Properties of the FhuA channel in the Escherichia coli outer membrane after deletion of FhuA portions within and outside the predicted gating loop.

Escherichia coli transports Fe3+ as a ferrichrome complex through the outer membrane in an energy-dependent process mediated by the FhuA protein. A FhuA deletion derivative lacking residues 322 to 355 (FhuA delta322-355) forms a permanently open channel through which ferrichrome diffused. This finding led to the concept that the FhuA protein forms a closed channel that is opened by input of energy derived from the electrochemical potential across the cytoplasmic membrane, mediated by the Ton system. In this study, we constructed various FhuA derivatives containing deletions inside and outside the gating loop. FhuA delta322-336 bound ferrichrome and displayed a residual Ton-dependent ferrichrome transport activity. FhuA delta335-355 no longer bound ferrichrome but supported ferrichrome diffusion through the outer membrane in the absence of the Ton system. FhuA delta335-355 rendered cells sensitive to sodium dodecyl sulfate and supported diffusion of maltotetraose and maltopentaose in a lamB mutant lacking the maltodextrin-specific channel in the outer membrane. Cells expressing FhuA delta70-223, which has a large deletion outside the gating loop, were highly sensitive to sodium dodecyl sulfate and grew on maltodextrins but showed only weak ferrichrome uptake, suggesting formation of a nonspecific pore through the outer membrane. FhuA delta457-479 supported Ton-dependent uptake of ferrichrome. None of these FhuA deletion derivatives formed pores in black lipid membranes with a stable single-channel conductance. Rather, the conductance displayed a high degree of current noise, indicating a substantial influence of the deletions on the conformation of the FhuA protein. FhuA also supports infection by the phages T1, T5, and phi80 and renders cells sensitive to albomycin and colicin M. Cells expressing FhuA delta322-336 were sensitive to albomycin and colicin M but were only weakly sensitive to T5 and phi480 and insensitive to T1. Cells expressing FhuA delta335-355 were resistant to all FhuA ligands. These results indicate different structural requirements within the gating loop for the various FhuA ligands. Cells expressing FhuA delta457-479 displayed a strongly reduced sensitivity to all FhuA ligands, while cells expressing FhuA delta70-223 were rather sensitive to all FhuA ligands except albomycin, to which they were nearly resistant. It is concluded that residues 335 to 355 mainly determine the properties of the gate with regard to FhuA permeability and ligand binding.     

Transport across the outer membrane of Escherichia coli K12 via the FhuA receptor is regulated by the TonB protein of the cytoplasmic membrane

Summary Point mutations in the “TonB box” offhuA were suppressed by point mutations in thetonB gene, suggesting both a functional and physical interaction between the FhuA receptor protein in the outer membrane and the TonB protein in the cytoplasmic membrane ofEscherichia coli K12. Mutations influA were classified into four types according to the extent by which they impaired mutant cells in their growth on ferrichrome as sole iron source and in their sensitivity to the antibiotic albomycin and to colicin M. ThetonB mutation with a glutamine to leucine replacement at position 165 was less efficient in restoring the FhuA functions than the glutamine to lysine exchange at the same position. The better the coupling between FhuA and TonB the poorer was the inhibition of phage T1 binding to FhuA by ferrichrome. A working model is proposed in which the TonB protein assumes different conformations in response to the energized state of the cytoplasmic membrane and thereby allosterically regulates the activity of the FhuA receptor. This model implies an intermembrane coupling between two proteins in adjacent membranes.     

FhuA, a transporter of the Escherichia coli outer membrane, is converted into a channel upon binding of bacteriophage T5.

