Preparation of Pseudomonas aeruginosa PAO1 Lysates

The strain of P. aeruginosa PAO1, subline Lausanne (PAO1-L), was used throughout this study. PAO1 is a serogroup O2/O5, type b flagellated strain [33 (link),34 (link)] exhibiting moderate virulence [35 (link)]. The genome of PAO1-L has been fully sequenced and found closest to Holloway’s original isolate (data not shown) [36 (link),37 (link)]. Mid-log phase cultures of PAO1-L (3 hour, h) in Luria-Bertani (LB) broth were washed with PBS without Ca²⁺ or Mg²⁺ (Sigma-Aldrich), resuspended in RPMI-1640 without phenol red (Sigma-Aldrich) such that cultures were 20x concentrated, and lysed using a French Press. Lysates were centrifuged to remove debris, filtered through a 0.2 μm filter, aliquoted, snap frozen on dry ice, and stored at -80°C until used. Total protein content in lysates was quantified using the bicinchoninic acid assay (Thermo Scientific).