Cultivation and Zinc Treatment of P. aeruginosa

PAO1 was purchased from American Type Culture Collection (#15692, ATCC; Manassas, VA, USA). P. aeruginosa containing a triple hemagglutinin tag on its czcR N-terminus (CzcR-HA PAO1) was kindly provided by Dr. Karl Perron (University of Geneva; Geneva, Switzerland) [41 (link)]. P. aeruginosa was cultured in LB (Alfa Aesar, #3339936; Haverhill, MA, USA) overnight at 200 rpm and 37 °C, then diluted to 5 × 10⁷ CFU/mL in LB (as a control) or LB containing zinc (800 µM, as ZnSO₄, except for growth curve as indicated), and incubated for 16 h at 37 °C (shaking at 200 rpm). Zinc concentration was confirmed using Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES; Agilent 5110; Agilent Technologies, Santa Clara, CA, USA) [66 (link)].