INTRODUCTION

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The FhuA outer membrane transport protein of Escherichia coli consists of 22 antiparallel -sheets that form a -barrel into which a globular domain is inserted from the periplasmic side. The globular domain seems to close the -barrel channel and prevent entry of even small molecules and was for this reason designated the "cork" (7) or "plug" (20). Ferrichrome, the natural substrate of FhuA, binds in a cavity located well above the outer membrane lipid bilayer. The cork domain and the -barrel domain contribute five and six amino acid side chains to the cavity, respectively, which are less than 4 Å away from the ferrichrome (7). It is thought that opening of the FhuA channel requires dislocation of the cork, resulting in a connection between the cavity exposed to the cell surface and the region exposed to the periplasm. Although binding of ferrichrome to FhuA moves the cork about 2 Å towards ferrichrome, this does not open the channel.

Energy provided by the cytoplasmic membrane in the form of the proton motive force (3) and the TonB-ExbB-ExbD protein complex are required for active transport through FhuA. Binding of ferrichrome results in the movement of Glu19 17 Å away from its former -carbon position, which probably facilitates binding of FhuA to TonB. This hypothesis is supported by the finding that chemical cross-linking of FhuA to TonB is enhanced in vivo upon binding of ferrichrome (25). An N-proximal region of FhuA, residues 7 to 11 (TonB box), interacts with a region around residue 160 of TonB, as shown by mutations in the TonB box that are suppressed by mutations in TonB (9, 30).

A similar suppression analysis revealed the same interacting regions in the BtuB vitamin B₁₂ transport protein and in TonB (11). Moreover, in vivo a segment of the TonB box of BtuB is chemically cross-linked via disulfide bonds with a segment around residue 160 of TonB (6). Cross-linking at several positions is increased when BtuB is loaded with vitamin B₁₂, and the cross-linking pattern changes in mutants containing amino acid substitutions in BtuB that impair TonB-dependent BtuB activity. Site-directed spin labeling and electron paramagnetic resonance assays have suggested that the TonB box of BtuB in the unliganded conformation is located in a helix that forms specific interactions with side chain residues of the periplasmic turns of the -barrel domain of BtuB (23). Binding of vitamin B₁₂ to BtuB converts this segment into an extended, disordered, and highly dynamic structure that likely extends into the periplasm to interact physically with TonB. A TonB-uncoupled TonB box mutant of BtuB shows a strongly altered electron paramagnetic resonance spectrum and no longer responds to the addition of vitamin B₁₂. These experiments strongly support the interaction of the transporter TonB box with the region around residue 160 of TonB.

In a previous study, we deleted the cork domain, including the TonB box, of E. coli FhuA. To our surprise, the protein FhuA5-160 was found in the outer membrane, although in amounts lower than that of wild-type FhuA; FhuA5-160 could still transport ferrichrome (at 30 to 40% the rate of wild-type FhuA) and albomycin in a TonB-dependent manner and conferred the same or almost the same degree of sensitivity as wild-type FhuA to the TonB-dependent colicin M and the phages T1 and 80 and to the TonB-independent phage T5 (4). Since FhuA5-160 lacks the TonB box, TonB must interact with other regions of FhuA, and this interaction suffices for TonB-dependent FhuA activities. FhuA5-160 mediates slow diffusion, since sensitivity to larger hydrophilic antibiotics to which the outer membrane normally forms a permeability barrier is only moderately increased and cells remain resistant to sodium dodecyl sulfate (SDS) and EDTA.

