Structure‐function analysis of the vitamin B12 receptor of Escherichia coli by means of informational suppression

Summary We describe a genetic analysis of the vitamin B12 receptor of Escherichia coli. Through the use of informational suppression, we have been able to generate a family of receptor variants, each identical save for a single, known substitution (Ser, Gln, Lys, Tyr, Leu, Cys, Phe) at a known site. We have studied 22 different mutants, 14 in detail, distributed throughout the length of the btuB gene. Most amino acid substitutions have a pleiotropic effect with respect to all ligands tested, the two colicins E1 and E3, the T5‐like bacteriophage BF23, and vitamin B12 (The dramatic effect of a single amino acid substitution is also well exemplified by the G142A missense change which renders the receptor completely non‐functional.) In some instances, however, we have been able to modify a subset of receptor functions (viz, Q62, Q150 and Q299 and the response to phage BF23). These data are summarized on a two‐dimensional folding model for the BtuB protein in the outer membrane (devised using both amphipathic β‐strand analysis and sequence conservation amongst the TonB‐dependent receptors). In addition, we report that the extreme C‐terminus of BtuB is vital for receptor localization and provide evidence for it being a membrane‐spanning β‐sheet with residue L588 situated on its hydrophobic surface. Two of the C‐terminal btuB mutations are located within the region of overlap with the recently identified dga (murl) gene.