The Escherichia coli outer membrane protein FhuA catalyzes the transport of Fe3+(-)ferrichrome and is the receptor of phage T5 and phi 80. The purified protein inserted into planar lipid bilayers showed no channel activity. Binding of phage T5 and FhuA resulted in the appearance of high conductance ion channels. The electrophysiological characteristics of the channels (conductance, kinetic behavior, substates, ion selectivity including the effect of ferrichrome) showed similarities with those of the channel formed by a FhuA derivative from which the 'gating loop' (delta 322-355) had been removed. binding of phage T5 to FhuA in E.coli cells conferred SDS sensitivity to the bacteria, suggesting that such channels also exist in vivo. These data suggest that binding of T5 to loop 322-355 of FhuA, which constitutes the T5 binding site, unmasks an inner channel in FhuA. Both T5 and ferrichrome bind to the closed state of the channel but only T5 can trigger its opening.     

Preparation of the FhuA (TonA) receptor protein from cell envelopes of an overproducing strain of Escherichia coli K-12.

A rapid and simple method for purification of the FhuA receptor protein from cell envelopes of a FhuA-overproducing strain of Escherichia coli K-12 was developed. The overproduction of FhuA was programmed by the thermoamplifiable plasmid pHK232, which carried the fhuACD genes of pLC19-19 of the Clarke and Carbon collection. At low temperature (27 degrees C), pHK232 specified the overproduction of FhuA to levels comparable to those of major outer membrane proteins OmpF, OmpC, and OmpA. The amount of these proteins in the outer membrane was reduced along with overproduction of FhuA. Upon runaway replication of pHK232 at 37 degrees C, the precursor of the FhuA protein, proFhuA, was also accumulated in the cell envelope in amounts similar to FhuA. For extraction of the FhuA protein, crude cell envelopes were washed with 2% Triton X-100-6 M urea to remove less tightly bound proteins. Then FhuA but not proFhuA was solubilized by treating Triton X-100-urea-washed membranes with 1% octylglucoside-1 mM EDTA. This procedure yielded FhuA protein free from other membrane proteins. The amount of lipopolysaccharide and phospholipids was low and ranged from 5 to 15% and 10 to 25% of the weight of the FhuA protein, respectively. As shown by direct inactivation and by competition assays, the isolated FhuA protein retained receptor activity for ferrichrome, albomycin, colicin M, and phages T5 and T1.     

Insertion derivatives containing segments of up to 16 amino acids identify surface- and periplasm-exposed regions of the FhuA outer membrane receptor of Escherichia coli K-12.

The FhuA receptor in the outer membrane of Escherichia coli K-12 is involved in the uptake of ferrichrome, colicin M, and the antibiotic albomycin and in infection by phages T1, T5, and phi 80. Fragments of up to 16 amino acid residues were inserted into FhuA and used to determine FhuA active sites and FhuA topology in the outer membrane. For this purpose antibiotic resistance boxes flanked by symmetric polylinkers were inserted into fhuA and subsequently partially deleted. Additional in-frame insertions were generated by mutagenesis with transposon Tn1725. The 68 FhuA protein derivatives examined contained segments of 4, 8, 12, 16, and 22 additional amino acid residues at 34 different locations from residues 5 to 646 of the mature protein. Most of the FhuA derivatives were found in normal amounts in the outer membrane fraction. Half of these were fully active toward all ligands, demonstrating proper insertion into the outer membrane. Seven of the 12- and 16-amino-acid-insertion derivatives (at residues 378, 402, 405, 415, 417, 456, and 646) were active toward all of the ligands and could be cleaved by subtilisin in whole cells, suggesting a surface location of the extra loops at sites which did not affect FhuA function. Two mutants were sensitive to subtilisin (insertions at residues 511 and 321) but displayed a strongly reduced sensitivity to colicin M and to phages phi 80 and T1. Four of the insertion derivatives (at residues 162, 223, 369, and 531) were cleaved only in spheroplasts and probably form loops at the periplasmic side of the outer membrane. The number and size of the proteolytic fragments indicate cleavage at or close to the sites of insertion, which has been proved for five insertions by amino acid sequencing. Most mutants with functional defects were affected in their sensitivity to all ligands, yet frequently to different degrees. Some mutants showed a specifically altered sensitivity to a few ligands; for example, mutant 511-04 was partially resistant only to colicin M, mutant 241-04 was reduced in ferrichrome and albomycin uptake and showed a reduced colicin M sensitivity, and mutant 321-04 was fully resistant to phage T1 and partially resistant to phage phi 80. The altered residues define preferential binding sites for these ligands. Insertions of 4 to 16 residues at positions 69, 70, 402, 530, 564, and 572 resulted in strongly reduced amounts of FhuA in the outer membrane fraction, varying in function from fully active to inactive. These results provide the basis for a model of FhuA organization in the outer membrane.     