In this study, we intended to corroborate our previous results with the E. coli FhuA5-160 protein by constructing FhuA5-160 derivatives of Salmonella paratyphi B, Salmonella enterica serovar Typhimurium, and Pantoea agglomerans; we have previously determined the fhuA nucleotide sequences of these strains (17). Comparison of the E. coli FhuA amino acid sequence with that of S. paratyphi B, Salmonella serovar Typhimurium and P. agglomerans revealed 94, 79, and 60% identity in the cork domain and 92, 74, and 58% identity in the -barrel domain, respectively. In addition, we exchanged the cork domains to determine whether cork domains insert into heterologous -barrel domains and whether the resulting FhuA hybrid proteins still respond to TonB and the proton motive force.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, and growth conditions. The E. coli strains and plasmids used are listed in Table 1. Cells were grown in TY medium (10 g of Bacto tryptone [Difco Laboratories]/liter, 5 g of yeast extract/liter, 5 g of NaCl/liter) or NB medium (8 g of nutrient broth/liter, 5 g of NaCl/liter, pH 7) at 37°C. To reduce the available iron of the NB medium, 2,2'-dipyridyl (0.2 mM) was added (NBD medium). The antibiotics ampicillin (40 µg/ml) and chloramphenicol (25 µg/ml) were added when required.

Recombinant DNA techniques. Isolation of plasmids, use of restriction enzymes, ligation, agarose gel electrophoresis, and transformation were performed according to standard techniques (29). All genetic constructions were examined by DNA sequencing using the dideoxy chain-termination method with fluorescence-labeled or unlabeled nucleotides (Auto Read Sequencing Kit, Pharmacia Biotech, Freiburg, Germany) and the ALF sequencer (Pharmacia).

Protein analytical methods. E. coli BL21 cells (optical density at 578 nm of 0.5) transformed with one of various plasmids encoding complete FhuA, corkless FhuA, or reconstituted FhuA hybrids were collected by centrifugation and resuspended in 1 ml of M9 salts (24) supplemented with 0.4% glucose, 0.01% methionine assay medium, 0.01% thiamine, and 1 mM IPTG (isopropyl--D-thiogalactopyranoside) to induce T7 RNA polymerase synthesis. After shaking the cells for 1 h at 37°C, rifamycin (10 µl of a 5-mg/ml solution in methanol) was added and incubation was continued at 37°C for 30 min. [³⁵S]methionine was added, and the suspension was incubated for 10 min. Cells were then collected by centrifugation and suspended in sample buffer. The radioactively labeled proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously (12). In addition, cells transformed with wild-type fhuA and mutant fhuA genes were grown in NB medium, the outer membrane fractions were isolated, and the proteins were separated by SDS-PAGE and stained with Serva blue.

Phenotype assays. All phenotype assays were carried out with freshly transformed E. coli K-12 strains 41/2 aroB fhuA, HK97 aroB fhuA fhuE, and HK99 aroB fhuA tonB. These strains carry the same four amino acid replacements and an amino acid deletion in fhuA and contain the mutated FhuA protein in the outer membrane (12). The plasmid-encoded fhuA genes in the transformants were transcribed from the fhuA promoter. The sensitivity of cells against the FhuA ligands (phages T1, T5, 80, and ES18, colicin M, microcin J25, rifamycin CGP 4832, and albomycin) was tested by spotting 10-fold-diluted solutions (4 µl) on TY agar plates overlaid with 3 ml of TY soft agar containing 10⁸ cells of the strain to be tested. The colicin M solution was a crude extract of a strain carrying plasmid pTO4 cma cmi (26). The microcin J25 solution was a supernatant of E. coli MC4100 carrying the plasmid pTUC203 mcjABCD (31) after growth of the transformants in brain heart infusion medium (37 g/liter; Difco Laboratories) at 37°C.

Transport and binding assays. E. coli K-12 strains 41/2 aroB fhuA, HK97 aroB fhuA fhuE, HK99 aroB fhuA tonB, and CH1857 fhuACDB tonB aroB freshly transformed with the plasmids to be tested were grown overnight on TY plates. Cells were washed and suspended in transport medium (M9 salts [24], 0.4% glucose), and the cell density was then adjusted to an optical density at 578 nm of 0.5. Free iron ions were removed by adding 25 µl of 10 mM nitrilotriacetate, pH 7.0, to 1 ml of cells. After incubation for 5 min at 37°C, transport or binding assays were started by adding 10 µl of 100 µM [⁵⁵Fe³⁺]ferrichrome. Only in the case of binding assays, a 150-fold surplus of nonradioactive ferrichrome was added as a chase after 19 min to show the specificity of the ferrichrome binding. Samples of 100 µl were withdrawn, and cells were harvested on cellulose nitrate filters (pore size, 0.45 µm; Sartorius AG, Göttingen, Germany) and washed twice with 5 ml of 0.1 M LiCl. The filters were dried, and the radioactivity was determined by liquid scintillation counting.

