Dissecting the Pathway for Colicin Ia Translocation across the E. coli Outer Membrane

Abstract

The bacterial protein toxins called colicins all share the common challenges both of binding to and crossing the E. coli outer membrane, regardless of their ultimate target in susceptible cells. All colicins have co-opted, as their primary binding receptors, one of a family of outer membrane proteins normally involved in the uptake of essential nutrients, such as siderophore-bound iron or cobalamin. Many colicins then use a porin, such as OmpF, as their translocator across the outer membrane. For another family of colicins, no translocator had ever been genetically or functionally identified. We recently showed that one of those colicins, colicin Ia, uses a second copy of its receptor, Cir, in an entirely different way_as its translocator. Here, we begin to dissect the steps by which the translocation domain of the colicin traverses the outer membrane through the Cir protein. Genetically attaching a folded protein at the C-terminus of the isolated T domain yields a protein that protects sensitive cells from killing by colicin Ia significantly more efficiently than does the purified T domain alone. Therefore, the chimeric T domain protein appears to stop or slow down translocation better than T domain. The effects of the interaction of T domain with the periplasmic protein, TonB, will be reported.