T β mca

For Lactobacillus whole cell assays, cells were centrifuged and washed twice with sterile PBS, pH 7.4, then resuspended in fresh media. T-β-MCA (Steraloids) was added to a concentration 500 nM, and cultures were incubated at 37°C for 90 mins. This concentration was chosen as it provided visible peaks by GCMS analysis and also allowed us to visualize concentration changes over time (see UPLC-ESI-QTOFMS analysis below). Cells were precipitated by centrifugation and 100 μL of supernatant was added directly to 100 μL of ice cold methanol and stored at -80°C for analysis by UPLC-ESI-QTOFMS. Cell lysate assays were used to assess BSH substrate specificity in transgenic E. coli C600 strains and were performed by washing overnight cultures twice in sterile PBS, pH 7.4, and resuspending the cells in 3 mM sodium acetate buffer, pH 5.2. Cells were lysed by vortexing with 0.1 mm glass beads (Mo Bio Laboratories, Inc.) according to the manufacturer’s instructions. Total protein was measured by Bradford assay and normalized to 0.100 mg/mL in 100 μL of buffer before adding a concentrated master solution of T-β-MCA, TCA, TDCA, TCDCA, GCA, and GCDCA (Sigma) to achieve a final concentration of 500 nM. Cell lysates were incubated at 37°C for 90 mins, quenched with 100 μL of ice cold methanol and stored at -80°C for analysis by UPLC-ESI-QTOFMS.

TαMCA, TβMCA, TωMCA, THCA, αMCA, βMCA, ωMCA, HCA, HDCA, MCA, GCDCA, GDCA, and GCA (Additional file 1: Table S6) were obtained from Steraloids (Newport, RI, USA).
TCA, CA, DCA, TCDCA, TDCA, TUDCA, TLCA, CDCA, UDCA, LCA, D₄-TCA, D₄-DCA, D₄-CA, D₄-TDCA, D₄-GLCA, D₄-GUDCA, D₄-GCDCA, D₄-GCA, and D₄-GDCA (Additional file 1: Table S6) were obtained from Isosciences (Ambler, PA, USA).
LC/MS grade acetonitrile (#A955-500), water (#W6500), and formic acid (#A117-50) were obtained from Thermo Fisher Scientific.