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...

The nucleotide sequence of the Escherichia coli colicin I receptor gene (cir) has been determined. The predicted mature protein consists of 599 amino acids and has a molecular weight of 67,169. Several previously noted characteristics of other E. coli outer membrane protein sequences were also identified in the sequence of Cir. These include an overall acidic nature, the absence of long hydrophobic stretches of amino acids, and a lack of predicted a-helical secondary structure. Because two classes of outer membrane proteins (the TonB-dependent transport proteins and the porins) share some structural features, protein sequences from both of these groups were aligned pairwise and scored for sequence similarity. Statistical evidence suggested that the porins were not related to the proteins in the TonB-dependent group; however, there was a significant relationship between the proteins in the TonB-dependent group. On the basis of the multiple progressive sequence alignment and the similarity scores derived from it, a tree representing evolutionary distance between five TonB-dependent outer membrane transport proteins was generated.

Synthesis of the colicin I receptor protein, encoded by the cir gene, was determined to be sensitive to control by the catabolite repression regulatory system. Under both high-and low-iron conditions for growth, mutants unable to produce cyclic AMP (cAMP) (cya) or functional cAMP receptor protein (crp) exhibited decreased membrane levels of the receptor relative to those of the wild-type strain. Exogenous addition of cAMP to the cya mutant restored maximal expression. cAMP-dependent changes in steady-state levels of cir mRNA suggested that the effect is mediated by control of transcript synthesis or stability. Potential mechanisms for regulation were examined by deletion and sequence analysis.