Conversion of the coprogen transport protein FhuE and the ferrioxamine B transport protein FoxA into ferrichrome transport proteins

The FhuA protein of Escherichia coli K-12 transports ferrichrome and the structurally related antibiotic albomycin across the outer membrane and serves as a receptor for the phages T1, T5, and φ80 and for colicin M. In this paper, we show that chimeric proteins consisting of the central part of FhuA and the N- and C-terminal parts of FhuE (coprogen receptor) or the N- and/or C-terminal parts of FoxA (ferrioxamine B receptor), function as ferrichrome transport proteins. Although the hybrid proteins contained the previously identified gating loop of FhuA, which is the principal binding site of the phages T5, T1, and φ80, only the hybrid protein consisting of the N-terminal third of FoxA and the C-terminal two thirds of FhuA conferred weak phage sensitivity to cells. Apparently, the gating loop is essential, but not sufficient for wild-type levels of ferrichrome transport and for phage sensitivity. The properties of FhuA-FoxA hybrids suggest different regions of the two receptors for ferric siderophore uptake.     

Stability studies of FhuA, a two-domain outer membrane protein from Escherichia coli

FhuA (MM 78.9 kDa) is an Escherichia coli outer membrane protein that transports iron coupled to ferrichrome and is the receptor for a number of bacteriophages and protein antibiotics. Its three-dimensional structure consists of a 22-stranded beta-barrel lodged in the membrane, extracellular hydrophilic loops, and a globular domain (the "cork") located within the beta-barrel and occluding it. This unexpected structure raises questions about the connectivity of the different domains and their respective roles in the different functions of the protein. To address these questions, we have compared the properties of the wild-type receptor to those of a mutated FhuA (FhuA Delta) missing a large part of the cork. Differential scanning calorimetry experiments on wild-type FhuA indicated that the cork and the beta-barrel behave as autonomous domains that unfold at 65 and 75 degrees C, respectively. Ferrichrome had a strong stabilizing effect on the loops and cork since it shifted the first transition to 71.4 degrees C. Removal of the cork destabilized the protein since a unique transition at 61.6 degrees C was observed even in the presence of ferrichrome. FhuA Delta showed an increased sensitivity to proteolysis and to denaturant agents and an impairment in phage T5 and ferrichrome binding.     

Inactivation of FhuA at the cell surface of Escherichia coli K-12 by a phage T5 lipoprotein at the periplasmic face of the outer membrane.

Inactivation of phage T5 by lysed cells after phage multiplication is prevented by a phage-encoded lipoprotein (Llp) that inactivates the FhuA outer membrane receptor protein (K. Decker, V. Krauel, A. Meesmann, and K. Heller, Mol. Microbiol. 12:321-332, 1994). Using FhuA derivatives carrying insertions of 4 and 16 amino acid residues and point mutations, we determined whether FhuA inactivation is caused by binding of Llp to FhuA and which regions of FhuA are important for inactivation by Llp. Cells expressing Llp were resistant not only to phage T5 but to all FhuA ligands tested, such as phage phi 80, colicin M, and albomycin, and they were strongly reduced in the uptake of ferrichrome. Most of the FhuA derivatives which were not affected by Llp were, according to a previously published FhuA transmembrane topology model, located in periplasmic turns and in the TonB box close to the periplasm. Since the ligands bind to the cell surface, interaction of FhuA with Llp in the periplasm may induce a FhuA conformation which impairs binding of the ligands. This conclusion was supported by the increase rather than decrease of colicin M sensitivity of two mutants in the presence of Llp. The only Llp-resistant FhuA derivatives with mutations at the cell surface contained insertions of 16 residues in the loop that determines the permeability of the FhuA channel and serves as the principal binding site for all FhuA ligands. This region may be inactivated by steric hindrance in that a portion of Llp penetrates into the channel. Outer membranes prepared with 0.25% Triton X-100 from cells expressing Llp contained inactivated FhuA, suggesting Llp to be an outer membrane protein whose interaction with FhuA was not abolished by Triton X-100. Llp solubilized in 1.1% octylglucoside prevented T5 inactivation by FhuA dissolved in octylglucoside.     

