Dpd ai 2

ITC measurements were carried out at 20 °C with a Nano ITC Standard Volume isothermal calorimeter (TA Instruments, New Castle, DE) controlled by the ITCRun software. The N-terminal His₆ tags of all purified proteins were removed before ITC analysis. For titrations with DPD/AI-2 (Omm Scientific), the tag-free proteins were dialyzed against a Tris buffer (25 mM Tris-HCl, 150 mM NaCl, pH 7.5) and diluted to 70 μM, while DPD/AI-2 was diluted with the same buffer to 700 μM. For titrations with 100 μM taurocholate, taurodeoxycholate (both Sigma-Aldrich), SicA, and its variants, the tag-free proteins subjected to the sample cell were dialyzed against the Tris buffer and diluted to 10 μM. Protein samples were dialyzed against the buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 5 mM MgCl₂, and diluted to 10 μM for titrations with 100 μM c-di-GMP, c-di-AMP, or cGMP dissolved in the same buffer. After being degassed, 1 ml of the protein and 250 μl of the ligand solution were added to the sample cell and the syringe, respectively. There were 25 injections per experiment and the stirring speed was 200 rpm. In control experiments, the ligand solution was titrated into the buffer in the sample cell to obtain the heat of dilution. Microcalorimetric data were corrected by subtracting the heats of dilution and fit to the independent binding site model using the NanoAnalyze software.

E. coli strain ATCC 15144 carrying T1 prophage was grown overnight in 5 ml TB at 37°C with shaking. Day cultures were prepared by diluting the overnight cultures 100 times in 2 ml TB and were grown at 37°C with shaking (220 rpm) for 1 h. Synthetic DPD/AI-2 (27 (link)) and glucose or 2-deoxy-d-glucose (Sigma-Aldrich, Germany) were added for final concentrations of 30 μM and 0.2%, respectively, followed by further incubation at 37°C with shaking. As an alternative to DPD/AI-2, 10 μl of E. coli W3110 cell-free supernatant was used. Prophage induction and the resulting cell lysis were visually observed after 60 to 130 min by the dramatic drop in the optical density (OD) of the test culture (OD at 600 nm [OD₆₀₀], approximately 0.1 to 0.2) compared to that of the control (OD₆₀₀, approximately 0.6 to 0.9). The phage titer after prophage induction and E. coli culture lysis was determined as the PFU level per milliliter using the Gratia method (50 ).