Modulation of flagellar rotation in surface-attached bacteria: A pathway for rapid surface-sensing after flagellar attachment
Fig 4
The carboxy terminus of FimV mediates interactions with FlhF and is required to stop rotation of tethered bacteria.
(A) FlhF interacts with the carboxy terminus of FimV. FlhF interactions with FimV and Vfr were assayed by bacterial two-hybrid. ω or Zif fusions were constructed as indicated, with interactions resulting in beta-galactosidase expression and activity (reported in Miller units). Bars show mean ± S.D. (n = 3) for a representative experiment. FlhF (FWT), which forms a homodimer, served as a positive control (black bar). FimV (VC) interacted with both wild-type (FWT) and R251G alleles (FR) of FlhF (red bars), but gave no signal when co-expressed with either the ω or Zif domain alone (white bars). Neither allele of FlhF interacted with a FimV construct lacking the final 150 aa (VCΔC, blue bars) nor with Vfr (green bars). (B) Surface-tethered bacteria expressing fimV(L7P) or fimV(ΔC) exhibit persistent rotation. Rotating bacteria identified 5 minutes after tethering on anti-FliC coated slides (t = 0) were observed for 45 min. The proportion of rotating cells (red), attached cells (black), and detached cells (white) was determined over 3–8 independent experiments. Survival curves of rotating fimV(L7P) and fimV(ΔC) differed significantly from PAK (p < 0.0001), but not from ΔflhF (p = 0.53 and p = 0.52, respectively; Mantel-Cox test). (C) fimV(L7P) and fimV(ΔC) assemble a unipolar flagellum. Cells were labelled with anti-FliC antibodies conjugated to Alexa Fluor 488 and visualized by phase and fluorescence microscopy.