An aspartate deletion mutation defines a binding site of the multifunctional FhuA outer membrane receptor of Escherichia coli K-12.

The FhuA protein of the outer membrane serves as a receptor for phages T5, T1, and phi 80, for colicin M, for the antibiotic albomycin, and for ferrichrome and related siderophores. To identify protein regions important for the multiple FhuA activities, fhuA genes of spontaneous chromosomal mutants which expressed wild-type amounts of the FhuA protein were sequenced. A mutant which was partially T5 sensitive but impaired in all other functions was missing aspartate residue 348 of the mature protein as a result of a three-base deletion. This aspartate residue is part of the hydrophilic sequence Asp-Asp-Glu-Lys. Replacement by site-specific mutagenesis of each of the Asp residues by Tyr, of Glu by Val, and of Lys by Met reduced FhuA activity but less than the Asp deletion did. Ferrichrome inhibited binding of phage phi 80 and of colicin M to these mutants in an allele-specific manner. A completely resistant derivative of the Asp deletion mutant contained, in addition, a leucine-to-proline substitution at position 106 and eight changed bases, converting at positions 576 to 578 an Arg-Pro-Leu sequence to Ala-Arg-Cys. The latter mutations and the Leu-to-Pro replacement alone did not alter sensitivity to the phages but reduced sensitivity to colicin M and albomycin 10- to 1,000-fold. The proline replacements probably disturb FhuA conformation and, in concert with the Asp deletion, inactivate FhuA completely. It is concluded that the Asp deletion site defines a region of FhuA which directly participates in binding of all FhuA ligands. Growth promotion studies on iron-limited media revealed that certain siderophores of the hydroxamate type, such as butylferrichrome, ferrichrysin, and ferrirubin, are taken up not only via FhuA but also via the FhuE outer membrane receptor protein.     

The β-barrel domain of FhuAΔ5-160 is sufficient for TonB-dependent FhuA activities of Escherichia coli

FhuA in the outer membrane of Escherichia coli serves as a transporter for ferrichrome, the antibiotics albomycin and rifamycin CGP4832, colicin M, and as receptor for phages T1, T5 and phi80. The previously determined crystal structure reveals that residues 160-714 of the mature protein form a beta-barrel that is closed from the periplasmic side by the globular N-proximal fragment, residues 1-159, designated the cork. In this study, deletion of the cork resulted in a stable protein, FhuADelta5-160, that was incorporated in the outer membrane. Cells that synthesized FhuADelta5-160 displayed a higher sensitivity to large antibiotics such as erythromycin, rifamycin, bacitracin and vancomycin, and grew on maltotetraose and maltopentaose in the absence of LamB. Higher concentrations of ferrichrome supported growth of a tonB mutant that synthesized FhuADelta5-160. These results demonstrate non-specific diffusion of compounds across the outer membrane of cells that synthesize FhuADelta5-160. However, growth of a FhuADelta5-160 tonB wild-type strain occurred at low ferrichrome concentrations, and ferrichrome was transported at about 45% of the FhuA wild-type rate despite the lack of ferrichrome binding sites provided by the cork. FhuADelta5-160 conferred sensitivity to the phages and colicin M at levels similar to that of wild-type FhuA, and to albomycin and rifamycin CGP 4832. The activity of FhuADelta5-160 depended on TonB, although the mutant lacks the TonB box (residues 7-11) previously implicated in the interaction of FhuA with TonB. CCCP inhibited tonB-dependent transport of ferrichrome through FhuADelta5-160. FhuADelta5-160 still functions as a specific transporter, and sites in addition to the TonB box are involved in the TonB-mediated response of FhuA to the proton gradient of the cytoplasmic membrane. It is proposed that TonB interacts with the TonB box of FhuA and with the beta-barrel to release ferrichrome from the FhuA binding sites and to open the channel in FhuA. For transport of ferrichrome through the open channel of FhuADelta5-160, interaction of TonB with the beta-barrel is sufficient to release ferrichrome from the residual binding sites at the beta-barrel and to induce the active conformation of the L4 loop at the cell surface for infection by the TonB-dependent phages T1 and phi80.     