Computer-assisted sequence analysis. Sequences were analyzed using the program package PC.GENE and the BLAST homology search (1).

	RESULTS

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FhuA5-160 corkless deletion derivatives display TonB-dependent activities. Precise excision of the cork domain of E. coli FhuA results in a stable barrel that is inserted into the outer membrane and exerts TonB-dependent FhuA activities. Deletions within the cork domain and deletions in the barrel domain, with the exception of the surface-exposed loops, frequently result in unstable FhuA derivatives (4). In this study, we excised the cork domain of FhuA from S. paratyphi B, Salmonella serovar Typhimurium, and P. agglomerans based on the E. coli FhuA crystal structure. The cork domain of all FhuA proteins used in this study have the same length as that of E. coli FhuA, except for FhuA of P. agglomerans, which contains a three-amino-acid insertion and a two-amino-acid deletion (17). In addition, the amino acid sequences of all four FhuA proteins are rather similar, which makes it likely that the cork domains comprise the same or nearly the same segment of the FhuA polypeptide.

View larger version (71K): [in this window] [in a new window]   FIG. 1.   Comparison of [35S]methionine-labeled FhuA proteins and FhuA barrel-cork hybrids after transformation of E. coli CH21 with plasmid pHK763 (lane 1), pEcBSpC (lane 2), pEcBStC (lane 3), pEcBPaC (lane 4), pBK7 (lane 5), p76Sp (lane 6), pSpBEcC (lane 7), pSpBStC (lane 8), pSpBPaC (lane 9), pSp5-160 (lane 10), p76St (lane 11), pStBEcC (lane 12), pStBSpC (lane 13), pStBPaC (lane 14), pSt5-160 (lane 15), p76Pa (lane 16), pPaBEcC (lane 17), pPaBSpC (lane 18), pPaBStC (lane 19), or pPa5-160 (lane 20). No other bands were seen outside the gel section represented here. Nomenclature used: EcBSpC means the -barrel (B) of E. coli FhuA and cork (C) of S. paratyphi B FhuA; the other designations follow the same rule. Ec, E. coli; Sp, S. paratyphi B; St, Salmonella serovar Typhimurium; Pa, P. agglomerans.

View larger version (51K): [in this window] [in a new window]   FIG. 2.   Stained proteins after SDS-PAGE of outer membrane fractions of E. coli HK97 fhuA transformed with the plasmids listed in Table 1 that encoded the FhuA proteins indicated in the figure. Solid arrows denote complete FhuA and reconstituted FhuA, and dotted arrows denote corkless FhuA. The molecular masses of standard proteins in kDa are indicated. The nomenclature used is described in the legend for Fig. 1.

View larger version (36K): [in this window] [in a new window]   FIG. 3.   Time-dependent transport of [55Fe3+]ferrichrome (1 µM) into E. coli HK97 fhuA fhuE aroB expressing the plasmid-encoded FhuA proteins and FhuA barrel-cork hybrids of E. coli (Ec, panel A), S. paratyphi (Sp, panel B), Salmonella serovar Typhimurium (St, panel C), and P. agglomerans (Pa, panel D) as indicated in the figure.

View larger version (19K): [in this window] [in a new window]   FIG. 4.   Binding of [55Fe3+]ferrichrome to E. coli CH1857 fhuABCD tonB aroB expressing the indicated FhuA proteins. After 19 min a 150-fold surplus of nonradioactive ferrichrome was added (marked by an arrow).

