Abstract
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 φ80. 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 φ80 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.
The multifunctional FhuA receptor protein in the outer membrane of Escherichia coli K-12 serves as a binding site for the phages T1, T5, φ80, and UC-1, for the entry of colicin M and microcin 25, and for the uptake of ferrichrome and the structurally related albomycin. With the exception of the infection by phage T5, killing by the phages and the toxins and transport of the iron complexes require the Ton system, which is composed of the proteins TonB, ExbB, and ExbD (6). It is thought that by consumption of energy, provided by the electrochemical potential of the cytoplasmic membrane, FhuA is converted from the ground state to the energized conformation, which is recognized by the Ton-dependent phages and triggers DNA release from the phage head. Inhibition of phage T5 binding by ferrichrome provided evidence for a conformational change of FhuA mediated by TonB. Much less ferrichrome was required to inhibit T5 binding by ferrichrome in unenergized cells and tonB mutants than in energized tonB+ cells (12). It is further assumed that energized FhuA forms an open channel through which the toxins and ferrichrome, which bind to FhuA independent of the Ton system, enter the cytoplasm. Evidence for a FhuA channel was obtained by excision of most of a surface-exposed loop (21), which converted FhuA into a permanently open channel through which ferrichrome, sodium dodecyl sulfate (SDS), and certain antibiotics diffused in vivo (15) and which rendered artificial bilayer membranes permeable to anions and cations (15). The single-channel conductance of the FhuA deletion mutant was at least three times larger than that of the E. coli OmpC and OmpF porins (15). From these results the region comprising residues 322 to 355, or the entire loop (residues 316 to 356), was proposed to form a gating loop that closed the FhuA channel unless it was moved by input of conformational energy provided by the TonB protein. Accessibility of the gating loop from the cell surface was derived from proteolytic cleavage of FhuA in viable cells within inserted 4- and 16-residue peptides after amino acid 321 (21), reaction with a monoclonal antibody (27) and an antibody directed against a C3 viral reporter epitope inserted after residue 321 (25), and labeling of cysteine 318 and cysteine 329 after reduction of the disulfide bridge and of an inserted cysteine (Asp336Cys) with biotin-maleimide and fluorescein-maleimide (4, 5). Interaction of FhuA with TonB was evidenced by suppressor mutations in TonB that partially restored activity of FhuA carrying certain point mutations in the TonB box, a conserved pentapeptide located close to the N terminus of all Ton-dependent outer membrane proteins and colicins (32). Furthermore, degradation of overexpressed TonB by cellular proteases was prevented by overexpressed FhuA (11), by chemical cross-linking of FhuA to TonB, and by retention of TonB on a nickel column loaded with histidine-tagged FhuA (26). Interaction of FhuA with TonB was increased by the ferrichrome homolog ferricrocin (26), suggesting induction of an FhuA conformation by ferricrocin that favors binding to TonB. Indeed, ferrichrome induced a conformational change of FhuA, as was deduced from inhibition of FhuA degradation by added proteases in the presence of ferrichrome (13, 28). In isolated FhuA phage T5 opens a channel without cellular energy input and involvement of the Ton system (3). The physical characteristics of the channel are similar to the channel formed by the FhuA deletion derivative (15). FhuA inserted into liposomes induced the release of phage T5 DNA and its transfer inside the vesicles (30). Phage T5 triggered release of ferrichrome trapped in proteoliposomes containing FhuA (23). These data prove beyond a doubt that FhuA contains a closed channel which is opened upon binding of phage T5. Binding of the other FhuA ligands is not sufficient for opening of the channel but requires in addition an input of energy. However, the differences between the way T5 opens the channel and the ways the other FhuA ligands do so may not be as serious as they seem. Certain tonB point mutations increase T5 infection of certain FhuA mutants 100-fold (18), and host range mutants of phage T1 infect tonB deletion mutants with high efficiency (12).
Further analysis of the FhuA gating loop by competitive peptide mapping revealed three subdomains involved in phage binding which are distributed over the entire loop (20). Deletion of residues 322 to 336 of the gating loop strongly reduced phage binding but not ferrichrome binding. FhuA with residues 322 to 336 deleted (FhuA Δ322-336) supported Ton-dependent ferrichrome transport at a somewhat reduced rate (16). In contrast, FhuA Δ335-355 did not bind ferrichrome and did not actively transport ferrichrome across the outer membrane. It formed a permanently open channel through which ferrichrome, SDS, and maltotetraose diffused into the periplasm (16). Apparently, the segment containing residues 335 to 355 mainly controls the permeability of FhuA.
If only half of the gating loop is essential for ferrichrome transport and the entire loop is required for phage sensitivity, analysis of the FhuA proteins of E. coli-related bacteria may disclose regions that determine specificity for ferrichrome transport and phage infection. Salmonella paratyphi, Salmonella typhimurium (10), and Pantoea agglomerans (formerly Erwinia herbicola) (2) transport ferrichrome and structurally related ferric hydoxamates (22), but only S. paratyphi is sensitive to the E. coli phages. Therefore, we sequenced the fhuA genes of these strains and found that the largest difference between the FhuA proteins is located in the predicted gating loops. The activities of the FhuA proteins are consistent with the proposal that ferrichrome uptake is mainly determined by the region equivalent to residues 335 to 355 of E. coli FhuA. In addition, deletion mutations in a predicted loop near the gating loop inactivated the ferrichrome transport activity of FhuA but left its role in phage infection and colicin M killing largely intact, which indicates its specific involvement in ferrichrome transport.
MATERIALS AND METHODS
Bacterial strains, cosmids, plasmids, and growth conditions.
