Taurodeoxycholate

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.

Standards for primary bile acids cholate (CA) and chenodeoxycholate (CDCA), conjugated primary bile acids glycocholate (GCA), taurocholate (TCA), glycochenodeoxycholate (GCDCA), and taurochenodeoxycholate (TCDCA), secondary bile acids lithocholate (LCA), deoxycholate (DCA), ursodeoxycholate (UDCA), and hyodeoxycholate (HDCA), and conjugated secondary bile acids glycolithocholate (GLCA), taurolithocholate (TLCA), glycodeoxycholate (GDCA), and taurodeoxycholate (TDCA) were purchased from Sigma.

Killing assays were performed as described previously [1 (link)]. Briefly, indicated bacterial strains were grown overnight on LB agar plates with appropriate antibiotics. Prey and predator were mixed at a 10:1 or 1:1 ratios with titers normalized by OD₆₀₀ readings. Mixtures were spotted on LB agar plates with indicated supplements and incubated for 4 h at 37°C. Bacteria were harvested and serial dilutions of rifampicin-resistant prey or streptomycin-resistant predator were selectively grown on plates overnight. Sodium deoxycholate, sodium cholate, taurodeoxycholate, glycodeoxycholate, taurocholate, glycocholate, and taurine were obtained from Sigma (St. Louis MO). Glycine was obtained from Thermo Fisher Scientific (Waltham MA). Difco™ Bile Salts No.3 was obtained from BD (Mississauga ON). For assays of anaerobic bacteria, indicated bacteria were spotted on LB agar plates supplemented with 1.2 mM bile acids and incubated for 2 days under anaerobic conditions. Bacteria were scraped from the plates and plates were treated for 15 min with chloroform and incubated for an additional 15 min at 37°C [47 (link)]. This procedure ensured that all anaerobic bacteria were dead, and only their metabolites remained. Killing assays were performed on these plates.