Conversion of the FhuA transport protein into a diffusion channel through the outer membrane of Escherichia coli.

The FhuA receptor protein is involved in energy-coupled transport of Fe3+ via ferrichrome through the outer membrane of Escherichia coli. Since no energy source is known in the outer membrane it is assumed that energy is provided through the action of the TonB, ExbB and ExbD proteins, which are anchored to the cytoplasmic membrane. By deleting 34 amino acid residues of a putative cell surface exposed loop, FhuA was converted from a ligand specific transport protein into a TonB independent and nonspecific diffusion channel. The FhuA deletion derivative FhuA delta 322-355 formed stable channels in black lipid membranes, in contrast to wild-type FhuA which did not increase membrane conductance. The single-channel conductance of the FhuA mutant channels was at least three times larger than that of the general diffusion porins of E. coli outer membrane. It is proposed that the basic structure of FhuA in the outer membrane is a channel formed by beta-barrels. Since the loop extending from residue 316 to 356 is part of the active site of FhuA, it probably controls the permeability of the channel. The transport-active conformation of FhuA is mediated by a TonB-induced conformational change in response to the energized cytoplasmic membrane. The ferrichrome transport rate into cells expressing FhuA delta 322-355 increased linearly with increasing substrate concentration (from 0.5 to 20 microM), in contrast to FhuA wild-type cells, which displayed saturation at 5 microM. This implies that in wild-type cells ferrichrome transport through the outer membrane is the rate-limiting step and that TonB, ExbB and ExbD are only required for outer membrane transport.     

Specific In Vivo Labeling of Cell Surface-Exposed Protein Loops: Reactive Cysteines in the Predicted Gating Loop Mark a Ferrichrome Binding Site and a Ligand-Induced Conformational Change of the Escherichia coli FhuA Protein

The FhuA protein of Escherichia coli K-12 transports ferrichrome, the antibiotic albomycin, colicin M, and microcin 25 across the outer membrane and serves as a receptor for the phages T1, T5, 80, and UC-1. FhuA is activated by the electrochemical potential of the cytoplasmic membrane, which probably opens a channel in FhuA. It is thought that the proteins TonB, ExbB, and ExbD function as a coupling device between the cytoplasmic membrane and the outer membrane. Excision of 34 residues from FhuA, tentatively designated the gating loop, converts FhuA into a permanently open channel. FhuA contains two disulfide bridges, one in the gating loop and one close to the C-terminal end. Reduction of the disulfide bridges results in a low in vivo reaction of the cysteines in the gating loop and no reaction of the C-terminal cysteines with biotin-maleimide, as determined by streptavidin-β-galactosidase bound to biotin. In this study we show that a cysteine residue introduced into the gating loop by replacement of Asp-336 displayed a rather high reactivity and was used to monitor structural changes in FhuA upon binding of ferrichrome. Flow cytometric analysis revealed fluorescence quenching by ferrichrome and albomycin of fluorescein-maleimide bound to FhuA. Ferrichrome did not inhibit Cys-336 labeling. In contrast, labeling of Cys-347, obtained by replacing Val-347 in the gating loop, was inhibited by ferrichrome, but ferrichrome quenching was negligible. It is concluded that binding of ferrichrome causes a conformational change of the gating loop and that Cys-347 is part of or close to the ferrichrome binding site. Fluorescence quenching was independent of the TonB activity. The newly introduced cysteines and the replacement of the existing cysteines by serine did not alter sensitivity of cells to the FhuA ligands tested (T5, 80, T1, colicin M, and albomycin) and fully supported growth on ferrichrome as the sole iron source. Since cells of E. coli K-12 display no reactivity to thiol reagents, newly introduced cysteines can be used to determine surface-exposed regions of outer membrane proteins and to monitor conformational changes during their function.     

