Point mutations in a conserved region (TonB box) of Escherichia coli outer membrane protein BtuB affect vitamin B12 transport.
Uptake of cobalamins and iron chelates in Escherichia coli K-12 is dependent on specific outer membrane transport proteins and the energy-coupling function provided by the TonB protein. The btuB product is the outer membrane receptor for cobalamins, bacteriophage BF23, and the E colicins. A short sequence near the amino terminus of mature BtuB, previously called the TonB box, is conserved in all tonB-dependent receptors and colicins and is the site of the btuB451 mutation (Leu-8----Pro), which prevents energy-coupled cobalamin uptake. This phenotype is partially suppressed by certain mutations in tonB. To examine the role of individual amino acids in the TonB box of BtuB, more than 30 amino acid substitutions in residues 6 to 13 were generated by doped oligonucleotide-directed mutagenesis. Many of the mutations affecting each amino acid did not impair transport activity, although some substitutions reduced cobalamin uptake and the Leu-8----Pro and Val-10----Gly alleles were completely inactive. To test whether the btuB451 mutation affects only cobalamin transport, a hybrid gene was constructed which encodes the signal sequence and first 39 residues of BtuB fused to the bulk of the ferrienterobactin receptor FepA (residues 26 to 723). This hybrid protein conferred all FepA functions but no BtuB functions. The presence of the btuB451 mutation in this fusion gene eliminated all of its tonB-coupled reactions, showing that the TonB box of FepA could be replaced by that from BtuB. These results suggest that the TonB-box region of BtuB is involved in active transport in a manner dependent not on the identity of specific side chains but on the local secondary structure.
Gudmundsdottir
A
A
Department of Microbiology, School of Medicine, University of Virginia, Charlottesville 22908.
Bell
P E
PE
Lundrigan
M D
MD
Bradbeer
C
C
Kadner
R J
RJ
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport; D002678:Chimera; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005838:Genotype; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D014805:Vitamin B 12 / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D003062:Codon; D015345:Oligonucleotide Probes; D014805:Vitamin B 12