Precise deletions of cell surface-exposed loops of FhuA resulted in mutants of Escherichia coli with distinct phenotypes. Deletion of loop 3 or 11 inactivated ferrichrome transport activity. Deletion of loop 8 inactivated receptor activity for colicin M and the phages T1, T5, and 80. The loop 7 deletion mutant was colicin M resistant but fully phage sensitive. The loop 4 deletion mutant was resistant to the TonB-dependent phages T1 and 80 but fully sensitive to the TonB-independent phage T5. The phenotypes of the deletion mutants revealed important sites for the multiple FhuA transport and receptor activities. The ligand binding sites are nonidentical and are distributed among the entire exposed surface. Presumably, FhuA evolved as a ferrichrome transporter and was subsequently used as a receptor by the phages and colicin M, which selected the same as well as distinct loops as receptor sites.

FhuA and FecA are two examples of energy-coupled outer membrane import proteins of gram-negative bacteria. FhuA transports iron complexed by the siderophore ferrichrome and serves as a receptor for phages, a toxic bacterial peptide, and a toxic protein. FecA transports diferric dicitrate and regulates transcription of an operon encoding five ferric citrate (Fec) transport genes. Properties of FhuA mutants selected according to the FhuA crystal structure are described. FhuA mutants in the TonB box, the hatch, and the beta-barrel are rather robust. TonB box mutants in FhuA FecA, FepA, Cir, and BtuB are compared; some mutations are suppressed by mutations in TonB. Mutant studies have not revealed a ferrichrome diffusion pathway, and tolerance to mutations in the region linking the TonB box to the hatch does not disclose a mechanism for how energy transfer from the cytoplasmic membrane to FhuA changes the conformation of FhuA such that bound substrates are released, the pore is opened, and substrates enter the periplasm, or how surface loops change their conformation such that TonB-dependent phages bind irreversibly and release their DNA into the cells. The FhuA and FecA crystal structures do not disclose the mechanism of these proteins, but they provide important information for specific functional studies. FecA is also a regulatory protein that transduces a signal from the cell surface into the cytoplasm. The interacting subdomains of the proteins in the FecA --> FecR --> FecI --> RNA polymerase signal transduction pathway resulting in fecABCDE transcription have been determined. Energy-coupled transporters transport not only iron and vitamin B12, but also other substrates of very low abundance such as sugars across the outer membrane; transcription regulation of the transport genes may occur similarly to that of the Fec transport genes.

The FhuA outer membrane protein of Escherichia coli actively transports ferrichrome, albomycin, and rifamycin CGP 4832, and confers sensitivity to microcin J25, colicin M, and the phages T1, T5, and 80. Guided by the FhuA crystal structure and derived predictions on how FhuA might function, mutants were isolated in the cork domain (residues 1 to 160) and in the ␤-barrel domain (residues 161 to 714). Deletion of the TonB box (residues 7 to 11) completely inactivated all TonB-dependent functions of FhuA. Fixation of the cork to turn 7 of the barrel through a disulfide bridge between introduced C27 and C533 residues abolished ferrichrome transport, which was restored by reduction of the disulfide bond. Deletion of residues 24 to 31, including the switch helix (residues 24 to 29), which upon binding of ferrichrome to FhuA undergoes a large structural transition (17 Å) and exposes the N terminus of FhuA (TonB box) to the periplasm, reduced FhuA transport activity (79% of the wild-type activity) but conferred full sensitivity to colicin M and the phages. Duplication of residues 23 to 30 or deletion of residues 13 to 20 resulted in FhuA derivatives with properties similar to those of FhuA with a deletion of residues 24 to 31. However, a frameshift mutation that changed QSEA at positions 18 to 21 to KKAP abolished almost completely most of FhuA's activities. The conserved residues R93 and R133 among energy-coupled outer membrane transporters are thought to fix the cork to the ␤-barrel by forming salt bridges to the conserved residues E522 and E571 of the ␤-barrel. Proteins with the E522R and E571R mutations were inactive, but inactivity was not caused by repulsion of R93 by R522 and R571 and of R133 by R571. Point mutations in the cork at sites that move or do not move upon the binding of ferrichrome had no effect or conferred only slightly reduced activities. It is concluded that the TonB box is essential for FhuA activity. The TonB box region has to be flexible, but its distance from the cork domain can greatly vary. The removal of salt bridges between the cork and the barrel affects the structure but not the function of FhuA.

The FhuA protein in the outer membrane of Escherichia coli actively transports ferrichrome and the antibiotics albomycin and rifamycin CGP 4832 and serves as a receptor for the phages T1, T5, and 80 and for colicin M and microcin J25. The crystal structure reveals a ␤-barrel with a globular domain, the cork, which closes the channel formed by the barrel. Genetic deletion of the cork resulted in a ␤-barrel that displays no FhuA activity. A functional FhuA was obtained by cosynthesis of separately encoded cork and the ␤-barrel domain, each endowed with a signal sequence, which showed that complementation occurs after secretion of the fragments across the cytoplasmic membrane. Inactive complete mutant FhuA and an FhuA fragment containing 357 N-proximal amino acid residues complemented the separately synthesized wild-type ␤-barrel to form an active FhuA. Previous claims that the ␤-barrel is functional as transporter and receptor resulted from complementation by inactive complete FhuA and the 357-residue fragment. No complementation was observed between the wild-type cork and complete but inactive FhuA carrying cork mutations that excluded the exchange of cork domains. The data indicate that active FhuA is reconstituted extracytoplasmically by insertion of separately synthesized cork or cork from complete FhuA into the ␤-barrel, and they suggest that in wild-type FhuA the ␤-barrel is formed prior to the insertion of the cork.The FhuA protein of Escherichia coli K-12 transports ferrichrome, the structurally related antibiotic albomycin, and the unrelated antibiotic rifamycin CGP 4832 across the outer membrane and serves as a receptor for colicin M, microcin J25, and the phages T1, T5, and 80 (7). Although extensive mutational analyses have been performed (6, 7, 33) and the crystal structure has been determined (15, 30), the molecular mechanism of FhuA transport is unclear.The protein consists of a ␤-barrel (residues 161 to 714) composed of 22 antiparallel ␤-strands that form a channel that is closed by a globular domain (residues 1 to 160), designated the cork or plug, that inserts from the periplasmic side into the ␤-barrel. Binding of ferrichrome elicits small, 1-to 2-Å movements of the cork domain relative to the ␤-barrel and a large 17-Å transition of E19 without opening the channel. Release of ferrichrome from the binding site formed by 10 amino acid residues located in the ␤-barrel and the cork, and opening of the channel, is thought to be triggered by interaction with the TonB protein, which requires the proton motive force of the cytoplasmic membrane to exert its action on FhuA. All FhuArelated activities, except infection by phage T5, depend on TonB.We previously reported that deletion of the cork (residues 5 to 160) results in a protein (FhuA⌬5-160) that still functions as an active TonB-dependent transporter and receptor (3,4,25). These experiments were performed with two fhuA mutant strains; mutant 41/2 contains a number of amino acid replacements and D348 is deleted (26), whereas PCR analysis indicated th...