Protein import through the E. coli cell envelope

The cell envelope of Gram-negative bacteria is a robust, adaptive ~30 nm structure that supports the colonization of a broad range of environments. Escherichia coli, for example, can grow in open water and soil as well as colonizing humans, animals and plants as a commensal or pathogen. The importance of the cell envelope is underscored by the fact that it is the site of action of many common antibiotics. Yet our understanding of cell envelope organisation and in particular how active processes at the outer membrane are driven is poor. I aim to address these issues through colicin import. Colicins are E. coli-specific bacteriocins (protein antibiotics), released by E. coli populations to kill competing bacteria, which translocate a cytotoxic domain across one or both membranes of the cell envelope. We have found ways of trapping and crystallizing colicin translocons, which are partially-translocated states containing both outer membrane and periplasmic proteins. In addition, using fluorescence microscopy, we have, for the first time, imaged single colicin molecules entering bacteria. These assays have also demonstrated that import across the outer membrane is dependent on the proton-motive force across the inner membrane, much speculated on in the literature but never before demonstrated. These new methods are the foundation of the present proposal which aims to address the central question of how colicins translocate through the E. coli cell envelope. The proposal has three objectives: 1. Determining the architecture and structure of outer membrane colicin translocons and/or receptor complexes using X-ray crystallography or chemical cross-linking. 2. Following the translocation paths of colicins by photoactivated cross-linking using colicins impregnated with non-native amino acids and synchronised for cell entry using disulfide bonds. 3. Using fluorescence microscopy to dissect the colicin import mechanism at the single molecule level