Motility assay

Swimming, swarming, twitching (for P. aeruginosa, V. vulnificus, B. subtilis, and S. enterica), and spreading motilities (for S. aureus) were measured as previously described^31–36. Swimming was assayed on the following media; LB and LBS media solidified with 0.3% (wt/vol) agar were used for P. aeruginosa and V. vulnificus, respectively. Nutrient broth (NB) supplemented with 0.5% glucose and solidified with 0.3% agar was used for S. enterica, and tryptone medium (1% tryptone, 0.5% NaCl) solidified with 0.3% agar was used for B. subtilis. Overnight cultures (3 μl) were inoculated into the middle of agar and incubated at 37 °C for 24 hours. Degrees of swimming motility are presented as the average of the largest and smallest diameters of swimming areas. Swarming was assayed on NB medium supplemented with 0.5% glucose and solidified with 0.5% agar for P. aeruginosa, B. subtilis, and S. enterica, or on Brain Heart Infusion (BHI) medium containing 2% NaCl and solidified with 0.3% agar for the V. vulnificus. To assess swarming motilities, 3 μl of overnight culture was dropped on the surfaces of agar plates and incubated at 37 °C for 24 hours. Degrees of swarming motility are presented in the same manner as swimming motility. Twitching was assayed on LB or LBS (for V. vulnificus) medium solidified with 1% agar. The strains were stab-inoculated with a sharp toothpick to the bottoms of plates and incubated for 24 hours at 37 °C. Twitching zones at plate-agar interfaces were visualized by staining with 1% crystal violet and their sizes were measured as described above. Spreading motility (for S. aureus) was assayed on tryptone medium solidified with 0.3% agar. Briefly, 5 μl of overnight cultures were dropped onto the surfaces of agar plates and incubated at 37 °C for 24 hours. Degrees of spreading were measured as described for swimming motility.