Diffusion through channel derivatives of the Escherichia coli FhuA transport protein.

FhuA is a multifunctional protein in the outer membrane of Escherichia coli that actively transports [Fe3+]ferrichrome, the antibiotics albomycin and rifamycin CGP 4832, and mediates sensitivity of cells to the unrelated phages T5, T1, phi80 and UC-1, and to colicin M and microcin J25. The energy source of active transport is the proton motive force of the cytoplasmic membrane that is required for all FhuA functions except for infection by phage T5. The FhuA crystal structure reveals 22 antiparallel transmembrane beta-strands that form a beta-barrel which is closed by a globular N-terminal domain. FhuA still displays active transport and sensitivity to all ligands except microcin J25 when the globular domain (residues 5-160) is excised and supports weakly unspecific diffusion of substrates across the outer membrane. Here it is shown that isolated FhuADelta5-160 supported diffusion of ions through artificial planar lipid bilayer membranes but did not form stable channels. The double mutant FhuADelta5-160 Delta322-336 lacking in addition to the globular domain most of the large surface loop 4 which partially constricts the channel entrance, displayed an increased single-channel conductance but formed no stable channels. It transported in vivo[Fe3+]ferrichrome with 45% of the rate of wild-type FhuA and did not increase sensitivity of cells to antibiotics. In contrast, a second FhuA double mutant derivative which in addition to the globular domain contained a deletion of residues 335-355 comprising one-third of surface loop 4 and half of the transmembrane beta-strand 8 formed stable channels in lipid bilayers with a large single-channel conductance of 2.5 nS in 1 m KCl. Cells that synthesized FhuADelta5-160 Delta335-355 showed an increased sensitivity to antibiotics and supported diffusion of maltodextrins, SDS and ferrichrome across the outer membrane. FhuADelta5-160 Delta335-355 showed no FhuA specific functions such as active transport of [Fe3+]ferrichrome or sensitivity to the other FhuA ligands. It is concluded that FhuADelta5-160 Delta335-355 assumes a conformation that is incompatible with any of the FhuA functions.     

Identification of the sid outer membrane receptor protein in Salmonella typhimurium SL1027.

Summary A protein of molecular weight 78,000 daltons, missing in albomycin and phage ES18 resistant mutants, has been identified in the outer membrane of Salmonella typhimurium SL1027. Mutants with a tonB like resistance and overproduction of outer membrane proteins due to iron shortage were also isolated. The mutation which leads to the protein deficiency maps in the sid gene region, the mutation related to overproduction of proteins maps near trp. Although the S. typhimurium and the E. coli protein mediate translocation of the iron complex ferrichrome and the structurally analogous antibiotic albomycin through the outer membrane no cross-reactivity exists in binding the phages T5, T1 and ES18 or colicin M.     

