Extended Data Fig. 2: Detergent or EDTA treatment causes dissolution of the outer membrane.

a, Description of the outer-membrane release assay used to measure the effect of detergent and EDTA on the outer membrane. See Methods section ‘Outer membrane release assay’ for a more detailed description. b, Absorbance of 410-nm light (left axis) in the supernatant from detergent-treated cell suspensions after performing the LAL LPS detection assay. Absorbance is correlated with the amount of LPS in the sample, and is linear below 1,000 EU ml⁻¹ (right axis). This experiment was performed once. c, Immunoblot with an antibody cross-reactive with several outer-membrane proteins (for example, LamB and OmpA) of the supernatant of cells treated with various concentrations of N-lauroyl sarcosine (detergent). This experiment was performed once. d, Absorbance of 410-nm light (left axis) in the supernatant from EDTA-treated cell suspensions after performing the LAL LPS detection assay. Absorbance is correlated with the amount of LPS in the sample, and is linear below 1,000 EU ml⁻¹ (right axis). This experiment was performed once. e, Montage of a representative plasmolysed cell expressing cytosolic GFP lysing upon detergent treatment. Cell expansion corresponds to the time of GFP disappearance. Data are mean ± s.d. from 32 cells in one experiment. f, Duration of swelling (left), of release of cytosolic GFP (centre), and of release of ribosomes upon lysis of plasmolysed cells (n = 22, 32, 12 cells, respectively; one experiment each). Error bars, ±1 s.d. The duration of swelling is approximately equal to the duration of the release of cytoplasmic contents. g, Ultra performance liquid chromatography of peptidoglycan composition of cell walls purified from untreated (red), sorbitol-treated (yellow), detergent-treated (green), and EDTA-treated (blue) cells. Mean glycan strain lengths (i) and percentage of various peptidoglycan subunit eluents (ii) are the same across all treatments. Dap–Dap, Dimers formed with double diaminopimelic acid bonds as opposed to diaminopimelic acid–d-alanine bonds; lipoprotein, subunits bound to Lpp. n = 4, 2, 2 and 1 sacculi preparations for untreated, sorbitol-treated, detergent-treated and EDTA-treated cells, respectively.