Cells of Escherichia coli pump cobalamin (vitamin B12) across their outer membranes into the periplasmic space, and it was concluded previously that this process is potentiated by the proton motive force of the inner membrane. The novelty of such an energy coupling mechanism and its relevance to other outer membrane transport processes have required confirmation of this conclusion by studies with cells in which cobalamin transport is limited to the outer membrane. Accordingly, I have examined the effects of cyanide and of 2,4-dinitrophenol on cobalamin uptake in btuC and atp mutants, which lack inner membrane cobalamin transport and the membrane-bound ATP synthase, respectively. Dinitrophenol eliminated cobalamin transport in all strains, but cyanide inhibited this process only in atp and btuC atp mutant cells, providing conclusive evidence that cobalamin transport across the outer membrane requires specifically the proton motive force of the inner membrane. The coupling of metabolic energy to outer membrane cobalamin transport requires the TonB protein and is stimulated by the ExbB protein. I show here that the tolQ gene product can partly replace the function of the ExbB protein. Cells with mutations in both exbB and tolQ had no measurable cobalamin transport and thus had a phenotype that was essentially the same as TonB-. I conclude that the ExbB protein is a normal component of the energy coupling system for the transport of cobalamin across the outer membrane.

Cells of Escherichia coli take up vitamin B 12 (cyano-cobalamin [CN-Cbl])and iron chelates by use of sequential active transport processes. Transport of CN-Cbl across the outer membrane and its accumulation in the periplasm is mediated by the TonB-dependent transporter BtuB. Transport across the cytoplasmic membrane (CM) requires the BtuC and BtuD proteins, which are most related in sequence to the transmembrane and ATP-binding cassette proteins of periplasmic permeases for iron-siderophore transport. Unlike the genetic organization of most periplasmic permeases, a candidate gene for a periplasmic Cbl-binding protein is not linked to the btuCED operon. The open reading frame termed yadT in the E. coli genomic sequence is related in sequence to the periplasmic binding proteins for iron-siderophore complexes and was previously implicated in CN-Cbl uptake in Salmonella. The E. coli yadT product, renamed BtuF, is shown here to participate in CN-Cbl uptake. BtuF protein, expressed with a C-terminal His 6 tag, was shown to be translocated to the periplasm concomitant with removal of a signal sequence. CN-Cbl-binding assays using radiolabeled substrate or isothermal titration calorimetry showed that purified BtuF binds CN-Cbl with a binding constant of around 15 nM. A null mutation in btuF, but not in the flanking genes pfs and yadS, strongly decreased CN-Cbl utilization and transport into the cytoplasm. The growth response to CN-Cbl of the btuF mutant was much stronger than the slight impairment previously described for btuC, btuD, or btuF mutants. Hence, null mutations in btuC and btuD were constructed and revealed that the btuC mutant had a strong impairment similar to that of the btuF mutant, whereas the btuD defect was less pronounced. All mutants with defective transport across the CM gave rise to frequent suppressor variants which were able to respond at lower levels of CN-Cbl but were still defective in transport across the CM. These results finally establish the identity of the periplasmic binding protein for Cbl uptake, which is one of few cases where the components of a periplasmic permease are genetically separated.The outer membrane (OM) of gram-negative bacteria forms a permeability barrier which restricts passage of both nutrients and toxic environmental agents (19,25). Most nutrients cross the OM into the periplasmic space by diffusion through general or specific porins, such as OmpF or LamB. Nutrients which are too large or scarce to enter efficiently through the porins are taken into the periplasm via specific, high-affinity active transport systems. The transport systems for passage across the OM of ferric iron complexed with siderophores, heme, or host iron-binding proteins, and of cobalamins (Cbls) such as vitamin B 12 (CN-Cbl), consist of a substrate-specific TonB-dependent OM transporter, the transperiplasmic energy-coupling protein TonB, and its ancillary proteins ExbB and ExbD in the cytoplasmic membrane (CM).Most nutrients are transported across the CM by active transport systems coupled to a ...