The E. coli strains and plasmids used in this study are listed in Table 1. Cells were grown in TY medium (Bacto Tryptone [10 g/liter; Difco Laboratories], yeast extract [5 g/liter], NaCl [5 g/liter]) or NB medium (nutrient broth [8 g/liter], NaCl [5 g/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 neomycin (50 μg/ml) were added when required.
TABLE 1.
E. coli K-12 strains and plasmids used in this study
| Strain, cosmid, or plasmid | Genotype or phenotype | Reference or source |
|---|---|---|
| Strains | ||
| HK97 | F−araD139 lacU169 rpsL150 relA1 flbB5301 deoC1 ptsF25 rbsR aroB thi fhuE::λplacMu53 fhuA | 17 |
| WM1576 | K38 HfrC pGP1-2 | 33 |
| Cosmids | ||
| pHC79 | Ampr | 14 |
| pUH62 | pHC79 containing the fhu operon of P. agglomerans | 22 |
| pUH65 | pHC79 containing the fhu operon of S. paratyphi | 22 |
| pUH66 | pHC79 containing the fhu operon of S. typhimurium | 22 |
| Plasmids | ||
| p75Pa | pT7-5 fhuA(Pa) (wild type) and partial fhuC(Pa) (wild type) | This study |
| p76Sp | pT7-6 fhuA(Sp) (wild type) and partial fhuC(Sp) (wild type) | This study |
| p76Sp/1 | pT7-6 fhuA(Sp) (wild type) and partial fhuC(Sp) (wild type)a | This study |
| p76St | pT7-6 fhuA(St) (wild type) and partial fhuC(St) (wild type) | This study |
| p5242 | pT7-6 fhuA(Ec) with C-terminal part of fhuA(Pa) | This study |
| p9489 | pT7-6 fhuA(Pa) with C-terminal part of fhuA(Ec) | This study |
| p1/2 | pT7-6 fhuA(Sp) with central and C-terminal part of fhuA(St) | This study |
| p3/2 | pT7-6 fhuA(St) with central and C-terminal part of fhuA(Sp) | This study |
| p9/9 | pT7-6 fhuA(Sp) with C-terminal part of fhuA(St) | This study |
| p11/15 | pT7-6 fhuA(St) with C-terminal part of fhuA(Sp) | This study |
| p5/21 | pT7-6 fhuA(St) with central part of fhuA(Sp) | This study |
| p7/24 | pT7-6 fhuA(Sp) with central part of fhuA(St) | This study |
| pB3/4 | pT7-6 fhuA(Ec) Δ236-243 | This study |
| pB4/5 | pT7-6 fhuA(Ec) Δ236-248 | This study |
| pHK763 | pT7-6 fhuA(Ec) (wild type) | 17 |
| pHK763B | pT7-6 fhuA(Ec) Nuc. 1597 G→Cb | This study |
| pAM11 | pSL1180 fhuA(Ec) Nuc. 1597 G→C | A. Mademidis |
| pT7-5 | Ampr | 33 |
| pT7-6 | Ampr | 33 |
p76Sp/1 differs from p76Sp by a shorter fhuC fragment at the 3′ end.
Nuc. 1597 G→C, G-to-C substitution.
Cosmid pUH62 was digested with AvaI/HindIII and ligated into the AvaI/HindIII-cleaved vector pT7-5, resulting in plasmid p75Pa, which carries wild-type P. agglomerans fhuA [fhuA(Pa)] and partial fhuC(Pa). Cosmids pUH65 and pUH66 were digested with AccI and ligated into AccI-cleaved vector pT7-6, resulting in plasmids p76Sp and p76St, which carry wild-type S. paratyphi B fhuA [fhuA(Sp)] and partial fhuC(Sp) and wild-type S. typhimurium fhuA [fhuA(St)] and partial fhuC(St), respectively.
Plasmid p76Sp was digested with SmaI and partially digested with HpaI. A 5,029-bp DNA fragment was recovered from the agarose with Qiaex (Qiagen, Hilden, Germany) and religated, resulting in plasmid p76Sp/1. Plasmid p76St was digested with Eco47III/BamHI (see Fig. 2 for the location of the restriction sites) and ligated into Eco47III/BamHI-cleaved plasmid p76Sp/1, resulting in plasmid p1/2. Plasmid p76Sp/1 was digested with Eco47III/BamHI and ligated into Eco47III/BamHI-cleaved plasmid p76St, resulting in plasmid p3/2. Plasmid p76St was digested with SnaBI/BamHI and ligated into HpaI/BamHI-cleaved plasmid p76Sp/1, resulting in plasmid p9/9. Plasmid p76Sp/1 was digested with HpaI/BamHI and ligated into SnaBI/BamHI-cleaved plasmid p76St, resulting in plasmid p11/15. Plasmid p76Sp/1 was digested with Eco47III/HpaI and ligated into Eco47III/SnaBI-cleaved plasmid p76St, resulting in plasmid p5/21. Plasmid p76St was digested with Eco47III/SnaBI and ligated into Eco47III/HpaI-cleaved plasmid p76Sp/1, resulting in plasmid p7/24.
FIG. 2.
Schematic representation of wild-type fhuA genes. The enzymes used to construct the chimeric receptor derivatives are indicated. The sensitivities to the phages T1, T5, φ80, and ES18, to colicin M (ColM), and to albomycin (Albo) are given as the last of a 10-fold dilution series which resulted in a clear zone of growth inhibition. Numbers in parentheses indicate zones of turbid growth inhibition. Dashes indicate no growth inhibition. Growth promotion by ferrichrome (Fc) was tested by placing filter paper disks supplemented with 10 μl of ferrichrome (1 mM) onto NBD plates overlaid with NB top agar containing 108 cells of the strain to be tested. The results are given as the diameter of the growth zone (in millimeters) around the filter paper disk (6 mm) (the diameter of the discs was not subtracted from the values given). Transport indicates ferrichrome transport rates in units of 1,000 Fe3+ ions transported per cell per minute (average of three determinations without corrections). The positions of the gating loop and of the loop containing residues 236 to 257 as well as the number of the exchanged amino acids of FhuA(St) and FhuA(Sp) are indicated. The gating loop of FhuA(Ec) and of FhuA(Sp) consists of 41 amino acids (aa), while the gating loop of FhuA(St) and of FhuA(Pa) consists of only 24 aa.
