The transport of cobalamin (Cb1) across the cell envelope of Escherichi coli includes the following sequence of events, Cb1 binding to the outer membrane Cb1 receptor, proton motive force-and TonB-dependent release of Cb1 from the receptor towards the interior of the cell, and BtuC-dependent Cb1 transport across the inner membrane. The main objectives of this proposal are more detailed descriptions of the molecular interactions involved in each of these steps. The structure of the Cb1 receptor protein from wild type and mutant strains will be compared. Ion-exchange chromatography, electrophoresis, and electrofoccusing will be used for purification of the protein, followed by various, specific, partial digestions and high pressure liquid chromatography for structural analysis. Cobalamins containing a nitrophenylazide residue will be used as photoaffinity labels to identify components of the Cb1-binding site. The purified receptor protein will also be used in reconstitution experiments, together with lipopolysaccharide, phospholipid, peptidoglycan, and possibly other outer membrane proteins, in attempts to regenerate full receptor function for Cb1 and for the other substrates that use this receptor, i.e., the E colicins and bacteriophage BF23. The mechanism of action of the TonB protein in Cb1 transport will be studied, and our hypothesis that this protein and the proton motive force generate a diffusible, periplasmic messenger that catalyzes release of Cb1 from the receptor, will be tested. Attempts will also be made to identify the BtuC gene product and study its role in Cb1 transport across the inner membrane. The procedures will include genetic manipulations to amplify the BtuC gene product and the use of photoaffinity labels to identify envelope components involved in Cb1 transport. The energy dependence of Cb1 transport across the inner membrane and the possible involvement of the periplasmic Cb1-binding protein in this process will also be examined.
| Gudmundsdottir, A; Bradbeer, C; Kadner, R J (1988) Altered binding and transport of vitamin B12 resulting from insertion mutations in the Escherichia coli btuB gene. J Biol Chem 263:14224-30 |
| Bradbeer, C; Reynolds, P R; Bauler, G M et al. (1986) A requirement for calcium in the transport of cobalamin across the outer membrane of Escherichia coli. J Biol Chem 261:2520-3 |
| de Veaux, L C; Clevenson, D S; Bradbeer, C et al. (1986) Identification of the btuCED polypeptides and evidence for their role in vitamin B12 transport in Escherichia coli. J Bacteriol 167:920-7 |
