Altered binding and transport of vitamin B12 resulting from insertion mutations in the Escherichia coli btuB gene.
The BtuB protein of Escherichia coli is a multifunctional outer membrane receptor required for the binding and uptake of vitamin B12, bacteriophage BF23, and the E colicins. The btuB gene was mutagenized by the insertion of 6-base pair linkers into each of ten HpaII sites distributed throughout the coding region. Receptor function was measured with the mutated genes present in single or multiple copies. All of the mutant proteins were found in the outer membrane in similar amounts, although two of them were susceptible to cleavage by endogenous proteolytic activity. The vitamin B12 transport activity mediated by five of the mutants was essentially identical to that of the wild type. Four mutations (insertions after amino acids 50, 252, and 412, and a duplication of residues 434-472) reduced uptake activity to less than 2% of parental, whereas insertions at residues 343 and 434 had less severe effect. The insertions at residues 50 and 252 appeared to slow the rate of cobalamin binding to the receptor; the defect in the former mutant was partially corrected by elevated calcium levels. The insertion at residue 412 did not affect the rate of substrate binding but slowed its release from the receptor. Most of the receptors conferred susceptibility to phage BF23 and the E colicins, although several mutants were altered in the degree of their sensitivity to the lethal agents. None of the mutations affected the entry of only one type of ligand. Thus, several receptor domains have been implicated in substrate binding and energy coupling.
Gudmundsdottir
A
A
Department of Microbiology, University of Virginia School of Medicine, Charlottesville 22908.
Bradbeer
C
C
Kadner
R J
RJ
D000483:Alleles; D001425:Bacterial Outer Membrane Proteins; D003087:Colicins / Q000494:pharmacology; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005796:Genes; D005798:Genes, Bacterial; D007700:Kinetics; D026901:Membrane Transport Proteins; D009154:Mutation; D011392:Proline / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D018000:Receptors, Peptide; D014805:Vitamin B 12 / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; C044592:BtuB protein, E coli; D003087:Colicins; D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D011956:Receptors, Cell Surface; D018000:Receptors, Peptide; D011392:Proline; D014805:Vitamin B 12