Plasmid pAM11 was digested with MluI/SalI and ligated into MluI/SalI-cleaved plasmid pHK763, resulting in plasmid pHK763B. Plasmid pHK763B was digested with HindIII/BamHI and ligated into HindIII/BclI-cleaved plasmid p75Pa, resulting in plasmid p5242. Plasmid p75Pa was digested with HindIII/BclI and ligated into HindIII/BamHI-cleaved plasmid pHK763B, resulting in plasmid p9489.
Recombinant DNA techniques.
Isolation of plasmids, use of restriction enzymes, ligation, agarose gel electrophoresis, and transformation were done by standard techniques (31). DNA was sequenced by 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 WM1576 transformed with various plasmids encoding the receptors were collected by centrifugation at an optical density at 578 nm of 0.4 and resuspended in 1 ml of M9 salts (24) supplemented with 0.4% glucose, 0.01% methionine assay medium, and 0.01% thiamine. After shaking the cells for 1 h at 27°C, T7 RNA polymerase synthesis was induced by shifting the temperature to 42°C for 15 min. Rifampin (10 μl; 5 mg/ml in methanol) was added, and incubation continued at 27°C for 20 min. [35S]methionine was added, and the suspension was incubated for an additional 10 min before cells were collected by centrifugation and suspended in sample buffer. The radioactively labeled proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously (17).
Phenotype assays.
All phenotype assays were performed with freshly transformed E. coli K-12 strain HK97 aroB fhuA fhuE. The sensitivity of cells to the FhuA ligands (phages T1, T5, and φ80 and colicin M and albomycin) was tested by spotting 10-fold diluted solutions (4 μl) on TY plates overlaid with 3 ml of TY soft agar that contained 108 cells of the strain to be tested. The colicin M solution was a crude extract of a strain that carried the plasmid pTO4 cma cmi (29). Competition experiments between ferrichrome and phage infection and colicin M killing, respectively, were done with final ferrichrome concentrations of 10 and 100 μM in the 3 ml of TY soft agar that contained 108 cells of the strain to be tested. The TY nutrient agar plates were incubated for 15 min at 37°C before the spot tests were performed as described above.
Growth promotion by siderophores was tested by placing filter paper disks containing 10 μl of a 1 mM siderophore solution on NBD agar plates overlaid with 3 ml of NB soft agar which contained 0.1 ml of an overnight culture of the strain to be tested. The diameter of growth and the growth density around the filter paper disk were determined after incubation overnight.
Transport assays.
E. coli K-12 HK97 aroB fhuA fhuE freshly transformed with the plasmids to be tested was grown overnight on TY plates. Cells were washed and suspended in transport medium (M9 salts [24], 0.4% glucose) before the cell density was 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 was started by adding 10 μl of 100 μM [55Fe3+]ferrichrome. Samples (100 μl) were withdrawn; 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.
Computer-assisted sequence analysis was performed with the program package PC.GENE and the BLAST homology search (1).
Nucleotide sequence accession number.
The nucleotide sequences reported in this study were deposited in the EMBL data bank under accession numbers Y14026 [fhuA(Pa) and partial fhuC(Pa)], Y14067 [fhuA(Sp) and partial fhuC(Sp)], and Y14025 [fhuA(St) and partial fhuC(St)].
RESULTS
Nucleotide sequences of fhuA genes and derived amino acid sequences.
fhuA(Pa), fhuA(Sp), and fhuA(St) have been cloned previously on the cosmids pUH62, pUH65, and pUH66, respectively (22). Restriction fragments were cloned on plasmids pT7-5 and pT7-6 and transformed into E. coli HK97 fhuA fhuE aroB to test sensitivity to albomycin and growth on ferrichrome as the sole iron source. The fhuA mutation of HK97 displays no polar effect on the transcription of the downstream-located fhuCDB genes, which are required for transport of albomycin and ferrichrome across the cytoplasmic membrane. Due to the aroB mutation, strain HK97 did not produce its own siderophore enterobactin, and therefore growth on NBD plates or transport of iron in M9 medium depended on the ability to take up ferrichrome.
A 2.5-kb AvaI-HindIII fragment of cosmid pUH62 (p75Pa) and 3-kb AccI fragments of cosmids pUH65 and pUH66 (p76Sp and p76St) complemented the FhuA− phenotype of HK97. Both strands of the cloned fragments [2,531, 2,937, and 2,879 bp for the fhu(Pa), fhu(Sp), and fhu(St) fragments, respectively] were sequenced. Upstream of the fhuA genes are typical −35 and −10 promoter regions, ribosome binding sites, and a site with a strong identity to the binding site of the Fe2+ Fur repressor. The identities to the Fur consensus sequence (Fur box) composed of 19 nucleotides (9) were 14 [fhuA(Ec)], 16 [fhuA(Sp)], 15 [fhuA(St)], and 15 [fhuA(Pa)] nucleotides. The open reading frames showed strong sequence identities to the E. coli fhuA gene [65.21% for fhuA(Pa), 82.93% for fhuA(Sp), and 74.57% for fhuA(St)].
