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JOURNAL OF BACTERIOLOGY, June 2001, p. 3476–3487 Vol. 183, No. 11
0021-9193/01/$04.00⫹0 DOI: 10.1128/JB.183.11.3476–3487.2001
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
FhuA Barrel-Cork Hybrids Are Active Transporters
and Receptors
HELMUT KILLMANN, MICHAEL BRAUN, CHRISTINA HERRMANN, AND VOLKMAR BRAUN*
Mikrobiologie/Membranphysiologie, Universität Tübingen, D-72076 Tübingen, Germany
Received 9 January 2001/Accepted 20 March 2001
The crystal structure of Escherichia coli FhuA reveals a -barrel domain that is closed by a globular cork
domain. It has been assumed that the proton motive force of the cytoplasmic membrane through the interac-
tion of the TonB protein with the TonB box of the cork opens the FhuA channel. Yet, deletion of the cork results
in an FhuA derivative, FhuA⌬5–160, that still displays TonB-dependent substrate transport and phage
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receptor activity. To investigate this unexpected finding further, we constructed FhuA⌬5–160 derivatives of
FhuA proteins from Salmonella paratyphi B, Salmonella enterica serovar Typhimurium, and Pantoea agglomerans.
The FhuA⌬5–160 proteins inserted correctly into the outer membrane, and with the exception of the P.
agglomerans protein, transported ferrichrome and albomycin. FhuA hybrids consisting of the -barrel of one
strain and the cork of another strain were active and showed higher TonB-dependent ferrichrome transport
rates than the corkless derivatives. Exceptions were the E. coli -barrel/Salmonella serovar Typhimurium cork
hybrid protein and the Salmonella serovar Typhimurium -barrel/P. agglomerans cork hybrid protein, both of
which were less active than the -barrels alone. Each of the FhuA mutant proteins displayed activity for each
of their ligands, except for phage T5, only when coupled to TonB. The hybrid FhuA proteins displayed a similar
activity with the E. coli TonB protein as with their cognate TonB proteins. Sensitivity to phages T1, T5, and
80, rifamycin CGP 4832, and colicin M was determined by the -barrel, whereas sensitivity to phage ES18
and microcin J25 required both the -barrel and cork domains. These results demonstrate that the -barrel
domain of FhuA confers activity and specificity and responds to TonB and that the cork domains of various
FhuA proteins can be interchanged and contribute to the activities of the FhuA hybrids.
The FhuA outer membrane transport protein of Escherichia mutations in the TonB box that are suppressed by mutations in
coli consists of 22 antiparallel -sheets that form a -barrel TonB (9, 30).
into which a globular domain is inserted from the periplasmic A similar suppression analysis revealed the same interacting
side. The globular domain seems to close the -barrel channel regions in the BtuB vitamin B12 transport protein and in TonB
and prevent entry of even small molecules and was for this (11). Moreover, in vivo a segment of the TonB box of BtuB is
reason designated the “cork” (7) or “plug” (20). Ferrichrome, chemically cross-linked via disulfide bonds with a segment
the natural substrate of FhuA, binds in a cavity located well around residue 160 of TonB (6). Cross-linking at several po-
above the outer membrane lipid bilayer. The cork domain and sitions is increased when BtuB is loaded with vitamin B12, and
the -barrel domain contribute five and six amino acid side the cross-linking pattern changes in mutants containing amino
chains to the cavity, respectively, which are less than 4 Å away acid substitutions in BtuB that impair TonB-dependent BtuB
from the ferrichrome (7). It is thought that opening of the activity. Site-directed spin labeling and electron paramagnetic
FhuA channel requires dislocation of the cork, resulting in a resonance assays have suggested that the TonB box of BtuB in
connection between the cavity exposed to the cell surface and the unliganded conformation is located in a helix that forms
the region exposed to the periplasm. Although binding of fer- specific interactions with side chain residues of the periplasmic
richrome to FhuA moves the cork about 2 Å towards fer- turns of the -barrel domain of BtuB (23). Binding of vitamin
richrome, this does not open the channel. B12 to BtuB converts this segment into an extended, disor-
Energy provided by the cytoplasmic membrane in the form dered, and highly dynamic structure that likely extends into the
of the proton motive force (3) and the TonB-ExbB-ExbD pro- periplasm to interact physically with TonB. A TonB-uncoupled
tein complex are required for active transport through FhuA. TonB box mutant of BtuB shows a strongly altered electron
Binding of ferrichrome results in the movement of Glu19 17 Å paramagnetic resonance spectrum and no longer responds to
away from its former ␣-carbon position, which probably facil- the addition of vitamin B12. These experiments strongly sup-
itates binding of FhuA to TonB. This hypothesis is supported port the interaction of the transporter TonB box with the
by the finding that chemical cross-linking of FhuA to TonB is region around residue 160 of TonB.
