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Obstruction of pilus retraction stimulates bacterial surface sensing

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Science  27 Oct 2017:
Vol. 358, Issue 6362, pp. 535-538
DOI: 10.1126/science.aan5706
  • Fig. 1 In C. crescentus, Tad pili are required for surface stimulation of holdfast synthesis.

    (A to D) Histogram plots showing the time of holdfast synthesis in single cells after surface contact, for two independent replicates of wild type (n = 115) (A), ΔmotB (n = 182) (B), ΔpilA (n = 8) (C), and ΔpilA ΔmotB (n = 33) (D). The total number of cells tracked, including cells arriving with holdfast already synthesized, was as follows: wild type, n = 241; ΔmotB, n = 566; ΔpilA, n = 93; and ΔpilA ΔmotB, n = 84.

  • Fig. 2 Tad pili undergo dynamic cycles of extension and retraction.

    (A) Time-lapse of synchronized PilAT36C swarmer cells extending and retracting pili after labeling with AF488-mal dye. The white arrow indicates the most prominent extension and retraction event for a single cell, although all cells shown extended and retracted pili. Scale bar, 2 μm. (B) Slices from tomograms and corresponding three-dimensional (3D) segmentations of wild type, PilAT36C, PilAT36 labeled with AF594-mal, and PilAT36C blocked with PEG5000-mal and labeled with AF594-mal. In 3D segmentation volumes, flagella are pink, pili are blue, the S-layer is gold, the outer membrane is yellow, and the inner membrane is red. Scale bars, 200 nm. (C) Measurements of the force of retraction of Tad pili in flagellar motor mutant (ΔmotB) strains, assessed by micropillars assay. Flagellar motor mutants exhibiting paralyzed flagella were used to ensure that all measurements were dependent solely on pilus activity. The mean (widest bar) ± SD (error bars) from 30 cells is indicated for each data set.

  • Fig. 3 Tad pilus retraction internalizes labeled pilins into a recyclable pool of subunits.

    (A) Representative images of wild-type or PilAT36C cells labeled with AF488-mal, BODIPY-mal, or AF488-mal after OM permeabilization with 20 mM EDTA (ethylenediaminetetraacetic acid). (B) Representative images of PilAT36C cells labeled with AF488-mal with or without PEG5000-mal. (C) Quantification of fluorescent cell bodies in populations of cells from images shown in (A) and (B). A minimum of 398 cells from each of three independent biological replicates were quantified. Means and SD (error bars) are shown.

  • Fig. 4 Resistance to Tad pilus retraction triggers surface stimulation of holdfast synthesis.

    (A) Representative TIRF images of (left) a cell with unperturbed pili (labeled with AF488-mal), exhibiting dynamic pilus activity, and (right) a cell for which pilus retraction is blocked (labeled with both AF488-mal and PEG5000-mal), exhibiting no dynamic pilus activity. In the top images, cell bodies are shown in gray with green fluorescent pili. The bottom images show green fluorescent pili surrounded by a pink MicrobeJ overlay used to measure changes in the fluorescence area of pili (square micrometers) over the time shown in (B). Scale bars, 2 μm. (B) Changes in the fluorescence area occupied by pili over time for cells shown in (A) (red, blue, and green are fluorescent area traces for individual cells). (C) Plot showing the correlation between the time of holdfast synthesis after surface contact and the time of cessation of dynamic pilus activity after surface contact for 19 cells. r, Pearson’s correlation coefficient. (D) Relative attachment assay showing the binding efficiency of strains lacking holdfast (HF–) and PilAT36C strains compared with that of the wild-type strain after 30 min binding, with or without PEG5000-mal. Data are representative of binding from three independent cultures, normalized to wild-type binding levels. Means and SD (error bars) are shown. PilAT36C plus PEG5000-mal is significantly different from all other treatments (P < 0.03; unpaired, two-tailed t test). (E) Representative TIRF microscopy images of cells with unperturbed (top) and blocked (bottom) pili upon surface contact (time = 0 s) in the presence of AF594-WGA. The cell body is gray, labeled pili are green, and holdfast is red (shown in the upper right inset in each image). White arrowheads indicate the first appearance of holdfast for the cell depicted. Scale bars, 2 μm. (F) Quantification of cells after labeling with AF488-mal (unperturbed); AF488-mal plus PEG5000-mal (blocked); or AF488-mal plus PEG5000 (unperturbed + PEG). A minimum of 30 cells from each of three independent biological replicates were quantified. Means and SD (error bars) are shown.

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.

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