulses for 700 μM DPD/AI-2 injected into 70 μM KinD-LBD (b) or rpHK1S-Z16-LBD (c), and the lower panels show the integrated data that were corrected for heat of dilution of the ligands into buffer and fit to a one-site binding model. Data shown are one representative of three independent experiments with similar results. The Kd and binding stoichiometry (n) were calculated using the NanoAnalyze software and presented as mean ±  s.d. of three independent experiments. d AI-2 stimulates the autokinase activity of KinD. Autophosphorylation of KinD was carried out in reactions with or without DPD/AI-2. Reactions without ATP were established as controls. KinD phosphorylation was detected by determining the ratio between phosphopeptide and total peptides using LC-MS/MS. Data are mean ±  s.e.m. of four independent experiments. e AI-2 induces the DGC activity of rpHK1S-Z16. Membrane fractions containing rpHK1S-Z16 were incubated with GTP in the presence or absence of DPD/AI-2 at 30 °C for 0, 30, and 60 min and the product was analyzed by HPLC (Supplementary Fig. 10). The level of c-di-GMP was determined from a standard curve established with a serially diluted c-di-GMP solution. Data are mean ± s.e.m. of three independent experiments. d, e P values were determined using the two-tailed unpaired Student’s t-test. p < 0.05 was considered to indicate a statistically significant difference.