enhanced in vivo upon binding of ferrichrome (25). An N- In a previous study, we deleted the cork domain, including
proximal region of FhuA, residues 7 to 11 (TonB box), inter- the TonB box, of E. coli FhuA. To our surprise, the protein
acts with a region around residue 160 of TonB, as shown by FhuA⌬5–160 was found in the outer membrane, although in
amounts lower than that of wild-type FhuA; FhuA⌬5–160
could still transport ferrichrome (at 30 to 40% the rate of
* Corresponding author. Mailing address: Mikrobiologie/Membran-
physiologie, Universität Tübingen, Auf der Morgenstelle 28, D-72076
wild-type FhuA) and albomycin in a TonB-dependent manner
Tübingen, Germany. Phone: (49) 7071 2972096. Fax: (49) 7071 295843. and conferred the same or almost the same degree of sensitiv-
E-mail: volkmar.braun@mikrobio.uni-tuebingen.de. ity as wild-type FhuA to the TonB-dependent colicin M and
3476VOL. 183, 2001 ENTEROBACTERIAL FhuA BARREL-CORK HYBRIDS 3477
TABLE 1. E. coli strains and plasmids used in this study
Strain or plasmid Genotype or phenotypea Reference or source
Strains
AB2847 aroB tsx malT thi 10
41/2 AB2847 cir fepA fhuA 10
HK99 AB2847 tonB fhuA 13
CH1857 AB2847 ⌬fhuACDB tonB 14
HK97 F⫺ araD139 lacU169 rpsL150 relA1 flbB5301 deoC1 ptsF25 rbsR aroB thi fhuE::placMu53 fhuA 12
BL21(DE3) omp8 F⫺ hsdSB(rB⫺ mB⫺) gal ompT dcm (DE3) ⌬lamB ompF::Tn5 ⌬ompA T7 polymerase under 27
lacUV5 control
CH21 BL21 omp8 fhuA This study
Plasmids
pHK763 pT7-6 fhuA (Ec) wild type 13
pAB pT7-6 fhuA (Ec) with BamHI E159D 16
pBK7 pT7-6 fhuA (Ec) ⌬5–160 E3D 4
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p76Sp pT7-6 fhuA (Sp) wild type 17
p76SpB pT7-6 fhuA (Sp) with BamHI E159D This study
pSp⌬5–160 pT7-6 fhuA (Sp) ⌬5–160 Q3D This study
p76St pT7-6 fhuA (St) wild type 17
p76StB pT7-6 fhuA (St) with BamHI E159D This study
pSt⌬5–160 pT7-6 fhuA (St) ⌬5–160 Q3D This study
p76Pa pT7-6 fhuA (Pa) wild type 17
p76PaB pT7-6 fhuA (Pa) with BamHI L159D R160P This study
pPa⌬5–160 pT7-6 fhuA (Pa) ⌬5–160 A3D E4P This study
pEcBSpC pT7-6 fhuA (Ec) with the first 160 aa of fhuA (Sp) This study
pEcBStC pT7-6 fhuA (Ec) with the first 160 aa of fhuA (St) This study
pEcBPaC pT7-6 fhuA (Ec) with the first 160 aa of fhuA (Pa) This study
pSpBEcC pT7-6 fhuA (Sp) with the first 160 aa of fhuA (Ec) This study
pSpBStC pT7-6 fhuA (Sp) with the first 160 aa of fhuA (St) This study
pSpBPaC pT7-6 fhuA (Sp) with the first 160 aa of fhuA (Pa) This study
pStBEcC pT7-6 fhuA (St) with the first 160 aa of fhuA (Ec) This study
pStBSpC pT7-6 fhuA (St) with the first 160 aa of fhuA (Sp) This study
pStBPaC pT7-6 fhuA (St) with the first 160 aa of fhuA (Pa) This study
pPaBEcC pT7-6 fhuA (Pa) with the first 160 aa of fhuA (Ec) This study
pPaBSpC pT7-6 fhuA (Pa) with the first 160 aa of fhuA (Sp) This study
pPaBStC pT7-6 fhuA (Pa) with the first 160 aa of fhuA (St) This study
pAM33 pHSG576 tonB (Ec) 21
p576St pHSG576 tonB (St) This study
p576Pa pHSG576 tonB (Pa) This study
pBK71 pT7-6 fhuA ⌬25–160 P24D This study
pTO4 pBR322 cma cmi 26
pTUC203 pACYC184 mcjABCD 31
pT7-6 Ampr 32
pHSG576 Cmr 33
a
Ec, E. coli; Sp, S. paratyphi; St, Salmonella serovar Typhimurium; Pa, P. agglomerans; B, -barrel; C, cork.
the phages T1 and 80 and to the TonB-independent phage T5 cork domains to determine whether cork domains insert into
(4). Since FhuA⌬5–160 lacks the TonB box, TonB must inter- heterologous -barrel domains and whether the resulting
act with other regions of FhuA, and this interaction suffices for FhuA hybrid proteins still respond to TonB and the proton
TonB-dependent FhuA activities. FhuA⌬5–160 mediates slow motive force.
