C. elegans killing assays on Pseudomonas aeruginosa PA14

The C. elegans killing assays were carried out on wild-type P. aeruginosa PA14 lawns that were incubated at 37°C for 12 hr, or 37°C for 24 hr followed by 25°C for 48 hr. The bacterial lawns were prepared as described above. For full-lawn killing assays, 20 µL of an overnight culture of P. aeruginosa PA14 variants grown at 37°C was spread on the complete surface of 3.5-cm-diameter SK plates. The plates were incubated at 37°C for 12 hr and then cooled to room temperature for at least 30 min before seeding with synchronized young gravid adult hermaphroditic animals. The killing assays were performed at 25°C, and live animals were transferred daily to fresh plates. Animals were scored at the times indicated and were considered dead when they failed to respond to touch. The killing assays with different P. aeruginosa PA14 mutants were carried out on full lawns.