The fhuA genes code for proteins consisting of 732 [FhuA(Pa)], 747 [FhuA(Sp)], and 729 [FhuA(St)] residues, and the molecular masses of the mature proteins are 77.16, 78.79, and 77.16 kDa, respectively (Fig. 1). A 78-kDa FhuA protein (then called Sid) of S. typhimurium SL1027 was previously identified by comparing the outer membrane profile of wild-type cells to those of mutants resistant to albomycin and phage ES18 (7). The FhuA proteins contain typical signal peptides of 33 [FhuA(Sp) and FhuA(St)] and 34 [FhuA(Pa)] residues. In all FhuA proteins, a phenylalanine residue is located at the C terminus and an arginine residue is located at position −11 relative to the C terminus, both of which positions are widely conserved among outer membrane proteins. The TonB box sequences close to the N terminus read ETITV [FhuA(Sp) and FhuA(St)] (Fig. 1), which are very similar to the TonB box of E. coli (DTITV), and ETMVV [FhuA(Pa)] (Fig. 1), the last of which is identical to the TonB box of the Cir outer membrane protein. FhuA(Sp) shows the highest sequence identity to the E. coli FhuA [FhuA(Ec)] (91.97%), followed by FhuA(St) (72.7%) and FhuA(Pa) (55.46%).
FIG. 1.
Sequence alignment of various FhuA proteins. Asterisks denote identical residues, and dots indicate similar amino acids. Amino acids representing the gating loop are shown in shaded boxes. The amino acids deleted in FhuA Δ236-243 (Δ8aa) and in FhuA Δ236-248 (Δ13aa) are indicated. The TonB box sequences are underlined.
The cloned fragments also contained the 5′ regions of the fhuC genes which encoded proteins of 48 [FhuC(Pa)] and 129 [FhuC(Sp) and FhuC(St)] amino acids. An alignment of the partially sequenced FhuC proteins with the E. coli FhuC protein (265 residues) revealed in all four proteins an identical Walker motif A (GHNGSGKST), which is typical for these presumptive ATPases.
The largest difference between the FhuA proteins is located in the predicted gating loops.
Alignment of the FhuA amino acid sequences, related to the FhuA(Ec) gating loop, disclosed a gap of 17 residues in FhuA(St) and a gap of 14 plus 3 residues in FhuA(Pa) (Fig. 1). There are a few other sequence gaps that are introduced to obtain the highest degree of sequence identity and homology between the FhuA proteins of which the largest consists of six residues. At this site FhuA(Pa) is six residues larger than FhuA(Sp), FhuA(St), and Fhu(Ec), the three of which are identical (Fig. 1). The sequence gaps are confined to the first half of the E. coli gating loop, for which a previous deletion analysis has revealed its importance for phage infection but not for the uptake of ferrichrome and albomycin (16).
Functional analysis of the predicted gating loop.
The activities of the FhuA proteins of S. parathyphi, S. typhimurium, and P. agglomerans were determined in E. coli HK97 fhuA fhuE aroB transformed with the cloned heterologous fhuA genes. E. coli HK97 fhuA(Sp) was as sensitive to the phages T1, T5, and φ80 and to colicin M and albomycin and grew as well on ferrichrome as the sole iron source as E. coli HK97 transformed with the E. coli fhuA(Ec) gene (Fig. 2). This result was expected because of the high sequence identity of FhuA(Sp) and FhuA(Ec). FhuA(St) took up albomycin and ferrichrome but conferred no sensitivity to the E. coli phages and to colicin M but did confer sensitivity to the S. typhimurium phage ES18 (Fig. 2). Similar results were obtained with E. coli fhuA(Pa), which was resistant to the E. coli and S. typhimurium phages and to colicin M but took up albomycin and ferrichrome (Fig. 2). The ferrichrome transport rates of E. coli HK97 transformed with fhuA(Ec), fhuA(Sp), fhuA(St), and fhuA(Pa) were very similar (Fig. 2 and 3). The initial transport rate of E. coli HK97 fhuA(Pa) was consistently higher than those of the other transformants, which could reflect a stronger binding of ferrichrome to FhuA(Pa) or the presence of somewhat larger amounts of FhuA(Pa) in the outer membrane. After transcription by the T7 RNA polymerase, the amounts of the [35S]methionine-labeled proteins were very similar and no degradation products were observed, as revealed by SDS-PAGE (data not shown), which indicated a similar expression of the genes and a high stability of the proteins.
FIG. 3.
Time-dependent transport of [55Fe3+]ferrichrome (1 μM) into E. coli HK97 fhuA fhuE aroB expressing the plasmid-encoded FhuA wild-type proteins of E. coli(pHK763), P. agglomerans(p75Pa), S. paratyphi(p76Sp), and S. typhimurium(p76St).
For further analysis of FhuA regions which are important for activity, fragments were exchanged between the various FhuA proteins. All FhuA hybrids between FhuA(Sp) and FhuA(St) were found in outer membrane isolations after SDS-PAGE in amounts similar to those of wild-type receptors (Fig. 4). FhuA hybrids between FhuA(Sp) and FhuA(St) displayed FhuA(Sp) activity if they contained about two-thirds of the N- or C-terminal portion of FhuA(Sp) (Fig. 2) (fhuA3/2 and fhuA9/9). FhuA5/21, composed of the central segment of FhuA(Sp) flanked by the N-terminal and C-terminal segments of FhuA(St), conferred full sensitivity only to phage T5, reduced sensitivity to albomycin and growth on ferrichrome, and a very low sensitivity to phage φ80 (Fig. 2). To confer sensitivity to phage ES18 the hybrid FhuA protein (p11/15) had to consist of the two-thirds of N-terminal FhuA(St) combined with C-terminal FhuA(Sp), whereas all other hybrids did not serve as phage ES18 receptors (Fig. 2).
