Report

Mechanical crack propagation drives millisecond daughter cell separation in Staphylococcus aureus

See allHide authors and affiliations

Science  01 May 2015:
Vol. 348, Issue 6234, pp. 574-578
DOI: 10.1126/science.aaa1511
  • Fig. 1 Daughter cell separation in S. aureus occurs within milliseconds with characteristics of mechanical crack propagation.

    (A) A schematic diagram of the cell wall before daughter cell separation. (B) Snapshots of S. aureus strain Newman “popping” (inset) and histogram of daughter cell separation duration captured by means of phase contrast microscopy at 1000 frames/s (n = 16 popping events). (C) Distribution of cumulative counts of popping events plotted over the 2-min oscillatory period for 200 mM osmotic shocks. The red line denotes the concentration of sorbitol in the medium, and the dashed line denotes average popping counts, assuming a uniform distribution (n = 400 popping events). (D) 3D SIM images of fixed Newman cells labeled with fluorescent WGA (WGA-488, green), which marks the outer wall and followed by 0 or 10 min of growth in the absence of WGA. Cell surfaces and septa were stained with an amine reactive dye (NHS-568, red). (E) Time-lapse epifluorescence images of Newman cells labeled with WGA (green) before (0 min) and after (3 min) popping. Corresponding phase-contrast (gray), membrane staining with FM 4-64 (red), and overlay of WGA and FM signals are also displayed. Two types of old wall geometry after popping were observed: hinged (left, ~80%) and nonhinged (right, ~20%). (F) Correlative light and SEM on Newman cells labeled with WGA followed by 10-min chase showing the two types of WGA labeling patterns as in (E). Scale bars, 1 μm.

  • Fig. 2 Cell volume increases continuously throughout the cell cycle.

    (A) Time-lapse images of S. aureus cells stained with FM 4-64 (left) and outlined by fitting with ellipses (right). (B) Average aspect ratio of S. aureus cells throughout the cell cycle (from immediately after previous popping to ready-to-pop) and overlay of the cell outlines (inset) from a typical cell at different points of the cell cycle colored from blue (early) to red (late). Error bars denote standard errors (n = 27 cells). Red bars on top indicate the time fraction into the cell cycle when septation starts (left, 0.35 ± 0.03 SEM) and completes (right, 0.77 ± 0.02 SEM), respectively (n = 26 cells). (C) Representative traces of cell volume as a function of time following a microcolony starting from a single cell; solid blue traces indicate cell volumes of individual cells before popping, and the dashed black line denotes the total cell volume of all the cells present at a given time. Cell volume and surface area were estimated from the 2D cell outlines by fitting to ellipses and assuming prolate cell shapes (that each cell was rotationally symmetric around the long axis). (D and E) Distribution of relative changes in (D) volume and (E) surface area during popping, after correcting for baseline growth rate. The black solid line represents kernel density estimate of the distribution, and the red dashed line denotes the average (2 ± 10% SD for volume, –11 ± 6.5% SD for surface area; n = 69 division events). (F) (Top) 3D SIM images and corresponding extracted data (fig. S5); (bottom) fraction of old surface before (0.71 ± 0.01 SD; n = 15 cells) and after (0.73 ± 0.03 SD; n = 36 cells) popping. Cells were modeled as ellipsoids, and the contribution of the old, WGA-labeled wall to the daughter cells’ total surface area was measured by fitting a plane to the old/new boundary (fig. S5A). Scale bars, 1 μm.

  • Fig. 3 High stress in the peripheral ring prepares the cell for popping.

    (A) von Mises stress distribution in the “ready-to-pop” S. aureus cell wall (fig. S6, state 3) modeled as a linear elastic material (details of model construction are provided in the supplementary materials). Color represents the relative magnitude of stress. The stress at the peripheral ring (red arrow), where the cell wall splits open during popping, is higher than elsewhere in the outer wall. (B) Enlarged views of a cut-through slice of the cell in (A) shows high von Mises stress at the peripheral ring (red arrow) as well as the stress distribution in the circumferential and axial directions, respectively.

  • Fig. 4 Correlative SEM reveals cell cycle–dependent early signs of mechanical fracture.

    (A to D) Representative correlative fluorescence (top) and SEM images (bottom) of S. aureus Newman cells stained with FM 4-64, showing the cell surface features at different stages of the cell cycle. Ninety-eight percent (n = 54 cells) of the cells with visible holes [blind analysis (supplementary materials, materials and methods)] had completed septa, whereas only 49% (n = 108 cells) of the cells with completed septa had holes.

Supplementary Materials

  • Mechanical crack propagation drives millisecond daughter cell separation in Staphylococcus aureus

    Xiaoxue Zhou, David K. Halladin, Enrique R. Rojas, Elena F. Koslover, Timothy K. Lee, Kerwyn Casey Huang, Julie A. Theriot

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

    Download Supplement
    • Materials and Methods
    • Supplementary Text
    • Figs. S1 to S14
    • Table S1
    • Full Reference List

    Images, Video, and Other Other Media

    Movie S1
    Ultrafast popping of S. aureus strain Newman. Representative time-lapse movies of S. aureus strain Newman "popping" captured by phase-contrast microscopy at 1 ms/frame.
    Movie S2
    Deflation of S. aureus Newman cells. Time-lapse movies of "ready-to-pop" S. aureus Newman cells treated with 5% sarkosyl as in Fig. S1A.
    Movie S3
    3D Visualization of the old-wall labeling pattern after popping. Volume view of the 3D SIM images of fixed Newman cells pulse labeled with WGA-488 and followed by 0 or 10 min chase growth in the absence of the dye (Fig. 1D).
    Movie S4
    Illustration of the 2D cell outline tracking. Time-lapse movie of S. aureus cells stained with FM 4-64 (left) and outlined by fitting with ellipses (right) as in Fig. 2A. Scale bar: 1 μm.
    Movie S5
    Tracking cell separation with cytoplasmic GFP in 3D. Time-lapse movie of S. aureus cells expressing cytoplasmic GFP acquired with 3D deconvolution microscopy (only one z slice was shown) and the corresponding z stacks before and after a popping event. Scale bar: 1 μm.
    Movie S6
    Illustration of cell growth during septum construction. Time-lapse movies of intermediate models of the cell and septum growth, interpolating between state 1 and state 2 (Fig. S6). The von Mises stress at the peripheral ring connecting the two daughter cells (arrow) becomes higher than elsewhere in the outer wall as the cell grows. In addition, the aspect ratio increases as the cell and septum grow.
    Movie S7
    Daughter cells do not fully round up during popping. Time-lapse movie of daughter cell separation captured at 3 min/frame (same cell as in Fig. 1E left). Cells were labeled with WGA-488 before mounted and the cell membrane was stained with FM 4-64.

Stay Connected to Science

Navigate This Article