Point mutations in a conserved region (TonB box) of Escherichia coli outer membrane protein BtuB affect vitamin B12 transport

Abstract

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.

Differential substrate-induced signaling through the TonB-dependent transporter BtuB

“…2C). This constitutive labeling behavior is not the result of a defect in substrate binding, because the V10P mutant still bound CNCbl (14) and E3R (Fig. 3C).…”

“…Sequences of mutagenic oligonucleotide primers are available on request. Mutant alleles were transferred by exchange of HindIII-BsiWI fragments into the corresponding sites of plasmid pAG1, a pUC8 derivative carrying the btuB ORF and regulatory region (14). The presence of expected sequence changes was verified by automated DNA sequencing.…”

“…The BtuB L8P and V10P substitutions confer the TonB-uncoupled phenotype (13,14). Both uncoupling mutations affected the dynamics of the Ton box and its interaction with TonB.…”

“…The TonB-uncoupled phenotype results from substitution of Pro or Gly at certain Ton box positions (13,14), which could interfere with backbone conformation or side-chain recognition. To test whether Pro substitutions elsewhere in the Ton box conferred similar behavior, the D6P or A12P substitutions were combined with the T7C or V9C substitutions.…”

“…A key factor in signaling and transport is the conserved sequence near the N terminus of TonB-dependent transporters called the Ton box (the sequence in BtuB is DTLVVTA, residues 6-12). This region is the site of mutations, such as the substitution of Pro for Leu-8 or Val-10 in BtuB, which result in loss of Cbl transport but do not affect Cbl binding or entry of the TonB-independent phage BF23 and E colicins (13,14). The transport defect in Ton box mutants is partially reversed by suppressor mutations affecting nearby residues in the Ton box or residue Gln-160 of TonB (15,16).…”