FIG. 4.
Comparison of outer membrane preparations of [35S]methionine-labeled FhuA hybrid proteins [hybrids of FhuA(St) and FhuA(Sp)] (lanes 2 to 7), FhuA(Sp) (lane 1), and FhuA(St) (lane 8) after SDS-PAGE (see Table 1 and Fig. 2 for details).
In addition, two hybrids of FhuA(Ec) with FhuA(Pa) were analyzed. Due to the use of natural restriction enzymes to construct these hybrids, the resulting chimeric FhuA receptors were not identical in size. Plasmid p9489 encoded a FhuA hybrid that consisted of residues 1 to 355 of FhuA(Pa) and residues 320 to 714 of FhuA(Ec). Despite the duplication of the gating loop region the FhuA hybrid conferred sensitivity to phage T5 [dilution titer, 103, compared to 104 for FhuA(Ec) in this experiment] and to colicin M (dilution titer, 101, compared to 103 for FhuA(Ec)]. No other FhuA activity was encountered. The proteins of the hybrids were found in isolated outer membranes after SDS-PAGE in reduced amounts compared to wild-type FhuA(Ec) and wild-type FhuA(Pa), and the gels showed strong degradation products (data not shown). Increase of the amount of the FhuA hybrid by transcription through the T7 RNA polymerase did not increase the sensitivity of the cells to the FhuA ligands, indicating that the measured activity was related to the structure of the FhuA hybrid protein and was not caused by the reduced amounts of the hybrid protein. Plasmid p5242 encoded a truncated FhuA hybrid that contained residues 1 to 319 of FhuA(Ec) and residues 356 to 698 of FhuA(Pa). This FhuA derivative was lacking the gating loop region and displayed no FhuA activity.
Identification of an additional site in FhuA for ferrichrome uptake.
The sequences of all four mature FhuA proteins are identical from residues 234 to 248 (Fig. 1). The amino acid sequence from residues 236 to 257 of FhuA(Ec) was predicted to form a surface-exposed loop adjacent to the gating loop (21). To examine whether this highly conserved region is involved in ligand binding, small (8- and 13-amino-acid) deletions were made in FhuA(Ec). The fhuA(Ec) deletion derivatives were constructed by ligating shortened PCR fragments using introduced BamHI restriction sites. The deletions extended from residues 236 to 243 (FhuA Δ236-243) and from residue 236 to 248 (FhuA Δ236-248) (Fig. 1) and comprised 8 and 13 FhuA amino acids, respectively, but the genetic technique used to construct these deletions introduced two residues (glycine and serine). In the outer membrane the proteins of both deletion mutants were found in amounts similar to those of wild-type FhuA(Ec) (data not shown). FhuA Δ236-243 and FhuA Δ236-248 were resistant to albomycin and showed no growth on ferrichrome (Table 2). This was confirmed by transport assays with [55Fe3+]ferrichrome, in which both deletion derivatives showed no transport and no binding of ferrichrome (Fig. 5). However, the deletion derivatives conferred full sensitivity to phages T1 and T5 and a reduced sensitivity to phage φ80 and to colicin M (Table 2). Phage and colicin sensitivity provided an additional means to measure ferrichrome binding, because ferrichrome competes with these FhuA ligands, except phage T5. Only TonB mutants are protected from T5 infection by ferrichrome unless high ferrichrome concentrations are used (12). Sensitivity of E. coli HK97 that synthesized wild-type FhuA(Ec) was reduced 10-fold to phage T1, φ80, and colicin M by 10 μM ferrichrome added to the soft agar and 1,000-fold to φ80 by 100 μM ferrichrome, whereas the sensitivity of cells that synthesized the FhuA deletion derivatives was not altered by ferrichrome (Table 2).
TABLE 2.
Properties of FhuA deletion mutantsa
| HK97 plasmidb | Fc concn (μM) | Sensitivity toc:
|
Growth on Fc (1 mM)d | ||||
|---|---|---|---|---|---|---|---|
| T1 | T5 | φ80 | ColM | Albo | |||
| pHK763 | 0 | 4 (5, 6) | 4 (5, 6) | 4 (5, 6) | 2 (3, 4) | 3 | 20 |
| pHK763 | 10 | 3 (4, 5, 6) | 4 (5, 6) | 3 (4, 5) | 1 (2) | —e | ND |
| pHK763 | 100 | 3 (4) | 4 (5, 6) | 1 (2, 3) | 1 | — | ND |
| pB3/4 | 0 | 4 (5, 6) | 4 (5, 6) | 3 (4) | 1 (2) | — | 0 |
| pB3/4 | 100 | 4 (5, 6) | 4 (5, 6) | 3 (4) | 1 (2) | — | ND |
| pB4/5 | 0 | 4 (5, 6) | 4 (5, 6) | 3 (4) | 1 (2) | — | 0 |
| pB4/5 | 100 | 4 (5, 6) | 4 (5, 6) | 3 (4) | 1 (2) | — | ND |
Sensitivities to the ligands and growth promotion by ferrichrome (Fc) were tested by using freshly transformed strain HK97 fhuA fhuE aroB.
 |
The sensitivities to phages T1, T5, and φ80, to colicin M (ColM), and to albomycin (Albo) were tested by spotting 10-fold dilutions (4 μl) onto TY agar plates overlaid with TY top agar containing ferrichrome concentrations as indicated and the strain to be tested. The results are given as the last of a 10-fold dilution series which resulted in a clear zone of growth inhibition. Numbers in parentheses indicate turbid growth zones.
