Fig. 5: flgI mutation analysis by motility and secretion assay.

a Motility assay in 0.35% soft agar plate of the flgI(K63A), flgI(K63D), flgI(K95A), flgI(K95D), flgI(K63A/K95A), flgI(K63A/K95D), flgI(K63D/K95A) and flgI(K63D/K95D) mutants. N.M. indicates non-motile. The letter V and WT refers to the empty expression vector (pET22b) and wild type flgI expression vector (pTY03) (Supplementary Table 1) for the negative and positive controls, respectively. The diameter of the motility ring of five colonies of each strain was measured after incubating 8.5 h under 30 °C. The average diameter of the motility rings of the wild-type strain WT was set to 1.0, and then relative diameters of the motility rings of mutant cells were normalized to WT (mean ± SD, n = 5). The relative diameter of V was 0.16 ± 0.03. Comparisons between datasets were performed using a two-tailed Student’s t test. A P value of <0.05 was considered to be statistically significant difference. *P < 0.05; ***P < 0.001; ND no statistical difference. b The cellular expression levels of FlgE, FliC, FlgH and FlgI (left) and the secretion levels of FlgE and FliC (right) by immunoblotting with polyclonal anti-FlgE, FliC, FlgH and FlgI antibodies. The positions of molecular mass markers (kDa) are shown on the left. The original immunoblots are shown in Supplementary Fig. 11 and then the contrast and brightness were adjusted using a software, Photoshop (Adobe). c Relative secretion levels of FlgE and FliC were quantified and presented in the bar diagrams (mean ± SD, n = 5). The average secretion level of the wild-type strain WT was set to 1.0. Comparisons between datasets were performed using a two-tailed Student’s t test. A P value of <0.05 was considered to be statistically significant difference. **P < 0.01; ***P < 0.001; ND no statistical difference.