diffusion, since sensitivity to larger hydrophilic antibiotics to
which the outer membrane normally forms a permeability bar- MATERIALS AND METHODS
rier is only moderately increased and cells remain resistant to Bacterial strains, plasmids, and growth conditions. The E. coli strains and
sodium dodecyl sulfate (SDS) and EDTA. plasmids used are listed in Table 1. Cells were grown in TY medium (10 g of
In this study, we intended to corroborate our previous re- Bacto tryptone [Difco Laboratories]/liter, 5 g of yeast extract/liter, 5 g of NaCl/
sults with the E. coli FhuA⌬5–160 protein by constructing 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
FhuA⌬5–160 derivatives of Salmonella paratyphi B, Salmonella
added (NBD medium). The antibiotics ampicillin (40 g/ml) and chloramphen-
enterica serovar Typhimurium, and Pantoea agglomerans; we icol (25 g/ml) were added when required.
have previously determined the fhuA nucleotide sequences of To construct plasmids p76SpB, pSp⌬5–160, p76StB, pSt⌬5–160, p76PaB, and
these strains (17). Comparison of the E. coli FhuA amino acid pPa⌬5–160, a BamHI restriction site was introduced into the fhuA gene of p76Sp
sequence with that of S. paratyphi B, Salmonella serovar Ty- (S. paratyphi), p76St (Salmonella serovar Typhimurium), and p76Pa (P. agglom-
erans) using PCR and the following primers (mismatches are underlined):
phimurium and P. agglomerans revealed 94, 79, and 60% iden- Sp_160for (5⬘-CCGACGACGGATCCGCTGAAAG-3⬘), Sp_160rev (5⬘-CTTTC
tity in the cork domain and 92, 74, and 58% identity in the AGCGGATCCGTCGTCGG-3⬘), and Sp_BamAnf (5⬘-CTTCTTTCGGATCCA
-barrel domain, respectively. In addition, we exchanged the CCGCCGC-3⬘) (BamHI in S. paratyphi); St_160for (5⬘-CCGACTACGGATCC3478 KILLMANN ET AL. J. BACTERIOL.
GCTGAAAGAAATTC-3⬘), St_160rev (5⬘-CTTTCAGCGGATCCGTAGTCG outer membrane (12). The plasmid-encoded fhuA genes in the transformants
GCCG-3⬘), and St_BamAnf (5⬘-GTTTCTTCTTTCGGATCCACCGCCGCCT were transcribed from the fhuA promoter. The sensitivity of cells against the
G-3⬘) (BamHI in Salmonella serovar Typhimurium); and Pa_160for (5⬘-CCAG FhuA ligands (phages T1, T5, 80, and ES18, colicin M, microcin J25, rifamycin
GAAACGGATCCCGAAGTGCAGTTCC-3⬘), Pa_160rev (5⬘-CTGCACTTCG CGP 4832, and albomycin) was tested by spotting 10-fold-diluted solutions (4 l)
GGATCCGTATCCTGGGTCGG-3⬘), and Pa_BamAnf (5⬘-GACCATCGTCG on TY agar plates overlaid with 3 ml of TY soft agar containing 108 cells of the
GATCCTGCGCGGCGTAAAG-3⬘) (BamHI in P. agglomerans). The primers of strain to be tested. The colicin M solution was a crude extract of a strain carrying
the complementary strands were pT7_ (5⬘-GCGAGGCCCAGCTGGCTTATC plasmid pTO4 cma cmi (26). The microcin J25 solution was a supernatant of E.
G-3⬘) and T7_uni (5⬘-GATTAAGCATTGGTAACTGTCAGACC-3⬘). All PCR coli MC4100 carrying the plasmid pTUC203 mcjABCD (31) after growth of the
products were purified by agarose gel electrophoresis and recovered from aga- transformants in brain heart infusion medium (37 g/liter; Difco Laboratories) at
rose using the EasyPure DNA purification kit (Biozym, Oldendorf, Germany). 37°C.
Each of the DNA fragments obtained with primers Sp_160rev, St_160rev, and Growth inhibition by SDS and various antibiotics was detected by placing filter
Pa_160rev was digested with HindIII and BamHI and ligated into paper disks supplemented with 10 l of the agents in concentrations as indicated
HindIII/BamHI-cleaved vector pT7-6, resulting in plasmids p76SpBN, p76StBN, on TY agar plates overlaid with 3 ml of TY soft agar containing 108 cells of the
and p76PaBN, respectively. The DNA fragments obtained with primers strain to be tested. Growth promotion by siderophores was tested by placing filter
Sp_160for, St_160for, and Pa_160for were digested with EcoRI and BamHI and paper disks containing 10 l of a siderophore solution concentrated as indicated
ligated into EcoRI/BamHI-cleaved plasmids p76SpBN, p76StBN, and p76PaBN, on NBD agar plates overlaid with 3 ml of NB soft agar containing 108 cells of the
respectively, resulting in plasmids p76SpB⬘, p76StB⬘, and p76PaB⬘. To avoid strain to be tested. After overnight incubation, the diameter and the growth
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complete sequencing of the fhuA genes, plasmids p76SpB⬘, p76StB⬘, and density around the filter paper disk were determined.
