(A) SEC chromatogram of ColE1-TR (red line) and TolC (purple line). The arrow indicates the co-elution (black line) fractions that were analyzed by SDS-PAGE. On the SDS-PAGE gels (right), red arrow indicates the presence of colicin E1 construct that has co-eluted with TolC (purple arrow). (B) Extracellular protease digestion assay with two colicin E1 truncation constructs incubated with Escherichia coli, each construct is labeled with a C-terminal His-Tag. Periplasmic SurA was used as a membrane integrity control. After incubation with colicin fragments, cells were left intact or lysed with lysozyme. Proteins degraded by trypsin in intact cells are not protected by the outer membrane. (C) Fluorescence image (right) of ColE1-TR-Cy3 overlaid on outlines of living E. coli cells from phase-contrast microscopy (left) for WT and ΔtolC. Red arrow points to a punctum. A larger variety of images is also available (Figure 4—figure supplement 1). Similar localization was seen for ColE1-TR-GFP (Figure 4—figure supplement 2) demonstrating that the effect was not caused by the fluorophore, and ColE1-TRΔ1–40-Cy3 (Figure 4—source data 1, Figure 4—figure supplement 3) demonstrating that the effect is not TolA dependent. The puncta were stable and were found to remain intact for more than 5 min. The punctum shown here remained intact for at least 30 s (Figure 4—video 1). (D) Cell counts where ColE1-TR-Cy3 punctum formation was observed for WT, ΔtolC, and ΔbtuB. Number of cells observed, n=111, 91, 105, respectively. SEC, size exclusion chromatography.