The objective of this proposal is the further elucidation of the mechanisms involved in the transport of vitamin B12 across the cell envelope of Escherichia coli. The B12 receptors in the outer membrane of the cell envelope also serve as receptors for the E colicins and for bacteriophage BF23. One part of the experimentation will examine the structural components that are necessary for full receptor activity towards each of these substrates. The receptor system will be purified by affinity chromatography, on columns of porous glass containing covalently linked cobalamin, of outer membranes that have been solubilized with buffers containing Triton X-100 and EDTA. The ability of the receptor to catalyse facilitated diffusion of vitamin B12 in vesicles containing phospholipid and lipopolysaccharide will also be studied. A second part of the experimentation will use photo-affinity labeling techniques to probe both the active site and the environment of the B12 receptor in the outer membrane, and to identify other possible B12 carriers in either the periplasmic space or the inner membrane. The photo-affinity labels will consist of a phenylazide residue covalently linked to various 3H-labeled corrinoid compounds and to isotopically-labeled colicins E1 and E3. The proteins that become labeled during these experiments will be identified by means of one-dimensional SDS-polyacrylamide gel electrophoresis, and two-dimensional electrofocussing and polyacrylamide gel electrophoresis. The environment of the receptor, and of the communication pathway between the receptor and the inner membrane, will be probed under different physiological conditions, and with strains known to have modifications of either the structure or the configuration of the receptor. The E. coli strains to be used will include those containing the mutations, btuA, btuC, tonB, tolA, tolB, tolC, tolD, and tolE. It is hoped that these experiments will give some elucidation of the biochemical basis of tolerance towards some of the E colicins.