Fig. 4: Functional analysis of FlhBL.
a Motility in soft agar of E. coli W ΔfliOPQR complemented with plasmids expressing fliOPQR with the indicated mutations in FliQ (left) and E. coli W ΔflhB complemented with plasmids expressing FlhB with the indicated mutations (middle and right). b Immunodetection of SctUFLAG on western blottings of SDS-PAGE-separated crude membrane samples of the indicated S. typhimurium SctS pBpa mutants (denoted with X). Each sample is shown with and without UV-irradiation to induce photocrosslinking of pBpa to neighbouring interaction partners. Crosslinks between SctSpBpa and SctUFLAG are indicated. Crosslinking analysis was performed in the wild type and in an ATP hydrolysis-deficient SctNK165E mutant that is incapable of type III secretion. c Immunodetection of SctUFLAG on western blottings of 2D blue native/SDS-PAGE-separated crude membrane samples of the indicated S. typhimurium SctS pBpa mutant. The sample is shown with and without UV-irradiation to induce photocrosslinking of pBpa to neighbouring interaction partners. Crosslinks between SctSpBpa and SctUFLAG in the SctRSTU assembly intermediate and in the assembled needle complex (NC) are indicated. d Immunodetection of the early T3SS substrate SctP on western blottings of SDS-PAGE-separated culture supernatants of the indicated S. typhimurium SctS pBpa mutants. e Structure of FliPQR–FlhB highlighting mutation sites that impaired motility or secretion in red and mutation sites that had no or only a small effect in yellow.