FhuA5-160 deletion derivatives display low open channel activities. For the determination of active transport, 1 µM [⁵⁵Fe³⁺]ferrichrome was used. At this ferrichrome concentration, growth on nutrient broth agar plates containing 0.2 mM dipyridyl to suppress low-affinity iron uptake (NBD plates) is not supported. For an estimation of ferrichrome uptake by diffusion across the outer membrane, E. coli HK99 fhuA tonB aroB transformed with plasmids carrying the genes for the corkless FhuA proteins was used. Ferrichrome at concentrations of 0.1, 0.3, 1, 3, and 10 mM was placed on filter paper disks, and growth promotion around the disks on NBD plates seeded with 10⁸ cells of the HK99 transformants was recorded. Slow growth of a small number of cells that synthesized FhuA5-160 of E. coli or P. agglomerans was observed with 0.1 mM ferrichrome. The same result was obtained with cells that synthesized FhuA5-160 of S. paratyphi B when a solution of 0.3 mM ferrichrome was used. At this concentration, cells that synthesized FhuA5-160 of E. coli or P. agglomerans showed a strong growth zone of 10 mm in diameter (6-mm disk diameter not subtracted). At 10 mM ferrichrome, cells synthesizing E. coli FhuA5-160, S. paratyphi B FhuA5-160, Salmonella serovar Typhimurium FhuA5-160, and P. agglomerans FhuA5-160 had growth zones of 18, 13, 18, and 22 mm, respectively.

Hybrid FhuA proteins consisting of -barrel domains and unrelated cork domains are active. The cork and -barrel domains of the enterobacterial FhuA proteins were mutually exchanged to determine whether complete FhuA can be reconstituted, exported across the cytoplasmic membrane, and inserted correctly into the outer membrane. Moreover, it was of interest to determine whether FhuA hybrids consisting of -barrel domains and unrelated cork domains display activity with some or all of the ligands and whether the reconstituted FhuA proteins still respond to TonB.

There is no preference for the TonB protein related to the FhuA cork or -barrel. We first determined the transport activities of all FhuA derivatives in E. coli, which means in combination with the E. coli TonB protein. We then wanted to find out whether it makes a difference in FhuA activity when the FhuA hybrids are combined with the TonB proteins of the same strains from which the FhuA hybrids were derived. In addition, since TonB apparently interacts with the cork and the -barrel it was of interest to determine whether the cork or the -barrel should be from the same strain as the TonB protein. We constructed combinations of tonB genes on a low-copy plasmid with the plasmid-encoded wild-type fhuA and mutated fhuA genes in E. coli HK99 fhuA tonB, with the exception of the tonB gene of S. paratyphi B, which was unavailable. All the combinations were active, and the absolute transport rates listed as 100% in Table 2 are similar to the highest transport rates shown in Fig. 3. No alterations of the FhuA activities were observed that could be related to homologous versus heterologous FhuA-TonB combinations or to the cork or the -barrel (Table 2). The FhuA activities of the E. coli FhuA -barrel derivatives combined with TonB of E. coli (Table 2, HK97 and HK99 1) differed only slightly from the E. coli FhuA -barrel derivatives combined with the TonB protein of Salmonella serovar Typhimurium and P. agglomerans (HK99 2). When the -barrel of S. paratyphi B was combined with TonB of Salmonella serovar Typhimurium, the FhuA activities were somewhat higher (HK99 1) than when combined with the E. coli TonB (HK97). This increase may be a result of the overexpression of plasmid-encoded TonB, although in other cases the FhuA -barrel derivatives showed lower FhuA activities when combined with plasmid-encoded TonB (HK99) than with chromosomally encoded E. coli TonB (HK97) (Table 2), which has been observed previously (22). There was a tendency of a higher FhuA activity with TonB combined with the related cork domain (HK99 2) than with TonB combined with the related -barrel domain (HK99 1). However, we doubt that the observed differences are large enough to suggest a stronger impact of the cork than the -barrel in the interaction of FhuA with TonB.

