Preparation of bacterial supernatants for AHL measurements

The wild type A. wodanis, ΔlitR and litR⁺ were cultivated in parallel at 6 and 12 °C. The cultures were diluted to a start OD₆₀₀ of 0.001 in a total volume of 60 ml LB2.5 in a 250 ml baffled flasks. The cultures were grown further at the selected temperatures and 220 rpm. Cultures of 1 ml (wild type, ΔlitR and litR⁺) were collected at seven different cell densities in total, six at the log phase (OD₆₀₀ of 0.5, 1.0, 2.0, 3.0, 4.0, 5.0) and one at the stationary phase (8.0). For ΔainS AHL measurements, samples were only harvested at the early stationary phase (OD₆₀₀ of 6.0). The cultures were centrifuged at 13,000× g for 2 min at 4 °C (Heraeus fresco 21; Thermo Scientific, Waltham, MA, USA). Seventy-five microliters of each supernatant were acidified with 4 µl of 1M HCl and stored in three technical replicates at −20 °C before measuring the AHLs. A commercial 3-OH-C10-HSL was used as a standard (Sigma-Aldrich, St. Louis, MO, USA). The sample preparations for High-Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MS/MS) were done as described by others (Purohit et al., 2013; Hansen et al., 2015). Briefly, the acidified supernatants were mixed with three volumes of ethyl acetate (225 µl) and vortexed. The ethyl acetate phase of the three technical replicates was pooled together into a 1 ml 96 well plate and dried in a rotary vacuum centrifuge at −90 °C for 2 h (SpeedVac Savant™ concentrator; Thermo Scientific). The dried samples were dissolved in 150 µl of 20% acetonitrile containing 0.1% formic acid and 660 ng/ml of internal standard 3-oxo-C12-HSL (Sigma-Aldrich, St. Louis, MO, USA).