Abstract
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.
Highlights
The BtuB protein of Escherichia coliis a multifunc- products has been deduced from the nucleotide sequence
The BtuB protein serves as surface receptor for bacteriophage BF23 and the bactericidal proteins, colicins E and colicin A [14,15,16]
An M, 66,412 receptor protein, encoded by the btuB gene at minute 88, binds externalCN-Cbl with high affinity (I&, 0.5 nM) and, In this paper, we describe the isolation of 10 mutations generated by the insertion of a 6-base pair linker intdoifferent HpaII sites of the gene
Summary
Cobalamin Transport-The effect of insertion mutations on cobalamin uptake was measured in strains carrying single copies of each mutation in the chromosome. These strains were constructed by forcing integration of the BBam plasmids into a partiallydeleted btuB gene in apolA host. Cobalamin Binding-The effects of these mutations on receptor function were studied flirtherby measurement of Cbl binding and release in cells carrying pBBam plasmids with amplified levels of the mutant receptors (TableI).These. Five of the mutants with depressed uptake activity (BBam 252 343 434, and du[434-472]) displayed markedly reduced levels Another mutation generated by this procedure, du[434-472], was a 117-base pair duplication of the region encoding amino acid residues 434-472, with a BamHI linker inserted at the duplication joint.
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