Extended Data Fig. 5: Comparative analysis of filament characteristics and protein secretion in wild-type and FliD mutant strains.
a, Quantification of the number of flagella per cell in the wild-type and FliD mutants using the pulsed flhDC induction setup. The number of flagella per cell (ℕ0) was determined for n > 100 individual bacteria per strain for one biological replicate. Violin plots represent the distribution of the data including the median (dashed line) and quartiles (dotted lines). b, Quantification of the filament length in µm in the wild-type and FliD mutants using the pulsed flhDC induction setup. The filament length was determined for n > 100 individual filaments per strain for one biological replicate. All data points shown, black lines indicate the means. Ser, single substitution to serine; Ser Ser, double substitution to serine; Arg, single substitution to arginine; Ser Arg, double substitution to serine and arginine. c,d, Immunoblotting of the cellular and secreted fractions using the pulsed flhDC induction setup of wild-type, the FliD mutants (V9R and F440R), a ΔflgKL strain and a secretion-deficient ΔfliP mutant using c, anti-FliC or d, anti-FliD antibodies. Relative secreted FliC or FliD levels report mean ± standard deviation, n = 3. DnaK serves as a loading and lysis control and was used to normalize the protein levels. e, Pulsed flhDC induction setup used to analyse filament number and filament length with fluorescence microscopy, and perform secretion, leakage and shearing assays. For secretion assays, the first Pellet and Supernatant samples of the Leakage assay were directly used for TCA precipitation and SDS-PAGE analysis, without the additional steps.