Growth promotion by ferrichrome was tested by placing filter paper disks supplemented with 10 μl of ferrichrome (1 mM) onto NBD plates overlaid with NB top agar containing 108 cells of the strain to be tested. The results are given as the diameter of the growth zone (in millimeters) around the filter paper disk (6 mm) (the diameter of the discs was not subtracted from the values given). ND, not determined.
—, no growth inhibition.
FIG. 5.
Time-dependent transport of [55Fe3+]ferrichrome (1 μM) into E. coli HK97 fhuA fhuE aroB transformed with plasmid pHK763, which encodes wild-type FhuA(Ec), or with plasmids pB3/4 and pB4/5, which encode the FhuA(Ec) deletion derivatives FhuA Δ236-234 and FhuA Δ236-248, respectively.
DISCUSSION
The kinetics of ferrichrome transport into E. coli are composed of two rates, high initial binding to FhuA and lower transport into cells. The site for binding and transport was localized to one-half of the gating loop by the deletion of amino acids 335 to 355, which reduced binding and transport to less than 10% of that of FhuA wild-type and opened unspecifically the FhuA channel for SDS and maltodextrins, whereas the deletion of residues 322 to 336 did not affect ferrichrome binding and reduced the transport rate to 60% (16). The conclusion that the region containing residues 335 to 355 mainly determines the permeability of the FhuA channel is supported by the structures and properties of the FhuA proteins described in this work. FhuA(Sp), FhuA(St), and FhuA(Pa) contain the region equivalent to the region containing residues 335 to 355 of FhuA(Ec) and transport ferrichrome. Lack of the region containing residues 322 to 336 in FhuA(St) and FhuA(Pa) indicates that this region is dispensable for binding and transport of ferrichrome and, in general terms, for closing and opening of the FhuA channel. Only FhuA(Sp) contains a surface loop of the size and sequence of FhuA(Ec). FhuA(Sp) expressed in E. coli HK97 fhuA confers the same degree of sensitivity to the E. coli phages and to colicin M as does FhuA(Ec). This result per se does not indicate that the region containing residues 322 to 355 is required for phage infection and colicin killing, since the overall identity of FhuA(Sp) and FhuA(Ec) is 92%, but it does agree with the previous data which demonstrate the importance of the entire E. coli gating loop for phage infection (17, 20). In the mature proteins only conservative amino acid replacements occur (replacement of E by Q, E by D, F by Y, S by A, T by V, K by R, V by I, and N by Q), except at five sites, where KGS of E. coli is replaced by DRA, KDGN is replaced by ENGK, RP is replaced by AA, PE is replaced by SA, and R is replaced by G in S. paratyphi B. In the proposed FhuA(Ec) transmembrane model (21), the nonconservative replacements are contained in surface loops, except one which is located in a cytoplasmic turn (RP replaced by AA). This finding is consistent with the rule that loops and turns are more variable and tolerate nonconservative amino acids replacements more easily than membrane-spanning regions. In addition, surface loops determine ligand binding specificity. The locations of the conservative and nonconservative amino acid replacements support the transmembrane topology model of FhuA(Ec) and suggest the same arrangement for FhuA(Sp), FhuA(St), and FhuA(Pa).
An additional site important for ferrichrome uptake was localized in the surface loop containing residues 236 to 257, with residues 236 to 248 having an identical amino acid sequence in all four FhuA proteins. Deletion of residues 236 to 243 and residues 236 to 248 in FhuA(Ec) inactivated ferrichrome transport and rendered cells resistant to albomycin. In contrast, the FhuA deletion derivatives conferred high phage and colicin sensitivity, which indicates that their conformation was not grossly altered compared to that of wild-type FhuA and that they were properly inserted into the outer membrane. The deletion derivatives also did not bind ferrichrome, as shown by ferrichrome transport assays and the failure to prevent phage and colicin M binding by ferrichrome, which suggests that this loop is part of the ferrichrome binding site or contributes to the conformation of the ferrichrome binding site. In a previous study, insertion of a tetrapeptide after residue 241 reduced ferrichrome uptake, rendered cells albomycin resistant and reduced colicin M sensitivity 32-fold. Insertion of a 16-residue peptide reduced ferrichrome uptake and reduced sensitivity to colicin M 128-fold, to phage φ80 10-fold, and to phage T5 100-fold (21). Insertion of a dipeptide after residue 239 reduced sensitivity to φ80 10-fold, to phage UC-1 100-fold, and to colicin M 8-fold (8). The inserted heterologous peptides may have reduced access of the ligands to their binding sites by steric hindrance or may have distorted the conformation of the binding sites. This loop is in the two-dimensional transmembrane model (21) adjacent to the gating loop and probably contributes to the channel structure.
The specificity of the loss of ferrichrome uptake in the FhuA deletion derivatives FhuA Δ236-248 and FhuA Δ322-355 is supported by mutations in another proposed surface loop (residues 454 to 477) close to the gating loop which did not affect ferrichrome uptake. Insertion of a 12-amino-acid peptide after residue 456 only reduced sensitivity to colicin M 64-fold (21), and deletion of residues 457 to 479 resulted in mutant cells in which the FhuA-related phages formed turbid plaques (16). Monoclonal antibodies directed to residues 417 to 450 inhibited only colicin M killing and inactivation of phage T5 by E. coli cells (27).
