Fig. 5.

ALI1 interacts with oxo-C14-HSL. a Pull-down analysis between the His-tagged ALI1 and oxo-C14-HSL (M4) and detection of retained ALI1 on beads bound to M4 but not on beads bound to biotin. The upper panel presents a western blot, in which either 1/100 of the input proteins (6xHis-ALI1 or 6xHis-SpvC, as negative control) or proteins resulting from a pull-down were separated, blotted, and probed with anti-His antibody. The pull-down setup is indicated above the panel. Pull-down analysis was performed in three independent experiments. A representative blot from those independent experiments is shown. The lower panel presents loading control for the pull-down setup, and input shows 1/100 of the protein amount used for the pull-down. b The tertiary structure of ALI1 protein. (Predicted structure of ALI1 with threading or fold recognition model after docking simulations with oxo-C14-HSL). c Protein threading or fold recognition predicted model of ALI1 with docked oxo-C14-HSL ligand, simulated in the online docking webserver SwissDock. d Luminescence activity assay indicating the binding of oxo-C14-HSL to ALI1. The binding capacity was assessed by determining the concentration of free oxo-C14-HSL using the E. coil LuxCDABE reporter strain after an overnight incubation with 6 nmol ALI1 or LuxR (positive control) and BSA (negative control) with different amounts of oxo-C14-HSL, as indicated. The individual data values for d in Additional File 1