Minimal Media Preparation for Bacterial Assays

The minimal media for plate and liquid assays was prepared using the following recipe: 800 mL of Milipore water (with no agar for liquid assays, with 0.625 % agar for swarming assays, with 1.857 % agar for hard agar assays), 200 mL of 5X stock phosphate buffer, 1mL of 1 M magnesium sulfate, 0.1 mL of magnesium sulfate, 25 mL of 200 g/L solution of casamino acids (Bacto^™ from BD, Sparks, MD). 1 L of 5X stock phosphate buffer was prepared by dissolving 12 g of Na₂HPO₄ (anhydrous), 15 of KH₂PO₄ (anhydrows) and 2.5 g of NaCl into 1 L of Milipore water. The final pH of medium was 6.7. When necessary, media composition was altered as described in the text. Autoinducers N-(3-Oxododecanoyl)-L-homoserine lactone (called HSL in the text) and N-Butyryl-DL-homoserine lactone (called C4HSL in the text) were acquired from Sigma-Aldrich (St. Louis, US). Each swarming plate was prepared by pouring exactly 20 mL of medium onto a Petri dish and allowed to cool upright for 30 min. The plates were then turned upside down and left at room temperature to dry for 15 h. Inocula were prepared from 1 mL of overnight cultures washed twice with PBS. Plates inoculation was carried out by spotting a 2 μL drop of pre washed culture at the center of the swarming plate and allowed to dry. Plates were then placed upside down at 37C for 24 h. Each swarming experiment was repeated nine times (three different days with three experimental replicates each). All liquid assays were carried out at 37C with shaking, in 96-well microtiter plates using the Safire 2 (Tecan US, Inc) with OD600 and green fluorescence measured at 10 minute intervals.