The -barrel domain of E. coli FhuA containing the TonB box is less active. The results reported here and in our previous papers (4, 9, 30) indicated that FhuA activity is mediated by TonB through interaction with the cork and the -barrel. Therefore, we examined whether the TonB box linked to the -barrel domain affects the activity of the -barrel. We constructed FhuA25-160, in which the N-proximal 23 residues of mature FhuA, including the TonB box, were linked to residue 161 of the -barrel domain. The genetic manipulation replaced Pro24 with Asp. E. coli 41/2 fhuA synthesizing FhuA25-160(P24D) was as sensitive to phage 80 as E. coli 41/2 fhuA synthesizing FhuA5-160 but was 10-fold less sensitive to phages T1 and T5 and to colicin M and was resistant to albomycin and microcin J25. Since TonB-independent infection by phage T5 was also reduced, the lower activity of FhuA25-160(P24D) cannot be ascribed to an unproductive binding of TonB to the TonB box of FhuA25-160(P24D). This interpretation is supported by the finding that phage T5 sensitivity is also reduced 10-fold in the HK99 tonB mutant. The ferrichrome transport rate was near zero. After 30 min, there were 3,000 ferrichrome molecules per cell compared to 140,000 in cells expressing wild-type FhuA and 45,000 in cells expressing FhuA5-160 in experiments run in parallel. In addition, binding of ferrichrome to FhuA25-160(P24D) was examined. Using E. coli CH21(pBK71), binding of ferrichrome amounted to about 3,000 molecules per cell compared to 20,000 molecules bound to wild-type FhuA of E. coli CH21(pHK763), which after a chase with a 150-fold surplus of unlabeled ferrichrome was reduced to 3,000 molecules per cell. Although the binding site (residue 161) of the N-proximal 24-residue peptide is exposed to the periplasm outside the channel formed by the -barrel, the N-proximal peptide appears to strongly impair the binding of ferrichrome, which occurs well above the cell surface. In addition to binding, the transport activity must also be impaired since FhuA5-160 also binds ferrichrome poorly but transports ferrichrome rather well. It should be stated that the relative amount of FhuA25-160(P24D) protein observed after SDS-PAGE was comparable to that of wild-type FhuA (data not shown).

	DISCUSSION

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Our previous finding of high and specific activities of corkless FhuA of E. coli (4) are supported by the results described in this paper with the corkless FhuA proteins of S. paratyphi B and Salmonella serovar Typhimurium. These corkless FhuA derivatives exhibit TonB-dependent ferrichrome transport, although at rates lower than that of the E. coli corkless FhuA. The amounts of the corkless derivatives were also lower (approximately 25% that of wild-type FhuA), which may have reduced the activities. The rates of 14 and 21% in comparison to the rates obtained with the complete FhuA of the same strain decreased to zero in the tonB mutant strain HK99 carrying the same fhuA mutation as that of E. coli HK97 fhuA used for the transport experiments. To rule out complementation of the mutated E. coli HK97 FhuA protein by the E. coli corkless FhuA mutant protein through formation of a functional oligomer, we previously carried out experiments with E. coli H1857 in which the fhuABCD genes are deleted (4). After transformation of E. coli H1857 with fhuA5-160 and the fhuBCD genes for transport across the cytoplasmic membrane, ferrichrome transport is even higher than transport into E. coli HK97 since E. coli H1857 synthesizes greater amounts of plasmid-encoded FhuBCD proteins than E. coli HK97. In addition, X-ray analysis does not support the formation of an FhuA oligomer as the FhuA crystals consisted of a monomer (7, 20).

FhuA5-160 of P. agglomerans was considered inactive, as it did not transport ferrichrome, conferred no sensitivity to albomycin, and showed the same sensitivity to rifamycin CGP 4832 as to rifamycin. Among the FhuA proteins studied, that of P. agglomerans exhibits the least sequence similarity to E. coli FhuA (59%). The construction of the deletion introduced the amino acid replacements A3D and E4P; the latter replacement may not affect FhuA5-160 activity, since similar replacements at the A3 site in FhuA5-160 of E. coli (E3D), S. paratyphi B (Q3D), and Salmonella serovar Typhimurium (Q3D) did not abolish activity.

FhuA of S. paratyphi B is the only non-E. coli FhuA that mediates sensitivities to phages T1, T5, and 80 and to colicin M and albomycin, and this specificity was retained in the S. paratyphi B corkless FhuA, although at 1 or 2 orders of magnitude lower than the sensitivity conferred by the complete FhuA. Sensitivity to these FhuA ligands was TonB-dependent, except for infection by phage T5, which occurs independent of TonB. FhuA5-160 of S. paratyphi, like that of E. coli, did not mediate sensitivity to microcin J25 and differed from the E. coli FhuA5-160 in that it did not enhance sensitivity to rifamycin CGP 4832.