p76PaB⬘ were digested with HindIII and Eco47III and ligated into Transport and binding assays. E. coli K-12 strains 41/2 aroB fhuA, HK97 aroB
HindIII/Eco47III-cleaved plasmids p76Sp, p76St, and p76Pa, respectively, result- fhuA fhuE, HK99 aroB fhuA tonB, and CH1857 ⌬fhuACDB tonB aroB freshly
ing in plasmids p76SpB, p76StB, and p76PaB. The exchanged HindIII/Eco47III transformed with the plasmids to be tested were grown overnight on TY plates.
fragments were completely sequenced. Cells were washed and suspended in transport medium (M9 salts [24], 0.4%
Each of the DNA fragments obtained with primers Sp_BamAnf, St_BamAnf, glucose), and the cell density was then adjusted to an optical density at 578 nm
and Pa_BamAnf was digested with HindIII and BamHI and ligated into of 0.5. Free iron ions were removed by adding 25 l of 10 mM nitrilotriacetate,
HindIII/BamHI-cleaved plasmids p76SpB, p76StB, and p76PaB, resulting in pH 7.0, to 1 ml of cells. After incubation for 5 min at 37°C, transport or binding
plasmids pSp⌬5–160, pSt⌬5–160, and pPa⌬5–160, respectively. assays were started by adding 10 l of 100 M [55Fe3⫹]ferrichrome. Only in the
Plasmid pAB was digested with HindIII and BamHI, and the obtained 992-bp case of binding assays, a 150-fold surplus of nonradioactive ferrichrome was
fragment was ligated into HindIII/BamHI-cleaved p76SpB, p76StB, and p76PaB, added as a chase after 19 min to show the specificity of the ferrichrome binding.
resulting in plasmids pSpBEcC, pStBEcC, and pPaBEcC, respectively. Plasmid Samples of 100 l were withdrawn, and cells were harvested on cellulose nitrate
p76SpB was digested with HindIII and BamHI, and the obtained 831-bp frag- filters (pore size, 0.45 m; Sartorius AG, Göttingen, Germany) and washed twice
ment was ligated into HindIII/BamHI-cleaved pAB, p76StB, and p76PaB, re- with 5 ml of 0.1 M LiCl. The filters were dried, and the radioactivity was
sulting in plasmids pEcBSpC, pStBSpC, and pPaBSpC, respectively. Plasmid determined by liquid scintillation counting.
p76StB was digested with HindIII and BamHI, and the obtained 828-bp fragment Computer-assisted sequence analysis. Sequences were analyzed using the
was ligated into HindIII/BamHI-cleaved pAB, p76SpB, and p76PaB, resulting in program package PC.GENE and the BLAST homology search (1).
plasmids pEcBStC, pSpBStC, and pPaBStC, respectively. Plasmid p76PaB was
digested with HindIII and BamHI, and the obtained 726-bp fragment was ligated
into HindIII/BamHI-cleaved pAB, p76SpB, and p76StB, resulting in plasmids RESULTS
pEcBPaC, pSpBPaC, and pStBPaC, respectively.
To construct plasmid pBK71, a BamHI restriction site was introduced into the FhuA⌬5–160 corkless deletion derivatives display TonB-de-
fhuA gene on pHK763 (E. coli) using PCR and the primer Bam23_fhuA (5⬘-C pendent activities. Precise excision of the cork domain of E.
AATAGTTGCAGGATCCCCCCATGCGCTTTC-3⬘). The primer of the com- coli FhuA results in a stable barrel that is inserted into the
plementary strand was pT7_(5⬘-GCGAGGCCCAGCTGGCTTATCG-3⬘). The
PCR fragment was digested with HindIII and BamHI and ligated into
outer membrane and exerts TonB-dependent FhuA activities.
HindIII/BamHI-cleaved pBK7, resulting in plasmid pBK71. Deletions within the cork domain and deletions in the barrel
Plasmid pGB312 was digested with HindIII and EcoRI and ligated into domain, with the exception of the surface-exposed loops, fre-
HindIII/EcoRI-cleaved vector pHSG576, resulting in plasmid p576St. quently result in unstable FhuA derivatives (4). In this study,
Strain CH21 was constructed by picking a phage T5-resistant clone of strain
we excised the cork domain of FhuA from S. paratyphi B,
BL21 (DE3) omp8.
Recombinant DNA techniques. Isolation of plasmids, use of restriction en- Salmonella serovar Typhimurium, and P. agglomerans based on
zymes, ligation, agarose gel electrophoresis, and transformation were performed the E. coli FhuA crystal structure. The cork domain of all
according to standard techniques (29). All genetic constructions were examined FhuA proteins used in this study have the same length as that
by DNA sequencing using the dideoxy chain-termination method with fluores- of E. coli FhuA, except for FhuA of P. agglomerans, which
cence-labeled or unlabeled nucleotides (Auto Read Sequencing Kit, Pharmacia
contains a three-amino-acid insertion and a two-amino-acid
Biotech, Freiburg, Germany) and the ALF sequencer (Pharmacia).
