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

To analyse pilus assembly, bacteria of E. coli strain BW25113 F’lacIQ 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 OD600nm 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 OD600nm 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)