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.

Uptake of cobalamins by the transporter protein BtuB in the outer membrane of Escherichia coli requires the proton motive force and the transperiplasmic protein TonB. The Ton box sequence near the amino terminus of BtuB is conserved among all TonB-dependent transporters and is the only known site of mutations that confer a transport-defective phenotype which can be suppressed by certain substitutions at residue 160 in TonB. The crystallographic structures of the TonB-dependent transporter FhuA revealed that the region near the Ton box, which itself was not resolved, is exposed to the periplasmic space and undergoes an extensive shift in position upon binding of substrate. Site-directed disulfide bonding in intact cells has been used to show that the Ton box of BtuB and residues around position 160 of TonB approach each other in a highly oriented and specific manner to form BtuB-TonB heterodimers that are stimulated by the presence of transport substrate. Here, replacement of Ton box residues with proline or cysteine revealed that residue side chain recognition is not important for function, although replacement with proline at four of the seven Ton box positions impaired cobalamin transport. The defect in cobalamin utilization resulting from the L8P substitution was suppressed by cysteine substitutions in adjacent residues in BtuB or in TonB. This suppression did not restore active transport of cobalamins but may allow each transporter to function at most once. The uncoupled proline substitutions in BtuB markedly affected the pattern of disulfide bonding to TonB, both increasing the extent of cross-linking and shifting the pairs of residues that can be joined. Cross-linking of BtuB and TonB in the presence of the BtuB V10P substitution became independent of the presence of substrate, indicating an additional distortion of the exposure of the Ton box in the periplasmic space. TonB action thus requires a specific orientation for functional contact with the Ton box, and changes in the conformation of this region block transport by preventing substrate release and repeated transport cycles.TonB function is crucial for the operation of energy-dependent transport systems across the outer membrane (OM) of gram-negative bacteria (reviewed in references 19, 30, and 32). These transport systems carry out the high-affinity uptake of corrinoids, such as vitamin B 12 (cyano-cobalamin [CN-Cbl]), and of iron complexes with siderophores or host iron-binding proteins. These nutrients are too large or scarce in natural environments to diffuse effectively through the porin channels, and they require specialized uptake mechanisms comprising a specific, high-affinity OM transporter and an ATPdependent periplasmic permease system for transport across the cytoplasmic membrane. Transport across the OM depends on energy coupling through the action of the TonB protein (15, 34). TonB spans the periplasmic space to interact with its accessory proteins ExbB and ExbD in the cytoplasmic membrane and with the OM transporters (23, 35).The cryst...

The first step in the transport of cyanocobalamin (CN-B 12 ) by cells of Escherichia coli was shown previously to consist of binding of the B 12 to specific receptor sites located on the outer membrane of the cell envelope. In this paper, evidence is presented that these B 12 receptor sites also function as the receptors for the E colicins, and that there is competition between B 12 and the E colicins for occupancy of these sites. The cell strains used were E. coli KBT001, a methionine/B 12 auxotroph, and B 12 transport mutants derived from strain KBT001. Colicins E1 and E3 inhibited binding of B 12 to the outer membrane B 12 receptor sites, and CN-B 12 protected cells against these colicins. Half-maximal protection was given by CN-B 12 concentrations in the range of 1 to 6 nM, depending upon the colicin concentration used. Colicin E1 competitively inhibited the binding of 57 Co-labeled CN-B 12 to isolated outer membrane particles. Functional colicin E receptor sites were found in cell envelopes from cells of only those strains that possessed intact B 12 receptors. Colicin K did not inhibit the binding of B 12 to the outer membrane receptor sites, and no evidence was found for any identity between the B 12 and colicin K receptors. However, both colicin K and colicin E1 inhibited the secondary phase of B 12 transport, which is believed to consist of the energy-coupled movement of B 12 across the inner membrane.