Protein analytical methods. E. coli BL21 cells (optical density at 578 nm of 0.5) deletion (17). In addition, the amino acid sequences of all four
transformed with one of various plasmids encoding complete FhuA, corkless FhuA proteins are rather similar, which makes it likely that the
FhuA, or reconstituted FhuA hybrids were collected by centrifugation and re- cork domains comprise the same or nearly the same segment
suspended in 1 ml of M9 salts (24) supplemented with 0.4% glucose, 0.01% of the FhuA polypeptide.
methionine assay medium, 0.01% thiamine, and 1 mM IPTG (isopropyl--D-
thiogalactopyranoside) to induce T7 RNA polymerase synthesis. After shaking
To examine whether the corkless FhuA derivatives were
the cells for 1 h at 37°C, rifamycin (10 l of a 5-mg/ml solution in methanol) was synthesized and to estimate their relative amounts, the E. coli
added and incubation was continued at 37°C for 30 min. [35S]methionine was strain CH21 [an fhuA mutant of E. coli BL21 (DE3) omp8] was
added, and the suspension was incubated for 10 min. Cells were then collected by transformed with plasmids encoding the various fhuA genes
centrifugation and suspended in sample buffer. The radioactively labeled pro-
which were specifically transcribed by phage T7 RNA polymer-
teins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) as
described previously (12). In addition, cells transformed with wild-type fhuA and ase, and the proteins were labeled with [35S]methionine. The
mutant fhuA genes were grown in NB medium, the outer membrane fractions proteins of whole cells were separated by SDS-PAGE, and
were isolated, and the proteins were separated by SDS-PAGE and stained with only bands in the region of FhuA were seen on autoradio-
Serva blue. graphs (Fig. 1 shows only the FhuA-containing section of the
Phenotype assays. All phenotype assays were carried out with freshly trans-
formed E. coli K-12 strains 41/2 aroB fhuA, HK97 aroB fhuA fhuE, and HK99
gel). As observed previously with E. coli (4), less protein of the
aroB fhuA tonB. These strains carry the same four amino acid replacements and corkless FhuA derivatives was present (Fig. 1, lanes 5, 10, 15,
an amino acid deletion in fhuA and contain the mutated FhuA protein in the and 20) than that of the complete FhuA proteins from whichVOL. 183, 2001 ENTEROBACTERIAL FhuA BARREL-CORK HYBRIDS 3479
corkless FhuA proteins in cells that were grown under the
same conditions as those under which the activity assays were
performed. The fhuA transformants of E. coli HK97 were
grown in NB medium. Transcription of the fhuA genes en-
coded by the same plasmids as those used for T7 transcription
proceeded by E. coli RNA polymerase and was controlled by
the fhuA promoters. The FhuA proteins in the isolated outer
membrane fractions were separated by SDS-PAGE. The FhuA
protein of E. coli HK97 carries three amino acid replacements
and one amino acid deletion and was not seen after SDS-
PAGE when cells were grown in NB medium. Protein bands
with electrophoretic mobilities corresponding to the calculated
molecular mass of 61 kDa were present in lanes to which outer
membranes of corkless fhuA transformants were applied (Fig.
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2, marked by dotted arrows) and absent in lanes to which outer
membranes of wild-type fhuA transformants were applied (Fig.
FIG. 1. Comparison of [35S]methionine-labeled FhuA proteins and 2, marked by solid arrows). The amounts of the corkless FhuA
FhuA barrel-cork hybrids after transformation of E. coli CH21 with proteins were lower than those of the wild-type FhuA proteins,
plasmid pHK763 (lane 1), pEcBSpC (lane 2), pEcBStC (lane 3), pEcB- which agrees with the results obtained after transcription of the
PaC (lane 4), pBK7 (lane 5), p76Sp (lane 6), pSpBEcC (lane 7), genes with T7 RNA polymerase (Fig. 1). Since the same rela-
pSpBStC (lane 8), pSpBPaC (lane 9), pSp⌬5–160 (lane 10), p76St
tive amounts were obtained with whole cells and outer mem-
(lane 11), pStBEcC (lane 12), pStBSpC (lane 13), pStBPaC (lane 14),
pSt⌬5–160 (lane 15), p76Pa (lane 16), pPaBEcC (lane 17), pPaBSpC brane fractions, the lower amounts of FhuA⌬5–160 may arise
(lane 18), pPaBStC (lane 19), or pPa⌬5–160 (lane 20). No other bands from a lower mRNA stability caused by the deletion or by
were seen outside the gel section represented here. Nomenclature proteolytic degradation of FhuA⌬5–160 in the cytoplasm.
used: EcBSpC means the -barrel (B) of E. coli FhuA and cork (C) of Transport of the corkless derivatives was determined in E.
