Sample preparation and the Phos-tag gel electrophoresis

For the detection of in vivo BfmR phosphorylation, P. aeruginosa MPAO1 and its derivatives were grown at 37°C for 24 hours on M8-glutamate minimal agar plate supplemented with 0.2% glucose. To prepare cell lysates for the Phos-tag (APExBIO, code no. F4003) gel assay, bacteria cells were scraped from the plate and immediately resuspended in 60 μl of lysis buffer [50 mM tris-Cl (pH 7.5), 150 mM NaCl, 1 mM MgCl₂, 0.1% Triton X-100, deoxyribonuclease I (DNase I) (15 μg/ml), 0.5 mM PMSF, and 1 mM DTT] with 0.1% (v/v) Lysonase. Sufficient lysis was achieved by repeated pipetting up and down for 10 s, followed by addition of 20 μl of 4× SDS loading buffer. Fifteen-microliter aliquots of the resulting cell lysates were immediately loaded onto a Phos-tag gel (10% acrylamide gels containing 25 μM acrylamide–Phos-tag ligand and 50 μM MnCl₂) (27).

For the detection of BfmR and BfmS phosphorylation in DK2 and AU15431 strains or in their derivatives, overnight cultures (LB) were washed twice and diluted 100-fold in fresh PB medium. The liquid cultures were grown in a 20-ml tube with a volume-to-medium ratio of 5:1, shaken at 250 rpm for 3 hours. After collection of the cells by centrifugation, the pellet was washed once with 1 ml of 1× PBS and suspended in 1 ml of lysis buffer [50 mM tris-HCl (pH 7.5), 50 mM NaCl, 1 mM DTT, and 5% Glycerinum] supplied with 1 μl of protease inhibitor cocktail (Thermo Fisher Scientific, catalog no. 78430). The mixture was homogenized by mechanical disruption (FastPrep FP2400 instrument, Qbiogene) and then centrifuging at 2300g for 5 min. The supernatant was collected and followed by addition of 5× SDS loading buffer, and 15-μl aliquots were loaded onto a Phos-tag gel as described above for electrophoresis.

After 3 hours of electrophoresis at 30 mA in tris-glycine-SDS running buffer [25 mM tris, 192 mM glycine, and 0.1% SDS (pH 8.4)] at 4°C, the Phos-tag gel was washed for 10 min at room temperature with transfer buffer [20% (v/v) methanol, 50 mM tris, and 40 mM glycine] supplied with 1 mM EDTA to remove Mn²⁺ from the gel, and then the gel was incubated at room temperature with gentle shaking for another 10 min in transfer buffer [20% (v/v) methanol, 50 mM tris, and 40 mM glycine] twice to remove EDTA.