Assembly of wild type and mutant variants of Enterohemorrhagic type IVa pili

To analyse pilus assembly, bacteria of E. coli strain BW25113 F’lacI^Q were transformed with plasmid pMS41 encoding the EHEC pilus assembly genes and pCHAP8565 or its mutant derivatives encoding PpdD variants (Luna Rico et al. 2019). Bacteria were cultured for 48-72 hours on M9 minimal agar plates (Miller 1972) containing carbenicillin (100 mg.mL-1) and chloramphenicol (25 mg.mL-1). Bacteria were harvested and resuspended in LB medium at OD_600nm of 1. The suspension was vortexed vigorously for 1 min to detach pili from bacteria. Bacteria were pelleted by 5-min centrifugation at 16000 g at 4°C and resuspended in SDS sample buffer at a concentration of 10 OD_600nm per 1 mL. The supernatant containing pili was subjected to another round of centrifugation for 10 min. Pili were precipitated with 10% tri-chloroacetic acid on ice for 30 min and pelleted by a 30-min centrifugation at 16000g at 4°C. Pili pellets were washed twice with ice-cold acetone, air-dried and resuspended in SDS-sample buffer at a concentration equivalent to that of the bacteria. The equivalent volumes (5 ul) of each fraction were analysed by denaturing SDS polyacrylamide gel electrophoresis (SDS-PAGE) on 10% polyacrylamide Tris-Tricine gels (Schägger and Von Jagow 1987). Proteins were transferred on nitrocellulose by Western blot and probed with anti-PpdD antibodies and secondary anti-rabbit antibodies coupled to HRP as described (Luna Rico et al. 2019). The fluorescent signal was recorded with a Typhoon FLA-9000 scanner (GE).