S. paratyphi B FhuA; the other designations follow the same rule. Ec,
E. coli; Sp, S. paratyphi B; St, Salmonella serovar Typhimurium; Pa, P. coli HK97 fhuA aroB cells transformed with the plasmids car-
agglomerans. rying the genes for the corkless FhuA proteins. The fhuA
mutations of E. coli HK97 exert no polar effect on the down-
stream fhuBCD genes required for ferrichrome transport
they were derived (Fig. 1, lanes 1, 6, 11, and 16). The radio- across the cytoplasmic membrane. FhuA⌬5–160 of S. paratyphi
activity of the major bands of the FhuA⌬5–160 proteins B and Salmonella serovar Typhimurium conferred ferrichrome
amounted on average to 25% of that of the complete proteins. transport (Fig. 3B and C) at rates of 14 and 21% the rate of
The faint bands above the major bands probably represent the their respective complete FhuA protein (Fig. 3B and C; Table
precursor form with uncleaved signal peptide. The majority of 2). Each rate was calculated using the value after 31 min of
FhuA is processed and presumably inserted into the outer transport minus the value after 1 min. These values were lower
membrane. than those obtained with E. coli FhuA⌬5–160 (Fig. 3A; Table
As the corkless FhuA proteins will be used to determine 2). No transport was observed in E. coli HK99 fhuA tonB
FhuA activities it was important to estimate the amounts of the transformed with plasmids carrying the genes for the corkless
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. 1.3480 KILLMANN ET AL. J. BACTERIOL.
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FIG. 3. Time-dependent transport of [55Fe3⫹]ferrichrome (1 M) 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.
FhuA proteins (data not shown). Transport of these transfor- fraction that is bound to FhuA. An example is given in Fig. 4
mants (FhuA⌬5–160 of S. paratyphi B and Salmonella serovar which shows ferrichrome binding to wild-type FhuA and
Typhimurium) was restored by transformation with a plasmid FhuA⌬5–160 of Salmonella serovar Typhimurium. The curves
encoding a wild-type tonB gene (data not shown). FhuA⌬5– with a higher value for the 1-min sample than for the following
160 of P. agglomerans did not transport ferrichrome (Fig. 3D; samples are representative for all experiments performed with
Table 2). wild-type and mutant FhuA proteins (data not shown). The
Reduction and lack of ferrichrome transport of the corkless data of this and further experiments are listed in Table 2. They
FhuA derivatives could result from impaired ferrichrome bind- show that ferrichrome binding to FhuA⌬5–160 of E. coli
ing, translocation, or both. An estimate of ferrichrome binding amounts to 5% that of wild-type FhuA, to S. paratyphi
to E. coli FhuA⌬5–160 was previously derived from the FhuA⌬5–160 is 5.2% that of the wild type, to Salmonella se-
amount of radioactive ferrichrome that was found associated rovar Typhimurium FhuA⌬5–160 is 3.7% that of the wild type,
with cells of transport-negative E. coli HK99 fhuA tonB and and to P. agglomerans FhuA⌬5–160 is 0% that of the wild type.
CH1857⌬fhuABCD tonB in time-dependent transport assays. The lack of ferrichrome binding to P. agglomerans FhuA⌬5–
It amounted to not more than 7% of that of wild-type FhuA 160 would account for the inability of this corkless derivative to
(4). In this study we measured the binding of 1 M radioactive transport ferrichrome. The values have not been quantitatively
ferrichrome to fhuA⌬5–160 transformants of CH1857 by tak- related to the amounts of the FhuA proteins; however, they
ing samples after 1, 7, 13, and 19 min, after which the cultures reflect the conditions under which transport was measured.
were chased with 150 M nonradioactive ferrichrome. The FhuA also transports the structurally related antibiotic al-
amount of ferrichrome that could be chased was taken as the bomycin. To examine the albomycin sensitivity of cells thatVOL. 183, 2001 ENTEROBACTERIAL FhuA BARREL-CORK HYBRIDS 3481
TABLE 2. Ferrichrome binding and ferrichrome transport rates of
complete FhuA, corkless FhuA, and reconstituted FhuA hybrid
proteins
Ferrichrome Ferrichrome transport rates
binding to (% wild type)e into
FhuA protein
CH1857a (iron
ions per cell) HK97b HK99 1c HK99 2d
FhuAEc (wild type) 10,322 100 100 100
FhuAEcBSpC 9,384 100 100 95
FhuAEcBStC 2,579 17 7 11
FhuAEcBPaC 84 84 88 86
FhuAEc⌬5–160 514 35 8 ND
FhuASp (wild type) 12,072 100 100 100
FhuASpBEcC 11,917 71 97 100 FIG. 4. Binding of [55Fe3⫹]ferrichrome to E. coli CH1857 ⌬fhuA-
FhuASpBStC 10,265 74 86 100 BCD tonB aroB expressing the indicated FhuA proteins. After 19 min
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FhuASpBPaC 857 67 80 100 a 150-fold surplus of nonradioactive ferrichrome was added (marked
FhuASp⌬5–160 629 14 9 ND by an arrow).
