Figure 4. Phenotypic effects of compound 1 on alternate secretion systems.

a, Motility assays of S. typhimurium and P. aeruginosa in the absence (−) or presence (+) of 380 µM of compound 1. Bacteria were stabbed into motility plates and incubated at 37°C for 6 hours. The diameter of the ring was measured for three independent replicates and the mean standard deviations are shown. b, Elastolytic activity for 18 hour culture supernatants of P. aeruginosa grown in the absence (WT; 1) or presence of 380 µM of compound 1 (compound 1; 3) was determined using elastin Congo Red as a substrate. Elastase activity was measured as a change in OD₄₉₅ and can be observed as an increase in the red color of the sample. As a negative control elastase activity was determined for the culture supernatant of a P. aeruginosa T2S mutant (pilD; 2). As a control for inhibition of elastase activity by the compound, compound 1 was added directly to culture supernatants from P. aeruginosa grown in the absence of compound and assayed for elastolytic activity (WT and compound 1; 4). Pictures and the corresponding OD₄₉₅ of samples are shown. c, Twitching assays of P. aeruginosa in either the absence (−) or presence (+) of 380 µM of compound 1. Bacteria were stabbed into LB plates with 1.0% agar and incubated for 2 days at room temperature. Growth media was removed and the bacteria were stained with crystal violet for better visualization.