The -barrel of E. coli FhuA without the cork mediates all FhuA functions except uptake of microcin J25 (4). Uptake of microcin J25 and infection of Salmonella serovar Typhimurium by phage ES18 may require both the cork and the -barrel. We have previously shown that the prominent loop of the FhuA -barrel (18), which is loop 4 in the E. coli FhuA crystal structure (7, 20) and lies above the cell surface, serves as the principal binding site of the phages and colicin M (13, 14, 15). This result implies that TonB, without the help of the cork, can change the conformation of loop 4 such that binding of phages T1 and 80 triggers DNA release from the phage head. This conformational change is not restricted to loop 4, since release of ferrichrome from its binding sites in the -barrel (residues Y244, W246, Y313, Y315, F391, and F693) probably also requires a conformational change of the -barrel, and none of the ferrichrome binding sites are located in loop 4. These binding sites are contained in the four corkless FhuA proteins, with the exception of Y315, which is replaced in Salmonella serovar Typhimurium and P. agglomerans by T and N, respectively, and F693, which is replaced in P. agglomerans by Y. Since aromatic residues play a major role in ferrichrome and albomycin binding, replacement of Y315 by these nonaromatic amino acids may well contribute to the lower transport activity of Salmonella serovar Typhimurium FhuA5-160 and the inactivity of P. agglomerans FhuA5-160. However, this cannot be the only cause since the transport activity of S. paratyphi FhuA5-160 is rather low (after 12 min, 18,000 ions per cell compared to 48,000 per cell with E. coli FhuA5-160), despite the identity of these residues with those of E. coli FhuA5-160.

TonB-dependent conformational changes of the -barrel may also widen the channel to facilitate diffusion of ferrichrome and albomycin once they are released from their binding sites and/or may properly position the amino acid side chains along which ferrichrome and albomycin diffuse through FhuA. These possibilities should be considered due to the low diffusion rates through the corkless FhuA proteins, as evidenced by the small increase in sensitivity to the antibiotics erythromycin, rifamycin, and vancomycin compared to the same E. coli strain synthesizing plasmid-encoded wild-type FhuA proteins.

If TonB interacts only with -barrel regions exposed to the periplasm, the conformational change must be transmitted across the entire FhuA molecule up to the cell surface. It is not known whether TonB inserts into the outer membrane. However, the observed shuttling of TonB between the outer membrane and the cytoplasmic membrane (19) excludes a firm integration of TonB in the outer membrane.

Fusions of cork domains with -barrel domains of different species were constructed to determine whether the corks are inserted into the -barrels, how they fit into the -barrels, and whether they restore the activities to those of complete wild-type homologous FhuA proteins. It was conceivable that the corks were not incorporated into the -barrels, that the hybrid proteins were rapidly degraded in the cytoplasm or the periplasm, that they stayed in the cytoplasm and were not exported across the cytoplasmic membrane, that they remained in the periplasm, or that they were inserted into the outer membrane in an inactive form. The heterologous corks could interact with the -barrels such that structural transitions in the -barrels and the corks upon binding of the ligands and TonB were blocked or aberrant. We did not have to investigate all these possibilities since we obtained FhuA hybrids present in processed form in amounts similar to those of the wild-type FhuA proteins. The exceptions were the FhuA hybrids which contained the P. agglomerans -barrel and heterologous cork domains, which formed several bands of which one was probably the genuine FhuA hybrid. However, the reduced amounts of these hybrids do not fully explain the low activity, as they were present in higher amounts than that observed with chromosomally encoded wild-type FhuA, which confers full FhuA activity (10, 12). FhuAPa5-160 is somewhat unstable, as the band pattern demonstrates, and the hybrid proteins appear to be even less stable. Nevertheless, the hybrid proteins exhibit ferrichrome transport activity, while the corkless mutant does not. Most FhuA hybrids transported ferrichrome with rates higher than those of the corkless FhuA proteins from which they were derived. FhuAEcBStC displayed a low transport rate (17% that of E. coli FhuA wild-type), which may have attributed to the protein's instability (Fig. 2). In contrast, FhuAStBPaC is inactive despite its high amounts in the outer membrane fraction (Fig. 2). In this hybrid protein the cork apparently does not fit into the -barrel to reconstitute an active FhuA protein. In all mutant FhuA proteins the degree of albomycin sensitivity correlated with the ferrichrome transport rates.