FhuASt (wild type) 11,188 100 100 100
FhuAStBEcC 11,327 84 76 89
FhuAStBSpC 10,225 99 100 100
FhuAStBPaC 0 1 1 1 fold higher than its sensitivity to rifamycin (Table 3). The CGP
FhuASt⌬5–160 417 21 5 ND 4823 sensitivity of E. coli 41/2 fhuA (pBK7) was approximately
FhuAPa (wild type) 7,925 100 100 100 10-fold higher, and its sensitivity to rifamycin was threefold
FhuAPaBEcC 0 37 14 21 higher. The higher sensitivity to CGP 4832 is a result of both
FhuAPaBSpC 253 18 4 13 active transport, since it depended on active TonB, and passive
FhuAPaBStC 0 20 4 8 diffusion through the FhuA⌬5–160 channel (data not shown).
FhuAPa⌬5–160 0 1 5 ND
An increase in sensitivity by diffusion through FhuA⌬5–160
a
E. coli CH1857 ⌬fhuABCD tonB was transformed with the plasmids listed in was evaluated in the E. coli HK99 tonB mutant transformed
Table 1 that encoded the FhuA proteins listed in the left panel.
b
E. coli HK97 fhuA expressing chromosomally encoded tonB was transformed
with pBK7, which for CGP 4832 was as high (threefold) as for
with plasmids that encoded the FhuA proteins listed in the left panel. rifamycin. Unexpectedly, sensitivity to both antibiotics was not
c
E. coli HK99 fhuA tonB was transformed with plasmids that encoded the increased in cells that synthesized FhuA⌬5–160 of S. paratyphi
FhuA proteins listed in the left panel and in addition carried low-copy plasmids
(Table 1) that encoded tonB genes of the strains from which the FhuA barrel was B, Salmonella serovar Typhimurium, or P. agglomerans (Table
derived, i.e., barrel of E. coli (EcB), Salmonella serovar Typhimurium (StB), and 3).
P. agglomerans (PaB) combined with tonB of E. coli, Salmonella serovar Typhi- FhuA of E. coli and S. paratyphi B renders cells sensitive to
murium, and P. agglomerans, respectively. The exception was the barrel S. para-
typhi (SpB), which was combined with tonB of Salmonella serovar Typhimurium. colicin M and microcin J25 (31). FhuA⌬5–160 of S. paratyphi B
d
As described above, but the tonB genes were from the strains from which the conferred sensitivity to colicin M which was 10-fold lower than
FhuA cork was derived, e.g., cork of E. coli (EcC) combined with tonB of E. coli.
ND, not determined.
that of complete FhuA, E. coli FhuA, and E. coli FhuA⌬5–160.
e
The percentage is related to the transport rates of the wild-type strains taken Both FhuA deletion derivatives were unable to mediate sensi-
as 100%. tivity to microcin J25 (Table 3). Cells expressing FhuA⌬5–160
of Salmonella serovar Typhimurium or P. agglomerans were as
resistant to colicin M and microcin J25 as cells expressing
produce the FhuA⌬5–160 proteins, E. coli 41/2 fhuA was trans- wild-type FhuA of these strains (Table 3).
formed with plasmids carrying the genes for the corkless FhuA FhuA of E. coli and S. paratyphi B serves as a receptor of the
proteins, and transformants were seeded on nutrient agar phages T1, T5, and 80. Sensitivity was tested by spotting a
plates to which 4 l of a series of threefold-diluted solutions of series of 10-fold-diluted phage solutions onto a lawn of E. coli
the antibiotic were spotted. Transformants carrying the gene 41/2 fhuA transformants that synthesized one of the FhuA⌬5–
for the corkless FhuA were sensitive to albomycin, although to 160 proteins. Cells synthesizing FhuA⌬5–160 of S. paratyphi B
different degrees (Table 3). Only P. agglomerans FhuA⌬5–160 were 10-fold less sensitive to phages T1 and T5 and 100-fold
did not confer albomycin sensitivity, which probably results less sensitive to phage 80 than the transformants synthesizing
from the lack of binding, as has been observed for ferrichrome. wild-type FhuA of S. paratyphi or E. coli or FhuA⌬5–160 of E.
Sensitivity depended on TonB, as shown by the albomycin coli (Table 3). Cells that synthesized FhuA⌬5–160 of Salmo-
resistance of TonB-negative fhuA⌬5–160 transformants (data nella serovar Typhimurium or P. agglomerans were resistant to
not shown). all the phages (Table 3). E. coli cells that synthesized FhuA⌬5–
Rifamycin CGP 4832, a chemically synthesized derivative of 160 of Salmonella serovar Typhimurium were resistant to
rifamycin, has a much higher activity than rifamycin because it phage ES18, which normally infects Salmonella serovar Typhi-
is actively transported across the outer membrane by FhuA murium via FhuA. Since E. coli cells that synthesized wild-type
(28). However, CGP 4832 is structurally unrelated to either FhuA of Salmonella serovar Typhimurium were sensitive to
ferrichrome or albomycin. E. coli 41/2 fhuA was found to be phage ES18, a 103-fold-diluted ES18 stock suspension formed
equally sensitive to either CGP 4832 or rifamycin (data not clear plaques, and a 105-fold-diluted suspension formed turbid
shown). However, the sensitivity of E. coli 41/2 fhuA carrying plaques, we conclude that ES18 infection requires the FhuA
plasmid pHK763 fhuA to CGP 4823 was approximately 100- cork domain and the -barrel domain.3482 KILLMANN ET AL. J. BACTERIOL.
