Figure 5. ColE9* K469C^AF647 translocation across the outer membrane is PMF‐dependent.

ColE9* denotes ColE9 H551A, an active site mutation that inactivates the toxin.

1. Widefield fluorescence microscopy images of E. coli JM83 cells labelled with ColE9* K469C^AF647 (1.5 µM) and Hoechst DNA stain (20 µM) for 30 min at 37°C with or without trypsin treatment with or without prior treatment with CCCP. Each panel shows the same cell in DIC (grey), Hoechst DNA stain (blue) and ColE9* K469C^AF647 fluorescence (red). Overlays of Hoechst and ColE9* K469C^AF647 fluorescence are also shown. ColE9* K469C^AF647 remains bound to the OM in the presence of CCCP but this signal is lost on treatment with trypsin. Trypsin treatment in the absence of CCCP yields some cell‐associated ColE9* K469C^AF647 fluorescence that likely represents internalised molecules (see text). Scale bar, 1 µm.
2. Normalised fluorescence intensity profiles across E. coli JM83 cell widths for CCCP and/or trypsin treated cells. Fluorescence was measured in ImageJ for 15 cells per condition and normalised to more clearly show distribution profiles for each condition. CCCP‐treated cells showed peripheral ColE9* K469C^AF647 fluorescence consistent with the colicin being bound at the OM. Trypsin treatment of cells in the absence of CCCP results in loss of this peripheral fluorescence but the presence of mid‐cell signal consistent with internalisation. Treatment with CCCP and trypsin removed all ColE9* K469C^AF647 fluorescence from cells, as in A. Error bars represent % SEM.
3. Box and whisker plots showing mean pixel intensities for ColE9* K469C^AF647 fluorescence per cell measured for the indicated cells and condition used, whiskers represent minimum and maximum mean pixel intensity, box shows 1^st and 3^rd quartile with the median shown as a line. From left to right: E. coli JM83 cells; E. coli btuB deletion strain showing loss of all ColE9* K469C^AF647 cell‐associated fluorescence; E. coli JM83 cells in the presence of CCCP; E. coli JM83 cells following trypsin treatment showing significant ColE9* K469C^AF647 fluorescence remains associated with cells indicative of import; E. coli JM83 cells treated with CCCP and trypsin showing the complete loss of internalised ColE9* K469C^AF647 fluorescence. n, number of cells used, typically from 2 to 4 biological replicates. **** indicates a t value < 0.05 in a Student’s t‐test as a statistically significant result, ns indicates no significant difference as determined by Student’s t‐test.