Despite 74% sequence identity, hybrid proteins of FhuA(Sp) and FhuA(St) were only fully active when large fragments were exchanged. FhuA3/2 and FhuA9/9, composed of two-thirds of FhuA(Sp) and one-third of FhuA(St), displayed full FhuA(Sp)-specific activity and no FhuA(St)-specific activity. FhuA5/21, which consists of the central portion of FhuA(Sp) (residues 213 to 482) that forms the overlapping region in FhuA3/2 and FhuA9/9, flanked on both sides by FhuA(St) fragments, conferred full sensitivity only to phage T5 and conferred a reduced sensitivity to albomycin (caused by a reduced uptake), as evidenced by a reduced growth promotion by ferrichrome, and a very weak sensitivity to phage φ80. Apparently, the gating loop of FhuA(Sp) and loops containing residues 236 to 257, 404 to 433, and 454 to 477 [according to the FhuA(Ec) model (21)] inserted in FhuA(St) are not sufficient to confer FhuA(Sp) activity. Full sensitivity to T5 and lack or reduction of activity to the other FhuA ligands point to a somewhat distorted interaction of the hybrid protein with TonB. Sensitivity to the S. typhimurium phage ES18 is observed when the hybrid protein (FhuA11/15) contains two-thirds of the N-terminal segment of FhuA(St) but not when it contains two-thirds of the C-terminal segment (FhuA1/2) of FhuA(St). Hybrid proteins of FhuA(Ec) and FhuA(Pa), which display 55.4% sequence identity, confer only a reduced sensitivity to phage T5 and colicin M when they contain residues 320 to 714 of mature FhuA(Ec). The inactive or partially active hybrid proteins may lack amino acid side chains required for binding of the ligands, or the active sites cannot adopt completely their native conformation.
Chimeric proteins consisting of the central part of FhuA(Ec), extending from amino acid 161 to 370, and the N- and C-terminal parts of FhuE (coprogen receptor) and FoxA (ferrioxamine B receptor), respectively, transported ferrichrome. The transport rates, relative to FhuA, were 38% for the FhuE-FhuA hybrid and 8% for the FoxA-FhuA hybrid (19). These results are consistent with the data presented in this paper, which localized the ferrichrome binding sites to the loops containing residues 236 to 257 and residues 316 to 356. Taken together these data and the results presented in this work demonstrate that several segments of FhuA are involved in ligand binding, in transport of ferrichrome, albomycin, and colicin M, and in phage infection and that these segments adopt certain conformations which are determined by the entire FhuA polypeptide.
ACKNOWLEDGMENTS
We thank K. A. Brune for critical reading of the manuscript.
This work was supported by the Deutsche Forschungsgemeinschaft (SFB323, project B1) and the Fonds der Chemischen Industrie.
REFERENCES
- 1.Altschul S F, Gish W, Miller W, Myers E W, Lipman D J. Basic local alignment search tool. J Mol Biol. 1990;215:403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
- 2.Berner I, Konetschny Rapp S, Jung G, Winkelmann G. Characterization of ferrioxamine E as the principal siderophore of Erwinia herbicola (Enterobacter agglomerans) Biol Met. 1988;1:51–56. doi: 10.1007/BF01128017. [DOI] [PubMed] [Google Scholar]
- 3.Bonhivers M, Ghazi A, Boulanger P, Letellier L. FhuA, a transporter of the Escherichia coli outer membrane, is converted into a channel upon binding of bacteriophage T5. EMBO J. 1996;15:1850–1856. [PMC free article] [PubMed] [Google Scholar]
- 4.Bös C, Braun V. Specific in vivo thiol-labeling of the FhuA outer membrane ferrichrome transport protein of Escherichia coli K-12: evidence for a disulfide bridge in the predicted gating loop. FEMS Microbiol Lett. 1997;153:311–319. doi: 10.1111/j.1574-6968.1997.tb12590.x. [DOI] [PubMed] [Google Scholar]
- 5.Bös C, Lorenzen D, Braun V. 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. J Bacteriol. 1998;180:605–613. doi: 10.1128/jb.180.3.605-613.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Braun V. Energy-coupled transport and signal transduction through the gram-negative outer membrane via TonB-ExbB-ExbD-dependent receptor proteins. FEMS Microbiol Rev. 1995;16:295–307. doi: 10.1111/j.1574-6976.1995.tb00177.x. [DOI] [PubMed] [Google Scholar]
- 7.Braun V, Hantke K, Stauder W. Identification of the sid outer membrane receptor protein in Salmonella typhimurium SL1027. Mol Gen Genet. 1977;155:227–229. doi: 10.1007/BF00393164. [DOI] [PubMed] [Google Scholar]
- 8.Carmel G, Hellstern D, Henning D, Coulton J W. Insertion mutagenesis of the gene encoding the ferrichrome-iron receptor of Escherichia coli K-12. J Bacteriol. 1990;172:1861–1869. doi: 10.1128/jb.172.4.1861-1869.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.de Lorenzo V, Wee S, Herrero M, Neilands J B. Operator sequences of the aerobactin operon of plasmid ColV-K30 binding the ferric uptake regulation (fur) repressor. J Bacteriol. 1987;169:2624–2630. doi: 10.1128/jb.169.6.2624-2630.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Graham A C, Stocker B A D. Genetics of sensitivity of Salmonella species to colicin M and bacteriophages T5, T1, and ES18. J Bacteriol. 1977;130:1214–1223. doi: 10.1128/jb.130.3.1214-1223.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Günter K, Braun V. In vivo evidence for FhuA outer membrane interaction with the TonB inner membrane protein of Escherichia coli. FEBS Lett. 1990;274:85–88. doi: 10.1016/0014-5793(90)81335-l. [DOI] [PubMed] [Google Scholar]
