C4 hsl

Concentrations of 3OC12-HSL were measured using a biological assay as described previously (10 (link)). To measure C4-HSL concentrations, we developed an mCherry reporter strain. This strain contains rhlR under the control of the tac promoter and a PrhlA-mCherry fusion. The reporter strain was grown overnight and was subcultured to an OD₆₀₀ of 0.025 into minimal A medium (5 (link)) with extracted AHLs. After 18 h, the samples were loaded into a 96-well plate (Costar assay; Corning Incorporated, Kennebunk, ME) and fluorescence was measured in a BioTek Synergy H1 plate reader (excitation, 587 nm; emission, 620 nm). Synthetic 3OC12-HSL and C4-HSL (Cayman Chemical) were used to generate standard curves.

pqsE and rhlR were expressed from the lac promoter and the pBAD promoter, respectively, in an E. coli strain containing the prhlA-luxCDADE fusion. C₄HSL (Cayman Chemical) was supplied at 500 nM. The protocol has been described previously (37 (link)).

3OC12-HSL was measured using a GFP-reporter strain, which contains mbaR under tac promoter control and a gfp transcriptional fusion to −421 bp upstream of the mbaI promoter in the pECP61.5 vector background. C4-HSL was measured using a mCherry-reporter strain as previously reported (Ding et al., 2018 (link)). Synthetic 3OC12-HSL and C4-HSL (Cayman Chemical, Ann Arbor, MI, USA) were used to generate standard curves.

Wild-type TAS2R receptors were tested for function using a cell-based Ca²⁺ flux assay described previously [12 (link)], using the following compounds: probenecid, phenylthiourea (PTC), saccharin sodium salt hydrate (all from Sigma, St. Louis, MO), denatonium benzoate and aloin (Alfa Aesar, Ward Hill, MA), 6-n-propylthiouracil (PROP) (Selleck Chemicals) and C4-HSL (N-butyryl-L-homoserine; Cayman Chemical). Briefly, cells were transfected with the appropriate expression vector and a plasmid expressing a Gα16 chimera (Gα16-gust44), containing the last 44 amino acids of rat gustducin) in poly-lysine coated, black 384-well plates with clear bottoms (Costar). Cells were incubated for 22 hours at 37 °C then washed twice and loaded with a calcium indicator dye in HBSS containing 20 mM HEPES (Calcium 4 Assay kit, Molecular Devices), incubated for 1.5 hours, then moved to a Flexstation II-384 (Molecular Devices) set at 32 °C. probenecid, a commonly used additive designed to improve dye-loading of cells, was not included for most incubations due to our previous demonstration of probenecid as a TAS2R16 inhibitor [12 (link)]. After a 10-minute temperature equilibration, ligand was injected (at t = 20 seconds) and fluorescence was measured for 60 seconds (reading every 3 seconds). Data sets were analyzed using Prism 5.0 software (GraphPad Software, Inc).