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. 2016 Mar 16;113(13):3633–3638. doi: 10.1073/pnas.1524025113

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Fig. S5. — Effect of mutations of residues found in the FliH–FliI interface. (A and B) Effect of a triple mutation of the positively charged residues in the N-terminal domain of FliI on FliH–FliI interaction. (A) Swarming motility of MKM30 (ΔfliI) cells harboring pTrc99A (V), pTrc99A/His-FliI (FliI_WT), and pTrc99A/His-FliI(R26A/R27A/R33A) (R26A/R27A/R33A). (B) Interaction of FliH with His-FliI(R26A/R27A/R33A) examined by pull-down assay using Ni-NTA affinity chromatography. The eluted fractions were analyzed by CBB (Coomassie Brilliant Blue) staining. (C–F) Effect of mutations of the residues involved in the interaction between FliH and the N1 helix of FliI. (C) Interactions of FliH with His-FliI mutant variants. (D) Interactions of His-FliI with FliH mutant variants. Interactions were examined by pull-down assay using Ni-NTA affinity chromatography, and the eluted fractions were analyzed by CBB staining. (E) Motility of fliI mutant cells. Swarming motility of MKM30 (ΔfliI) cells transformed with pET19b-based plasmids: V, pET19b; FliI_WT, pMM1701; R4A, pYU21; R7A, pYU22; R4A/R7A, pYU121; W8A, pYU23; L12A, pYU24; F15A, pYU25; E16A, pYU26. (F) Motility of FliH mutant cells. Swarming motility of MKM11 (ΔfliH) cells transformed with pET22b-based plasmids: V, pET22b; FliH_WT, pMM307; D117A, pYU102; D120A, pYU104; D117A/D120A, pYU106; I123A, pYU114; R126A, pYU115; L127A, pYU116.