- 12.Hantke K, Braun V. Functional interaction of the tonA/tonB receptor system in Escherichia coli. J Bacteriol. 1978;135:190–197. doi: 10.1128/jb.135.1.190-197.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Hoffmann H, Fischer E, Schwarz H, Braun V. Overproduction of the proFhuA outer membrane receptor protein of Escherichia coli K-12: isolation, properties and immunocytochemical localization at the inner side of the cytoplasmic membrane. Arch Microbiol. 1986;145:334–341. doi: 10.1007/BF00470867. [DOI] [PubMed] [Google Scholar]
- 14.Hohn B, Collins J. A small cosmid for efficient cloning of large DNA fragments. Gene. 1980;11:291–298. doi: 10.1016/0378-1119(80)90069-4. [DOI] [PubMed] [Google Scholar]
- 15.Killmann H, Benz R, Braun V. Conversion of the FhuA transport protein into a diffusion channel through the outer membrane of Escherichia coli. EMBO J. 1993;12:3007–3016. doi: 10.1002/j.1460-2075.1993.tb05969.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Killmann H, Benz R, Braun V. Properties of the FhuA channel in the Escherichia coli outer membrane after deletion of FhuA portions within and outside the predicted gating loop. J Bacteriol. 1996;178:6913–6920. doi: 10.1128/jb.178.23.6913-6920.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Killmann H, Braun V. An aspartate deletion mutation defines a binding site of the multifunctional FhuA outer membrane receptor of Escherichia coli K-12. J Bacteriol. 1992;174:3479–3486. doi: 10.1128/jb.174.11.3479-3486.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Killmann H, Braun V. Energy-dependent receptor activities of Escherichia coli K-12: mutated TonB proteins alter FhuA receptor activities to phages T5, T1, φ80 and to colicin M. FEMS Microbiol Lett. 1994;119:71–76. doi: 10.1111/j.1574-6968.1994.tb06869.x. [DOI] [PubMed] [Google Scholar]
- 19.Killmann H, Braun V. Conversion of the coprogen transport protein FhuE and the ferrioxamine B transport protein FoxA into ferrichrome transport proteins. FEMS Microbiol Lett. 1998;161:59–67. doi: 10.1111/j.1574-6968.1998.tb12929.x. [DOI] [PubMed] [Google Scholar]
- 20.Killmann H, Videnov G, Jung G, Schwarz H, Braun V. Identification of receptor binding sites by competitive peptide mapping: phages T1, T5, and φ80 and colicin M bind to the gating loop of FhuA. J Bacteriol. 1995;177:694–698. doi: 10.1128/jb.177.3.694-698.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Koebnik R, Braun V. 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. J Bacteriol. 1993;175:826–839. doi: 10.1128/jb.175.3.826-839.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Koebnik R, Hantke K, Braun V. The TonB-dependent ferrichrome receptor FcuA of Yersinia enterocolitica: evidence against a strict coevolution of receptor structure and substrate specificity. Mol Microbiol. 1993;7:383–393. doi: 10.1111/j.1365-2958.1993.tb01130.x. [DOI] [PubMed] [Google Scholar]
- 23.Letellier L, Locher K P, Plancon L, Rosenbusch J P. Modeling ligand-gated receptor activity. FhuA-mediated ferrichrome efflux from lipid vesicles triggered by phage T5. J Biol Chem. 1997;272:1448–1451. doi: 10.1074/jbc.272.3.1448. [DOI] [PubMed] [Google Scholar]
- 24.Miller J H. Experiments in molecular genetics. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press; 1972. [Google Scholar]
- 25.Moeck G S, Bazzaz B S, Gras M F, Ravi T S, Ratcliffe M J, Coulton J W. Genetic insertion and exposure of a reporter epitope in the ferrichrome-iron receptor of Escherichia coli K-12. J Bacteriol. 1994;176:4250–4259. doi: 10.1128/jb.176.14.4250-4259.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Moeck G S, Coulton J W, Postle K. Cell envelope signaling in Escherichia coli. Ligand binding to the ferrichrome-iron receptor FhuA promotes interaction with the energy-transduction proteion TonB. J Biol Chem. 1997;272:28391–28397. doi: 10.1074/jbc.272.45.28391. [DOI] [PubMed] [Google Scholar]
- 27.Moeck G S, Ratcliffe M J, Coulton J W. Topological analysis of the Escherichia coli ferrichrome-iron receptor by using monoclonal antibodies. J Bacteriol. 1995;177:6118–6125. doi: 10.1128/jb.177.21.6118-6125.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Moeck G S, Tawa P, Xiang H, Ismail A A, Turnbull J L, Coulton J W. Ligand-induced conformational change in the ferrichrome-iron receptor of Escherichia coli K-12. Mol Microbiol. 1996;22:459–471. doi: 10.1046/j.1365-2958.1996.00112.x. [DOI] [PubMed] [Google Scholar]
- 29.Ölschläger T, Schramm E, Braun V. Cloning and expression of the activity and immunity genes of colicins B and M on pColBM plasmids. Mol Gen Genet. 1984;196:482–487. doi: 10.1007/BF00436196. [DOI] [PubMed] [Google Scholar]
- 30.Plancon L, Chami M, Letellier L. Reconstitution of FhuA, an Escherichia coli outer membrane protein, into liposomes. Binding of phage T5 to FhuA triggers the transfer of DNA into the proteoliposomes. J Biol Chem. 1997;272:16868–16872. doi: 10.1074/jbc.272.27.16868. [DOI] [PubMed] [Google Scholar]
- 31.Sambrook J, Fritsch E F, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press; 1989. [Google Scholar]
- 32.Schramm E, Mende J, Braun V, Kamp R M. Nucleotide sequence of the colicin B activity gene cba: consensus pentapeptide among TonB-dependent colicins and receptors. J Bacteriol. 1987;169:3350–3357. doi: 10.1128/jb.169.7.3350-3357.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Tabor S, Richardson C C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci USA. 1985;82:1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]