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. 2011 May 10;9(5):e1000616. doi: 10.1371/journal.pbio.1000616

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Minamino et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Motility of SJW1103 cells (wild-type) transformed with pET19b (indicated as Low-V), pTrc99A (indicated as High-V), pGMK4000 (pET19b/His-FliG(ΔPAA), indicated as Low-FliG(ΔPAA)), and pGMM4500 (pTrc99A/His-FliG(ΔPAA), indicated as High-FliG(ΔPAA)) in semi-solid agar plates. (B) Expression levels of FliG and His-FliG(ΔPAA). Immunoblotting, using polyclonal anti-FliG antibody, of whole cell proteins. Lane 1, MKM1/pET19b (indicated as ΔfliG/V); lane 2, SJW1103/pET19b (indicated as WT/V); lane 3, SJW1103/pGMK4000 (indicated as WT/Low-His-FliG(ΔPAA)). Arrows indicate positions of FliG and His-FliG(ΔPAA). (C) Measurement of CCW and CW rotation of the flagellar motor by bead assays. We used SJW46 (fliC(Δ204–292)) as a host because it produces flagellar motors with the sticky flagellar filaments, which are easily labeled with polystyrene beads. CCW, counterclockwise rotation; CW, clockwise rotation. Upper panel: SJW46 carrying pET19b. Middle panel: SJW46 carrying pGMK4000. Bottom panel: SJW46 carrying pGMM4500. (D) Effect of FliG(ΔPAA) on motility of a ΔcheA-Z mutant. Upper panel: Motility of SJW3076 (ΔcheA-Z) transformed with pET19b, pGMK3000 (pET19b/His-FliG), or pGMK4000 in semi-solid agar. Middle panel: measurement of CCW and CW rotation of the flagellar motor of MM3076iC/pGMK4000. Bottom panel: measurement of CCW and CW rotation of the flagellar motor of MM3076iC/pGMM4500.