Supplementary Materials

Obstruction of pilus retraction stimulates bacterial surface sensing

Courtney K. Ellison, Jingbo Kan, Rebecca S. Dillard, David T. Kysela, Adrien Ducret, Cecile Berne, Cheri M. Hampton, Zunlong Ke, Elizabeth R. Wright, Nicolas Biais, Ankur B. Dalia, Yves V. Brun

Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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  • Materials and Methods
  • Figs. S1 to S12
  • Table S1
  • Caption for Table S2
  • Caption for Movies S1 to S10
  • References
Table S2

Images, Video, and Other Media

Movie S1
Sequential slices through the cryo-ET reconstruction and segmentation of a PilAT36C cell labeled with AF594-mal and PEG5000-mal shown in Figure 2B, panel 4. In the segmented volume, flagellum is pink, pili are blue, s-layer is gold, outer-membrane is yellow, and inner membrane is red. Scale bar is 200 nm.
Movie S2
Time-lapse of labeled, synchronized PilAT36C cells shown in Fig. 2A extending and retracting pili after labeling with AF488-mal dye. Capture rate is 3 sec/frame. Scale bar is 2 μm.
Movie S3
Time-lapse of cells in micropillars assay with representative pillar movement analyzed for force measurements for the non-motile ΔmotB strain. The microscope is focused on the top of the micropillars that are dark circles. The cells are on another focal plane and appear white and slightly blurry. Binding of two adjacent pillars by a cell’s pili and pilus retraction with sufficient force causes micropillar bending, as depicted in Fig. S7A. The deflection of the pillars is used to calculate the force of pili retraction as the micropillars are independently calibrated.
Movie S4
Time-lapse of cells in micropillars assay with representative pillar movement analyzed for force measurements for the non-motile ΔmotB PilAT36C strain labeled with AF488-mal. The microscope is focused on the top of the micropillars that are dark circles. The cells are on another focal plane and appear white and slightly blurry. Binding of two adjacent pillars by a cell’s pili and pili retraction with sufficient force causes micropillar bending, as depicted in Fig. S7A. The deflection of the pillars is used to calculate the force of pili retraction as the micropillars are independently calibrated.
Movie S5
Time-lapse of cells in micropillars assay with representative pillar movement analyzed for force measurements for the non-motile ΔmotB PilAT36C strain labeled with AF488-mal. The microscope is focused on the top of the micropillars that are dark circles. The cells are on another focal plane and appear white and slightly blurry. Binding of two adjacent pillars by a cell’s pili and pili retraction with sufficient force causes micropillar bending, as depicted in Fig. S7A. The deflection of the pillars is used to calculate the force of pili retraction as the micropillars are independently calibrated
Movie S6
Time-lapse of cells in micropillars assay with representative pillar movement analyzed for force measurements for the negative control non-motile non-piliated ΔmotB ΔpilA strain. The microscope is focused on the top of the micropillars that are dark circles. The cells are on another focal plane and appear white and slightly blurry. Binding of two adjacent pillars by a cell’s pili and pili retraction with sufficient force causes micropillar bending, as depicted in Fig. S7A. The deflection of the pillars is used to calculate the force of pili retraction as the micropillars are independently calibrated.
Movie S7
Time-lapse of cells in micropillars assay with representative pillar movement analyzed for force measurements for the negative control non-motile non-piliated ΔmotB PilAT36C strain with the addition of PEG5000-mal. The microscope is focused on the top of the micropillars that are dark circles. The cells are on another focal plane and 24 appear white and slightly blurry. Binding of two adjacent pillars by a cell’s pili and pili retraction with sufficient force causes micropillar bending, as depicted in Fig. S7A. The deflection of the pillars is used to calculate the force of pili retraction as the micropillars are independently calibrated.
Movie S8
TIRF microscopy time-lapse of cell unperturbed for pilus retraction exhibiting dynamic pilus activity shown in Fig. 4A left panel. Cell body is gray and pili labeled with AF488-mal are shown in green. Capture rate is 30 sec/frame. Scale bar is 2 μm.
Movie S9
TIRF microscopy time-lapse of cells unperturbed for pilus retraction upon surface contact in the presence of AF594-WGA. The cell body is gray, labeled AF488- mal pili are green, and HF are red. Capture rate is 30 sec/frame. Scale bar is 2 μm.
Movie S10
TIRF microscopy time-lapse of cells blocked for pilus retraction upon surface contact in the presence of AF594-WGA. The cell body is gray, labeled AF488- mal pili are green, and HF are red. Capture rate is 30 sec/frame. Scale bar is 2 μm.