Figure 3
Effect of the FlhA(F459A) mutation on flagellar protein export. (a) Effect of the FlhA(F459A) mutation on motility. Motility of SJW1103 (WT), NH001 (∆flhA), MMA459 [flhA(F459A)], MMHI0117 (∆fliHI flhB*), NH004 (∆fliHI flhB* ∆flhA) and MMHI0117-1 [∆fliHI flhB* flhA(F459A)] in soft agar. Plates were incubated at 30 °C. (b) Effect of the FlhA(F459A) mutation on flagellar protein export in the presence (left panels) and absence (right panels) of FliH and FliI. Whole cell proteins (Cell) and culture supernatant fractions (Sup) were prepared from the above strains. An 8 μl solution of each protein sample, which was normalized to an optical density of OD600, was subjected to SDS-PAGE and was analyzed by CBB staining (first row) and immunoblotting with polyclonal anti-FlgD (second row), anti-FlgE (third row), anti-FliK (fourth row), anti-FlgK (fifth row) or anti-FlgL (sixth row) antibody. CBB-stained gels and immunoblots were cropped from original image data shown in Fig. S5 in the Supplemental information and were processed using Photoshop CS6. The positions of molecular mass markers (kDa) are indicated on the left. SipA, which is secreted by the SPI-1 virulence-associated type III secretion apparatus51, serves as a loading control.
