Fig. 1: Effects of the mutations in flaJ or degQ genes on flagellins, flagella, and motility.

a Effect of FlaJ on swimming motility on the soft-agar plate. Five microliters of the freshly grown cultures (at an OD of 1.0) of V. vulnificus strains of wild type and ΔflaJ carrying a broad-host-range vector (pRK415) or a pRK415 harboring the intact flaJ gene (pRK-flaJ) were spot-inoculated on a soft-agar plate made of LBS containing 0.3% agar, tetracycline (3 μg/ml), and IPTG (1.0 mM), and incubated at 30 °C for 6 h. As a control for the inoculum size of each strain, the same volumes of the bacterial suspensions were spotted on the LBS-1.5% agar plate supplemented with tetracycline (3 μg/ml) and IPTG (1.0 mM). b Effect of flaJ deletion on the cellular levels of flagellins. Cell lysates were prepared from the wild type carrying pRK415, ΔflaJ carrying a pRK415, and ΔflaJ carrying a pRK415- flaJ. Ten micrograms of the crude cell extracts were subjected to SDS-PAGE and subsequently western blot analysis using the polyclonal antibodies reacting with the flagellins produced by V. vulnificus, as previously described¹². An arrow indicated the V. vulnificus flagellin subunits. c Effect of DegQ on swimming motility on the soft-agar plate. V. vulnificus strains of wild type carrying pRK415 and ΔdegQ carrying pRK415 or pRK-degQ were spot-inoculated on the LBS plates containing agar at a concentration of 0.3 or 1.5%, as described in (a). d Effect of degQ deletion on the cellular levels of flagellins. Ten micrograms of cell extracts of wild type carrying pRK415, ΔdegQ carrying pRK415, and ΔdegQ carrying pRK415-degQ were subjected to SDS-PAGE and western blot analysis, as described in (b). e, f Effects of flaJ/degQ-double mutations on motility and flagellin levels. V. vulnificus strains of wild type, ΔflaJ, ΔdegQ, and ΔflaJ/ΔdegQ were spot-inoculated on a soft-agar plate and incubated at 30 °C for 8.5 h (e), as described in (a). Ten micrograms of cell extracts of the four strains were subjected to SDS-PAGE and western blot analysis (f), as described in (b). g, h Effects of FlaJ and DegQ on flagellation. Individual cells of V. vulnificus strains of wild type, ΔflaJ, and ΔdegQ were observed under TEM to examine the polar flagellum (g), as described in “Methods”. Lengths of flagella on the TEM images were measured from 121 cells of wild type and 115 cells of ΔdegQ (h). The distribution of flagellar lengths was plotted with the medians (marked with a horizontal line) and their standard deviations (marked with a vertical line) of each strain and the P-value derived from comparison of two strains (Student’s t-test).