Interbacterial Antagonism via T6SS

P. aeruginosa strains used in this study were derived from the sequenced strain PAO1³³ . All deletions were in-frame and unmarked, and were generated by allelic exchange. E. coli growth curves were conducted using BL21 pLysS cells harboring expression plasmids for tse and tsi genes. Intercellular self-intoxication and interbacterial competition assays were performed by spotting mixed overnight cultures on a nitrocellulose membrane placed on a 3% agar growth medium. Samples were incubated at 37°C (P. aeruginosa-P. aeruginosa) or 30°C (P. aeruginosa-P. putida) for 12 or 24 hours. Tse1-catalyzed P. aeruginosa lysis was measured by placing cells in a minimal buffer ± 1.5 mM EDTA containing either Tse1, Tse1* or lysozyme. The change in optical density at 600 nm following 5 min of incubation was used to calculate lysis. For determination of Tse1 and Tse3 activity, isolated E. coli peptidoglycan sacculi were incubated with the purified enzymes (100 μg/mL). The resulting peptidoglycan and soluble fragments released by the enzymes were separated by HPLC and their identities were determined using MS as described previously³⁴ .