Energy-coupled reactions of the Escherichia coli outer membrane transport proteins BtuB and Cir require the tonB product. Some point mutations in a region of btuB and cir that is highly conserved in TonB-dependent transport proteins led to loss of TonB-coupled uptake of vitamin B12 and colicin Ia, whereas binding was unaffected. Most other point mutations in this region had no detectable effect on transport activity. Mutations in tonB that suppressed the transport defect phenotype of these btuB mutations were isolated. All carried changes of glutamine 165 to leucine, lysine, or proline. The various tonB mutations differed markedly in their suppression activities on different btuB or cir mutations. This allele specificity of suppression indicates that TonB interacts directly with the outer membrane transport proteins in a manner that recognizes the local conformation but not specific side chains within this conserved region. An effect of the context of the remainder of the protein was seen, since the same substitution (valine 1O-*glycine) in btuB and cir responded differently to the suppressors. This finding supports the proposal that TonB interacts with more of the transport proteins than the first conserved domain alone.The outer membrane of Escherichia coli contains several high-affinity, active-transport systems for substrates, such as ferric-siderophore complexes and cobalamins, which are too large to diffuse effectively through the nonspecific porin channels. Transport is mediated by minor outer membrane proteins that bind these substrates with high affinity and specificity and that can serve as receptors for the adsorption and entry of specific bacteriophages and bacteriocins. The BtuB polypeptide is responsible for the uptake of vitamin B12 and other cobalamins, bacteriophage BF23, and colicins A and E. The FepA protein transports ferric enterochelin and colicins B and D. The iron-repressible Cir protein was initially identified as the receptor for colicin I, has been implicated in the uptake of catechol-substituted cephalosporins, and may mediate uptake of ferric complexes of other 2,3-dihydroxybenzoate derivatives (4).Active transport of most of the substrates listed above also requires functioning of the tonB product and is stimulated by the products of exbB and exbD (1, 7-9, 12). In the absence of tonB function, the outer membrane transport proteins are produced in normal or larger amounts and still bind their substrates effectively, but they are unable to carry out the energy-dependent steps of substrate accumulation (7). Analysis of cobalamin uptake in btuC mutants blocked in the cytoplasmic membrane transport system suggested that tonB-dependent active transport across the outer membrane is driven by the proton motive force (23). It was suggested that TonB couples energy from the proton motive force across the cytoplasmic membrane to the outer membrane transporters (7,12,23).The deduced sequences of tonB-dependent transport proteins possess short, highly conserved regions separated by long variable...

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.

Several anaerobic bacteria isolated from the sediments of Contrary Creek, an iron-rich environment, produced magnetite when cultured in combinations but not when cultured alone in synthetic iron oxyhydroxide medium. When glucose was added as a carbon source, the pH of the medium decreased (to 5.5) and no magnetite was formed. When the same growth medium without glucose was used, the pH increased (to 8.5) and magnetite was formed. In both cases, Fe2+ was released into the growth medium. Geochemical equilibrium equations with Eh and pH as master variables were solved for the concentrations of iron and inorganic carbon that were observed in the system. Magnetite was predicted to be the dominant iron oxide formed at high pHs, while free Fe2+ or siderite were the dominant forms of iron expected at low pHs. Thus, magnetite formation occurs because of microbial alteration of the local Eh and pH conditions, along with concurrent reduction of ferric iron (direct biological reduction or abiological oxidation-reduction reactions).

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