Colicin E2 is still in contact with its receptor and import machinery when its nuclease domain enters the cytoplasm

Collicins reach their targets in susceptible Escherichia coli strains through two envelope protein systems: the Tol system is used by group A colicins and the TonB system by group B colicins. Collicin E2 (CoIE2) is a cytotoxic protein that recognizes the outer membrane receptor BtuB. After gaining access to the cytoplasmic membrane of sensitive Escherichia coli cells, CoIE2 enters the cytoplasm to cleave DNA. After binding to BtuB, CoIE2 interacts with the Toll system to reach its target. However, it is not known if the entire colicin or only the nuclease domain of CoIE2 enters the cell. Here I show that preincubation of CoIE2 with Escherichia coli cells prevents binding and translocation of pore-forming collicins of group A but not of group B. This inhibition persisted even when cells were incubated with CoIE2 for 30 min before the addition of pore-forming colicins, indicating that CoIE2 releases neither its receptor nor its translocation machinery when its nuclease domain enters the cells. These competition experiments enabled me to estimate the time required for CoIE2 binding to its receptor and translocation.