Isolation,solubilization and reaggregation of outer membrane of Escherichia coli

Cytoplasmic (inner) and outer membranes of Escherichia coli K-12 were isolated with fair separation from each other, and their chemical, biological and morphological properties were compared. The outer membrane isolated was composed of protein, phospholipid and lipopolysaccharide as major high molecular weight components in a ratio of 100:82:34 (by wt), and was solubilized in 1% sodium dodecyl sulfate without any sediments. In polyacrylamide disc gel electrophorsis with the sodium dodecyl sulfate-solubilized outer membrane, six proteins were found to be major. Removal of sodium dodecyl sulfate from the sodium dodecyl sulfate-solubilized outer membrane by dialysis induced a self-assembly to form a membrane structure which has similar properties in chemical composition, density and morphology to those of the original outer membrane.     

Bacterial Phage Receptors, Versatile Tools for Display of Polypeptides on the Cell Surface

Four outer membrane proteins of Escherichia coli were examined for their capabilities and limitations in displaying heterologous peptide inserts on the bacterial cell surface. The T7 tag or multiple copies of the myc epitope were inserted into loops 4 and 5 of the ferrichrome and phage T5 receptor FhuA. Fluorescence-activated cell sorting analysis showed that peptides of up to 250 amino acids were efficiently displayed on the surface of E. coli as inserts within FhuA. Strains expressing FhuA fusion proteins behaved similarly to those expressing wild-type FhuA, as judged by phage infection and colicin sensitivity. The vitamin B(12) and phage BF23 receptor BtuB could display peptide inserts of at least 86 amino acids containing the T7 tag. In contrast, the receptors of the phages K3 and lambda, OmpA and LamB, accepted only insertions in their respective loop 4 of up to 40 amino acids containing the T7 tag. The insertion of larger fragments resulted in inefficient transport and/or assembly of OmpA and LamB fusion proteins into the outer membrane. Cells displaying a foreign peptide fused to any one of these outer membrane proteins were almost completely recovered by magnetic cell sorting from a large pool of cells expressing the relevant wild-type platform protein only. Thus, this approach offers a fast and simple screening procedure for cells displaying heterologous polypeptides. The combination of FhuA, along with with BtuB and LamB, should provide a comprehensive tool for displaying complex peptide libraries of various insert sizes on the surface of E. coli for diverse applications.     

Identification of a New Site for Ferrichrome Transport by Comparison of the FhuA Proteins of Escherichia coli, Salmonella paratyphi B, Salmonella typhimurium, and Pantoea agglomerans

The fhuA genes of Salmonella paratyphi B, Salmonella typhimurium, and Pantoea agglomerans were sequenced and compared with the known fhuA sequence of Escherichia coli. The highly similar FhuA proteins displayed the largest difference in the predicted gating loop, which in E. coli controls the permeability of the FhuA channel and serves as the principal binding site for the phages T1, T5, and 80. All the FhuA proteins contained the region in the gating loops required in E. coli for ferrichrome and albomycin transport. The three subdomains required for phage binding were contained in the gating loop of S. paratyphi B which is infected by the E. coli phages, whereas two of the subdomains were deleted in S. typhimurium and P. agglomerans which are resistant to the E. coli phages. Small deletions in a surface loop adjacent to the gating loop, residues 236 to 243 and 236 to 248, inactivated E. coli FhuA with regard to transport of ferrichrome and albomycin, but sensitivity to T1 and T5 was fully retained and sensitivity to 80 and colicin M was reduced 10-fold. Full-size FhuA hybrid proteins of S. paratyphi B and S. typhimurium displayed S. paratyphi B FhuA activity when the hybrids contained two-thirds of either the N- or the C-terminal portions of S. paratyphi B and displayed S. typhimurium FhuA activity to phage ES18 when the hybrid contained two-thirds of the N-terminal region of the S. typhimurium FhuA. The central segment of the S. paratyphi B FhuA flanked on both sides by S. typhimurium FhuA regions conferred full sensitivity only to phage T5. The data support the essential role of the gating loop for the transport of ferrichrome and albomycin, identified an additional loop for ferrichrome and albomycin uptake, and suggest that several segments and their proper conformation, determined by the entire FhuA protein, contribute to the multiple FhuA activities.     

