Fig. 5: In vitro proteolysis of FlaB and its truncated polypeptides by DegQ.

a V. vulnificus flagellins show to have the domains of ND0, ND1, ND2, D3, CD2, CD1, and CD0, as shown in other bacterial flagellar subunits⁶⁵. Based upon the domain information on the FlaB (VVMO6_00808), the overexpression plasmids for seven different truncated FlaB polypeptides were constructed (Supplementary Table 1): ΔN0 (from L54 to G377; 37.5 kDa), ΔN1 (from K162 to G377; 30.6 kDa), ΔC0 (from M1 to S332; 37.8 kDa), ΔC1 (from M1 to A287; 34.5 kDa), ΔN0C0 (from L54 to S332; 35.4 kDa), ΔN1C0 (from K162 to S332; 28.5 kDa), and ΔN1C1 (from K162 to A287; 25.2 kDa). b Proteolysis of flagellin polypeptides by DegQ. Recombinant FlaB (1 μM) was incubated with various concentrations of rDegQ (0, 0.12, 0.3, 0.48, 0.6, 0.72, 0.9, 1.08, and 1.2 μM) as described in the “Methods”. Various derivatives of FlaB (1 μM each), of which ND0-, ND1-, CD0-, and/or CD1-domains were truncated (FlaB_ΔN0, FlaB_ΔN1, FlaB_ΔC0, FlaB_ΔC1, FlaB_ΔN0C0, FlaB_ΔN1C0, and FlaB_ΔN1C1), were incubated in the same condition for proteolysis reaction. The resultant reaction mixtures were resolved in SDS-PAGE (the left gels), and the relative intensities of flagellin bands were obtained by densitometric reading, which were then plotted against the given concentrations of rDegQ with the concentration resulting in 50% proteolysis (EC₅₀) (the right graphs). nd, not determined. c Protection of flagellin polypeptides from DegQ proteolysis by FlaJ. The proteolysis assays of rFlaB and its truncated forms were conducted in the reaction mixtures containing 1.08 μM rDegQ and various concentrations of rFlaJ (0, 0.56, 1.11, 1.67, 2.22, 3.34, 4.45, and 6.67 μM). Proteins in the reaction mixtures resolved in SDS-PAGE (the left gels) were subjected to quantitative determination using densitometry. The relative intensities of flagellin bands compared to the intensity of flagellins in the control (the second lane in each gel) were plotted against the given concentrations of rFlaJ. d EC₅₀ values for the DegQ proteolysis. The average values of EC₅₀ for proteolysis of each flagellin by rDegQ were obtained from three independent proteolysis assays. The error bars represent standard deviation. The P-values for comparison with the original FlaB were indicated (Student’s t-test; **P < 0.005; ns, not significant).