AHL and AI-2 bioreporter assays.

R. radiobacter carrying pZLR4 was grown overnight at 30°C. Cultures were diluted to an OD₆₂₀ of 0.1 in 1 ml of BHI medium. A 1-μl volume of filter-sterilized supernatants of Y. pestis or 10 μl of filter-sterilized BALF, previously collected from mice intranasally infected with wild-type Y. pestis (26), was added. Tubes were incubated at 30°C with shaking at 250 rpm for 5 h. Cultures were pelleted and resuspended in 500 μl of Z-buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mM MgSO₄, pH 7.0). A 25-μl volume of chloroform and a 12.5-μl volume of 0.1% SDS were used to lyse the bacteria for 5 min. A 100-μl volume of 2-nitrophenyl-β-d-galactopyranoside (ONPG; 4 mg/ml) was added to initiate the galactosidase reaction, which was stopped by the addition of 250 μl of 1 M Na₂CO₃. The reaction mixtures were centrifuged for 5 min at 13,000 rpm, and 500 μl was added to a cuvette to record the OD₄₂₀. Relative beta-galactosidase activity levels were expressed as ratios of OD₄₂₀/OD₆₂₀.

For TLC, 100-μl volumes of culture supernatants or BALF were extracted twice with 100 μl of ethyl acetate and concentrated to 20 μl in a 60-Hz Savant SpeedVac DNA 100 concentrator (Thermo Fisher Scientific). Volumes of 1 μl of culture supernatants or 7.5 μl of BALF samples were spotted onto aluminum-backed C18-W silica plates (Sorbent Technologies) and developed in 60% methanol–40% water as described previously (55).

To measure AI-2 concentrations, overnight cultures of MM32 were diluted 1:500 and grown at 30°C for 1 h. A 180-μl volume was added to a 96-well plate with 20 μl of either culture supernatant or BALF. Plates were incubated at 30°C with shaking for 5 h. Optical density and luminescence were recorded on a Molecular Devices Spectramax M5 microplate reader. Reported data are from 3 h of incubation (culture supernatants) and 4 h of incubation (BALF).