Increased sensitivity to rifamycin CGP 4832, compared to rifamycin and sensitivity to microcin J25 were only mediated by FhuA hybrids containing the -barrel of E. coli or S. paratyphi B. The binding site of CGP 4832 in FhuA, as derived from the FhuA cocrystal structure (A. D. Ferguson, J. Ködding, G. Walker, C. Bös, J. W. Coulton, K. Diederichs, V. Braun, and W. Welte, unpublished data), largely overlaps with the ferrichrome and albomycin (8) binding site. The same amino acid residues contribute to binding of ferrichrome, albomycin, and CGP 4832 in the E. coli and S. paratyphi B FhuA proteins, except for a single, functionally equivalent ED exchange in S. paratyphi B. Of the total of 16 residues that bind CGP 4832 to E. coli FhuA, FhuA of Salmonella serovar Typhimurium and P. agglomerans deviate by 4 and 8 residues, respectively. The number of amino acid replacements may explain why the FhuA proteins of Salmonella serovar Typhimurium and P. agglomerans do not show increased sensitivity to CGP 4832. Two out of the 10 residues that in E. coli FhuA bind ferrichrome are different in Salmonella serovar Typhimurium FhuA, and 4 out of 10 differ in P. agglomerans FhuA. These sites also bind albomycin and CGP 4832 in E. coli FhuA.

In addition to the ligand binding sites, the data indicate that other regions are important for the transport activities of the hybrid FhuA proteins. For example, insertion of the Salmonella serovar Typhimurium FhuA cork decreases the activity of the E. coli FhuA -barrel; however, the E. coli cork strongly increases the transport activity of the Salmonella serovar Typhimurium -barrel. The Salmonella serovar Typhimurium cork fused to the S. paratyphi B -barrel results in a highly active transporter. The P. agglomerans cork increases the transport activities when inserted into the E. coli and S. paratyphi B -barrels but fails to complement the Salmonella serovar Typhimurium -barrel. These results show that incorporation of a cork into a barrel is not sufficient to restore transport activity; rather, intimate interactions between the cork and the -barrel must occur in order to form an active transporter. In a previous study, prior to the determination of the FhuA crystal structure, we had replaced the N-proximal 160 amino acids of E. coli FhuA with the first 150 amino acids of FoxA, the ferrioxamine transport protein of Yersinia enterocolitica (16). This FhuA hybrid conferred only low phage T5 sensitivity 4 orders of magnitude below that of wild-type FhuA and even lower phage T1 and 80 sensitivity and a reduced growth with ferrichrome as the sole iron source. In addition to the presumably impaired fitting of the FoxA cork into the FhuA -barrel because of the low sequence identity (36%), the approximately 10 residues of the cork that remain on the -barrel may also have inactivated the FhuA hybrid.

The FhuA hybrid proteins examined here showed nearly wild-type levels of ferrichrome binding, with the exception of those -barrels that contained the cork of P. agglomerans. However, weak binding did not necessarily result in low transport, as demonstrated by FhuAEcBPaC, FhuASpBPaC, and the corkless FhuA derivatives.

Impairment of the E. coli -barrel activity by the Salmonella serovar Typhimurium cork (FhuAEcBStC) also abolished the receptor activity for phage T1 and reduced phage 80 receptor activity 1,000-fold. The sensitivity of FhuAEcBStC to colicin M remained the same as the sensitivity mediated by the -barrel (and wild-type FhuA). In contrast, cells that synthesized FhuAEcBPaC displayed reduced colicin M sensitivity but full phage sensitivity and low ferrichrome binding but high ferrichrome transport. The data collectively reveal different structural requirements of FhuA for the various FhuA activities.

