Subcellular Fractionation of P. aeruginosa

For subcellular fractionation, a modified version of the protocol described by Wang et al.⁴³ was used. P. aeruginosa strains were grown in LB medium overnight. The cultures were inoculated into fresh LB with the OD₆₀₀ adjusted to 0.05 and subcultivated at 37 °C with shaking to an OD₆₀₀ = 1.5. 10 ml of each strain were harvested by centrifugation at 4500 × g for 10 min. The cell pellets were resuspended in 500 µl sucrose-EDTA solution (2.5 mM EDTA and 20% (w/v) sucrose in PBS, pH 7.3) and incubated at room temperature for 20 min. In total, 500 µl ice-cold H₂O were added and the samples were incubated for 5 min at 4 °C with gentle shaking (550 rpm). After centrifugation for 20 min at 4 °C and 7000 × g, the supernatant containing all periplasmic proteins was removed and filtered through a syringe filter with 0.2 µm pore size. To obtain the cytosolic and membrane-bound proteins, the pellets were resuspended in 375 µl H₂O and 125 µl 4x Laemmli solution with 10% β-mercaptoethanol and then incubated for 10 min at 95 °C.
To obtain the periplasmic proteins, 250 µl of 14.3% aqueous trichloroacetic acid solution (w/v in H₂O) were added to 1 ml of the supernatants containing the periplasmic proteins and incubated on ice for 30 min. After centrifugation for 5 min at 4 °C and 14,000 × g, the supernatant was discarded. The pellets were washed twice by adding 400 µl acetone, centrifuging at 14,000 × g and 4 °C for 5 min and discarding the acetone. The pellets were dried at 95 °C for 1 min, then resuspended in 36 µl H₂O. 12 µl 4x Laemmli buffer with 10% β-mercaptoethanol were added and the samples incubated for 10 min at 95 °C prior to loading on an SDS polyacrylamide gel.