E, First-order cell death kinetics for wild-type ColE9 or ColE9 K469C labelled with different AF dyes (AF488, AF568, AF647) against E. coli JW2203 with OmpF in the outer membrane. Cultures were incubated with 80 nM toxin at pH 7.5 and 37 oC and at various time points the reaction stopped using trypsin at 37 °C for 30 min, cells plated out and CFUs recorded. All time courses were conducted in the presence of chloramphenicol (20 µg/ml) to reversibly block cell division, which would otherwise compete with cell-killing. Control data shown are for cells with no ColE9 added but where growth was inhibited by the presence of chloramphenicol. Cell-killing half-lives, obtained from the fitted first-order plots shown (error bars represent the standard deviation across two biological replicates), were as follows: wild-type ColE9, 2.5 min; ColE9 K469CAF488, 4.1 min; ColE9 K469CAF647, 29.6 min; ColE9 K469CAF568, 78.8 min. The data show that the chemical nature of the fluorophore in the C-terminal DNase domain of ColE9 has a dramatic effect on the cell-killing kinetics of the colicin. The schematic shown alongside the kinetic data is a surface representation of AF568 (model Generated in coot), the fluorophore with the biggest impact on OmpF-mediated killing, manually grafted onto the structure of ColE9 OBS2 bound within subunit 2 of OmpF. The slow cell-death kinetics of ColE9 K469CAF568 likely reflects the time taken to pass this bulky molecule through the narrow eyelet of the porin. 