Fig. 6: Tme effectors disrupt membrane integrity upon delivery to the periplasm.
From: A comparative genomics methodology reveals a widespread family of membrane-disrupting T6SS effectors
a, b Toxicity of Tme in bacteria. Growth of E. coli BL21 (DE3) containing arabinose-inducible vectors for the expression of cytoplasmic (cyto) or periplasmic (peri) versions of Tme1 (a) and Tme2 (b). c, d Rescue of Tme-mediated toxicity by cognate Tmi. Growth of E. coli BL21 (DE3) containing an arabinose-inducible vector for the expression of periplasmic Tme1 (c) or Tme2 (d) together with an arabinose-inducible vector for the expression of Tmi1 or Tmi2. Empty expression vectors were used as the control. In a–d, data represent the mean ± S.D. (n = 4). Arrows denote the time at which L-arabinose was added. e Tme effectors dissipate membrane potential. E. coli BL21 (DE3) expressing periplasmic Tme1, Tme2, or the V. cholerae pore-forming effector VasX (used as a positive control) from arabinose-inducible vectors, were analyzed using flow cytometry following staining using the BacLight Membrane Potential Kit. The red/green fluorescence ratio of the dye DiOC2(3) was calculated for each condition. CCCP was used as a positive control. Data shown in the left panel represent the mean ± S.D. of four independent experiments. Asterisks denote statistical significance compared to pEmpty samples by one-way repeated measures ANOVA with the Dunnett test (*P < 0.01; **P < 0.001; ***P < 0.0005). Data shown in the right panel represent the distribution of red/green ratios for cells analyzed in one of the experiments shown on the left. f Tme effectors increase membrane permeability. E. coli cultures, like in (e), were stained with the membrane-impermeable, intercalating DNA dye propidium iodide (PI) and analyzed using flow cytometry. Pre-treatment with ethanol (EtOH) was used as a positive control. Data shown in the left panel represent the geometric mean fluorescence intensity (MFI) ± S.D. of three repeats from a representative experiment. Asterisks denote statistical significance compared to pEmpty samples by one-way repeated measures ANOVA with the Dunnett test (**P < 0.001; ***P < 0.0005). Data shown in the right panel represent the distribution of PI fluorescence intensities for cells analyzed in one repeat of the experiment shown on the left. Source data are provided as a source data file.