Export and activity of hybrid FhuA''-''Iut receptor proteins and of truncated FhuA'' proteins of the outer membrane of Escherichia coli

Summary The FhuA protein in the outer membrane of Escherichia coli serves as a multifunctional receptor for the phages T5, T1, 80, for colicin M, for ferrichrome (Fe³⁺-siderophore) and for the structurally related antibiotic, albomycin. To determine structural domains required for these receptor functions and for export, a fusion protein between FhuA and Iut (receptor for Fe³⁺-aerobactin and cloacin DF13) was constructed. In the FhuA-Iut hybrid protein, 24 amino acids of FhuA were replaced by 19 amino acids, 18 of which were from Iut. The number of plaque forming units of phage T5 and T1 on cells expressing FhuA-Iut was nearly as high as on cells expressing plasmid-encoded wild-type FhuA. However, 10⁷-fold higher concentrations of phage 80 and 10³ times more colicin M were required to obtain a zone of growth inhibition. Truncated FhuA proteins in which the last 24 amino acids at the carboxy-terminus were replaced by 16 (FhuA2) or 3 (FhuAT) amino acids could hardly be detected on polyacrylamide electrophoretograms of outer membrane proteins, due to proteolytic degradation. Sensitivity of cells expressing FhuA2 to phage T5 and T1 was reduced by several orders of magnitude and sensitivity to phage 80 and colicin M was totally abolished. In contrast, cells expressing FhuAT were nearly as sensitive to phage T5, T1, and 80 and to colicin M as cells containing FhuA-Iut. None of the constructs could grow on ferrichrome as sole iron source and none was sensitive to albomycin. Ferrichrome did not inhibit binding of T5 to TonB^– cells expressing FhuA-Iut (as it did in FhuA⁺ cells) due to the lack of ferrichrome binding. It is concluded that very small deletions (relative to the size of FhuA with 714 amino acids) at the C-terminal end render FhuA susceptible to proteolytic cleavage. The C-terminal alterations affect sensitivity to FhuA-specific agents very differently. Apparently, the C-terminus is a very important part of FhuA regarding stability and activity. Expression of active FhuA and partially inactive FhuA derivatives in the same cell revealed no negative complementation, suggesting a FhuA monomer as functional unit.     

Transport of iron across the outer membrane

Summary The TonB protein is involved in energy-coupled receptor-dependent transport processes across the outer membrane. The TonB protein is anchored in the cytoplasmic membrane but exposed to the periplasmic space. To fulfill its function, it has to couple the energy-providing metabolism in the cytoplasmic membrane with regulation of outer membrane receptor activity. Ferrichrome and albomycin transport, uptake of colicin M, and infection by the phages T1 and80 occur via the same receptor, the FhuA protein in the outer membrane. Therefore, this receptor is particularly suitable for the study of energy-coupled TonB-dependent transport across the outer membrane. Ferrichrome, albomycin and colicin M bind to the FhuA receptor but are not released into the periplasmic space of unenergized cells, ortonB mutants. In vivo interaction between FhuA and TonB is suggested by the restoration of activity of inactive FhuA proteins, bearing amino acid replacements in the TonB box, by TonB derivatives with single amino acid substitutions. Point mutations in thefhuA gene are suppressed by point mutations in thetonB gene. In addition, naturally occurring degradation of the TonB protein and its derivatives is preferentially prevented in vivo by FhuA and FhuA derivatives where functional interaction takes place. It is proposed that in the energized state, TonB induces a conformation in FhuA which leads to the release of the FhuA-bound compounds into the periplasmic space. Activation of FhuA by TonB depends on the ExbBD proteins in the cytoplasmic membrane. They can be partially replaced by the TolQR proteins which show strong sequence similarity to the ExbBD proteins. A physical interaction of these proteins with the TonB protein is suggested by TonB stabilization through ExbB and TolQR. We propose a permanent or reversible complex in the cytoplasmic membrane composed of the TonB protein and the ExbBD/TolQR proteins through which TonB is energized.