Extended Data Fig. 5: EDTA weakens the E. coli cell envelope.

a, Length of the cell wall versus time for 7 representative B. subtilis cell chains during treatment with detergent with treatment using detergent and 10 mM EDTA (n = 68 cell chains). Although detergent caused lysis, subsequent addition of EDTA did not affect cell-wall rest length. b, Lengths of the cell wall of B. subtilis cell chains versus time during 1-M oscillatory osmotic shocks, which caused cell lysis (for example, red arrows), with treatment using 10 mM EDTA (dashed arrow; n = 127 cell chains). EDTA did not affect the rest length of the cell walls. c, Top, length of the B. subtilis cell wall versus time through a hyperosmotic shock (3 M sorbitol, solid arrow) and subsequent treatment with 10 mM EDTA (dotted arrow) for four representative cell chains (n = 61 cell chains). Cell-wall length did not decrease after detergent, as it did for E. coli. Bottom, micrograph of a two-cell chain expressing cytosolic (strain HA405, left) and a kymograph showing fluorescence intensity along the dotted red line during the experiment in the top graph. The cell chain in the kymograph corresponds to the bottom-most length trace in the top graph. Red arrows demonstrate that the discrete increases in length observed after EDTA treatment correspond to cell-lysis events, when fluorescence within the cells begin to decrease. d, Population-averaged E. coli cell-wall length contraction upon EDTA application after plasmolysis increased with increasing concentration of EDTA (n = 131, 193, 225, 138, 81, 94, 36, 28, 72 cells, respectively). Error bars indicate ±1 s.d. e, Length of the cell wall versus time during hyperosmotic shock (3 M sorbitol, solid arrow) and subsequent treatment with 10 mM EDTA and 50 mM MgCl₂ (dotted arrow) for representative E. coli cells (n = 91 cells). f, Length of the cell walls of representative E. coli cells during 100-mM oscillatory shocks with 2-min period (n = 243 cells). g, Length of the cell walls of representative E. coli cells during a 100-mM oscillatory shock with 2-min period and 10 mM EDTA (n = 284 cells). h, Population-averaged elongation rate of the E. coli cell wall during 100-mM oscillatory shocks with 2-min period for untreated (black line) and 10 mM EDTA-treated cells (n = 284 cells). i, Effective population-averaged cell length (l_eff), calculated by integrating the population averaged elongation rate in h during 100-mM oscillatory shocks with 2-min period for untreated (black line) and 10 mM EDTA-treated cells (n = 284 cells). Dotted lines are the respective time-averaged l_eff using a rolling-window averaging filter with a 2-min window (equal to the period of oscillations). j, Deviation of the effective population-averaged length in i from the respective time-averaged trace. k, The mean amplitude of oscillation was found by averaging the peak-to-peak amplitude in j over cycles (n = 10 cycles). Error bars indicate ±1 s.d. The P value was calculated using a Student’s two-sided t-test. l, Amplitude of cell-wall length oscillations (ratio with respect to the untreated wild type; Fig. 2j) versus cell-wall stiffness calculated from plasmolysis–lysis experiments (ratio with respect to the untreated wild type; Fig. 2g). Solid line, linear best fit for only perturbations to the outer membrane (red circles; linear regression: R² = 0.71, F = 9.7, P = 0.0356). Dashed line, best fit when additionally considering perturbations to protein linkages between the outer membrane and cell wall (dashed circles; linear regression: R² = 0.25, F = 1.4, not significantly different from horizontal). For the O8-expressing strain, we conservatively used a stiffness ratio of 1.5 for the fits.