Quorum-sensing assays

To quantify the function of the CqsS::3XFLAG construct in V. cholerae, overnight cultures of V. cholerae strains carrying luxCDABE were diluted to OD₆₀₀ = 0.005 in fresh medium and grown at 30^°C with shaking. Every hour, bioluminescence and OD₆₀₀ were measured on a Tri-Carb 2810 TR scintillation counter and DU800 spectrophotometer, respectively. Samples with OD₆₀₀ > 0.7 were back-diluted 10-fold prior to analysis to ensure measurements could be made within the linear range of the spectrophotometer. CAI-1 was dissolved in DMSO and AI-2 was dissolved in water. The autoinducers were added at the indicated concentrations at the time of dilution. DMSO was used as the negative control and 100 μM of boric acid was added to cultures containing AI-2.

For relative CAI-1 activity assessment, an overnight culture of the V. cholerae CAI-1 reporter strain (WN1102: ΔcqsA, ΔluxQ/pBB1) was diluted 1:10 into sterile medium and 30% (v/v) cell-free culture fluids prepared from the indicated V. cholerae strains were added to the diluted reporter strain. The plasmid pBB1 carries the V. harvyei luxCDABE genes. Bioluminescence and OD₆₀₀ were measured after the cultures were incubated at 30^°C for 2.5 h with shaking. To quantify absolute CAI-1 concentration, the bioluminescence from the CAI-1 reporter strain supplemented with 30% cell-free culture fluids from WT V. cholerae was compared to the bioluminescence from the reporter strain supplemented with known concentrations of synthetic CAI-1 in 30% cell-free ΔcqsA V. cholerae cell-free culture fluids. DMSO was added to the cell-free WT culture fluids as a negative control. The concentration of endogenously produced CAI-1 in the cell-free culture fluids was extrapolated from the log (agonist) vs. response variable slope calculation for the synthetic CAI-1-directed bioluminescence output using Prism software.

The relative phosphatase activities of the QS receptors were measured in WT V. cholerae and in the ΔcqsA ΔluxS double autoinducer synthase mutant using a qrr4-luxCDABE transcriptional fusion. Overnight cultures were diluted 1:20 in fresh medium in the presence and absence of exogenous autoinducers and incubated 4 h at 30^°C with shaking. Bioluminescence was measured on a Tri-Carb 2810 TR scintillation counter. Bioluminescence was normalized to OD₆₀₀, which was measured on a DU800 spectrophotometer.

To determine the EC₅₀ of synthetic CqsS agonists, the CAI-1 reporter strain WN1102 was diluted 1:20 in fresh medium. The indicated concentrations of CAI-1 or synthetic agonist were added in triplicate to 96-well plates and incubated for 4 h at 30^°C with shaking. Bioluminescence and OD₆₀₀ were measured on an Envision 2103 Multilabel Reader (Perkin Elmer). We note that the baseline for bioluminescence is lower on the plate reader compared to that from the scintillation counter. We use relative light units rather than absolute light output, which circumvents issues arising from the different sensitivities of the instruments. The EC₅₀ of each compound was calculated using Prism software. To assess the ability of synthetic agonists to control QS genes, we used a qrr4-gfp construct in an assay that has been described [31].

To assay qrr4 regulation of cqsS, the relative fluorescence of translational fusions (VCA0107B-GFP and CqsS-mKATE2) was measured in E. coli BWR1. E. coli carrying one of these plasmids also contained either an empty plasmid (pZA31) or pZA31 carrying anhydrous tetracycline-inducible qrr4. Qrr4-mediated repression of CqsS-mKATE2 was assessed. VCA0107-GFP is a known Qrr4-repressed target and was used as a positive control to show the system was functional. Overnight cultures were diluted 1:50 into fresh medium and aliquotted in triplicate into 96 well plates. Concentrations of anhydrous tetracycline from 0.4 to 100 ng/μl were added to induce qrr4 expression and the plates were incubated overnight at 30^°C with shaking. OD₆₀₀ and fluorescence were assessed using an Envision 2103 Multilabel Reader (Perkin Elmer).