Our results disclose a rather high tolerance of FhuA -barrels for different FhuA corks. The -barrel domains largely determine the activity and specificity of the FhuA proteins. The E. coli cork domain did not confer sensitivity to the E. coli-specific phages, colicin M, rifamycin CGP 4832, and microcin J25 when incorporated into the -barrels of FhuA proteins of Salmonella serovar Typhimurium and P. agglomerans that did not confer these sensitivities. Conversely, the S. paratyphi and P. agglomerans corks, with the exception of the Salmonella serovar Typhimurium cork, left phage sensitivity of the E. coli -barrel unaltered and reduced the colicin M sensitivity of FhuAEcBPaC, synthesizing cells only slightly. In this context, it is interesting to note that the E. coli -barrel fused to the N-terminal 24 FhuA residues bound ferrichrome very poorly and had a low ferrichrome transport rate. It is difficult to predict the effect of the peptide on FhuA -barrel structure since the fusion site is exposed to the periplasm outside the -barrel channel. We expected that the peptide might extend into the periplasm and not affect FhuA activity unless TonB binds to it in an unproductive way and is no longer available for activating FhuA. Since the mutant showed a 10-fold reduced sensitivity to phage T5 independent of the presence of TonB, the properties of this FhuA derivative cannot be explained by a locking of the altered FhuA protein in an inactive conformation through binding to TonB. Apparently, the peptide interacts with the -barrel in a way that affects binding of ferrichrome far away from the peptide binding site, infection by phages, and sensitivity to colicin M, albomycin, and microcin J25. Such long-distance effects have also been observed with the lipoprotein of phage T5 that interacts with periplasmic sites of FhuA but inhibits phage infection, colicin M and albomycin sensitivity, and transport of ferrichrome (5).

We examined whether TonB preferentially activates FhuA of the same strain, and if so, whether interaction with the cork or the -barrel domain is more important. Therefore, we combined TonB with FhuA and corkless FhuA of the same strain and other strains and with FhuA hybrids of which the cork or the -barrel was from the same strain as TonB. Only the results collected with HK99 transformed with the various tonB genes on the same low-copy plasmid can be compared, since the level of TonB expression affects TonB-mediated activities (22). We obtained no ferrichrome transport activity pattern that would be consistent with preference for the same strain. The only and nearly consistent tendency found concerns the higher FhuA activities when TonB was derived from the same strain as the cork (Table 2, HK99 2) than when TonB was derived from the same strain as the -barrel (Table 2, HK99 1). However, the differences in the FhuA activities are not so strong that firm conclusions can be drawn.

It seems to us that FhuA behaves like an allosteric protein, and most of the allosteric transitions induced by binding of TonB, the phages, and ferrichrome take place in the -barrel, although the B-factors of the crystallographic analysis suggest a rather rigid -barrel structure and the multiple conformational changes occur independent of the cork. The cork functions in closing the -barrel channel to avoid uncontrolled diffusion of harmful compounds into the periplasm. It also contributes to ferrichrome binding in that 4 out of 10 ferrichrome binding sites are located in the cork. The strong binding of ferrichrome to FhuA (K_d < 0.1 µM) allows a sufficient iron supply with an iron source present in the culture medium at very low concentrations. For transport, ferrichrome has to be released from the binding sites and the cork has to move to open the channel. This presumably occurs through the concerted action of the cork and the -barrel, both of which respond to the TonB-mediated energy transfer from the cytoplasmic membrane through direct interaction with TonB. The inactive TonB box mutants of FhuA (30), BtuB (11), and Cir (2) suggest that interaction of TonB with the -barrel is not sufficient to open the channel of the wild-type proteins, but interaction with the TonB box of the cork has to occur as well. Our studies with the corkless FhuA mutants of E. coli, S. paratyphi B, and Salmonella serovar Typhimurium indicate important TonB-mediated FhuA activities through interaction of TonB with the -barrel and the cork.