TABLE 3. Sensitivity of E. coli 41/2 fhuA transformed with the indicated plasmids
Sensitivity to:a
Plasmid
Phage T1 Phage T5 Phage 80 Coicin M Microcin J25 Albomycin CGP 4832 Rifamycin
pHK763 (wild type) 4 4 4 3 3 3 6 2
pEcBSpCb 4 4 4 3 3 3 4 2
pEcBStC ⫺ 3 1 3 2 2 2 2
pEcBPaC 4 4 4 2 ⫺ 3 2 2
pBK7 (Ec⌬5–160) 4 4 4 3 ⫺ 2 4 3
p76Sp (wild type) 4 4 4 3 3 3 4 2
pSpBEcC 4 4 4 3 3 3 5 2
pSpBStC 4 4 4 3 2 3 4 2
pSpBPaC 4 4 4 3 ⫺ 3 2 2
pSp⌬5–160 3 3 2 2 ⫺ 1 2 2
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p76St (wild type) ⫺ ⫺ ⫺ ⫺ ⫺ 3 2 2
pStBEcC ⫺ ⫺ ⫺ ⫺ ⫺ 3 2 2
pStBSpC ⫺ ⫺ ⫺ ⫺ ⫺ 2 1 2
pStBPaC ⫺ ⫺ ⫺ ⫺ ⫺ 1 1 2
pSt⌬5–160 ⫺ ⫺ ⫺ ⫺ ⫺ 1 1 2
p76Pa (wild type) ⫺ ⫺ ⫺ ⫺ ⫺ 3 2 2
pPaBEcC ⫺ ⫺ ⫺ ⫺ ⫺ 1 2 2
pPaBSpC ⫺ ⫺ ⫺ ⫺ ⫺ 1 2 2
pPaBStC ⫺ ⫺ ⫺ ⫺ ⫺ 2 2 2
pPa⌬5–160 ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ 2 2
a
Sensitivities to the ligands were tested by using E. coli 41/2 aroB fhuA freshly transformed with the plasmids indicated. The sensitivities were tested by spotting 4
l of 10-fold or 3-fold (for microcin J25, rifamycin CGP 4832, and rifamycin) dilutions onto TY agar plates overlaid with TY top agar containing the strain to be tested.
The results are given as the last of a 10-fold or 3-fold dilution series that resulted in a clear zone of growth inhibition. For example, a value of 4 indicates that the phage
solution could be diluted 104-fold to yield a clear zone of cell lysis. ⫺, no growth inhibition and no phage plaques.
b
Ec, E. coli; Sp, S. paratyphi; St, Salmonella serovar Typhimurium; Pa, P. agglomerans; B, -barrel; C, cork.
FhuA⌬5–160 deletion derivatives display low open channel HK99 synthesizing E. coli FhuA⌬5–160 compared to complete
activities. For the determination of active transport, 1 M FhuA, measured as zones of growth inhibition, increased from
[55Fe3⫹]ferrichrome was used. At this ferrichrome concentra- 8 to 14 mm, 15 to 19 mm, and 9 to 12 mm, respectively. The
tion, growth on nutrient broth agar plates containing 0.2 mM sensitivity of E. coli HK99 tonB fhuA synthesizing P. agglom-
dipyridyl to suppress low-affinity iron uptake (NBD plates) is erans FhuA⌬5–160 to these antibiotics compared to complete
not supported. For an estimation of ferrichrome uptake by FhuA increased from 8 to 13 mm, 15 to 20 mm, and 9 to 11
diffusion across the outer membrane, E. coli HK99 fhuA tonB mm, respectively. The sensitivity of the FhuA⌬5–160 deriva-
aroB transformed with plasmids carrying the genes for the tives of S. paratyphi B and Salmonella serovar Typhimurium to
corkless FhuA proteins was used. Ferrichrome at concentra- antibiotics was not increased significantly. The parental strains
tions of 0.1, 0.3, 1, 3, and 10 mM was placed on filter paper AB2847 and 41/2 displayed the same sensitivities to the three
disks, and growth promotion around the disks on NBD plates antibiotics as the pHK763 (wild-type fhuA) transformants.
seeded with 108 cells of the HK99 transformants was recorded. Hybrid FhuA proteins consisting of -barrel domains and
Slow growth of a small number of cells that synthesized unrelated cork domains are active. The cork and -barrel
FhuA⌬5–160 of E. coli or P. agglomerans was observed with 0.1 domains of the enterobacterial FhuA proteins were mutually
mM ferrichrome. The same result was obtained with cells that exchanged to determine whether complete FhuA can be re-
synthesized FhuA⌬5–160 of S. paratyphi B when a solution of constituted, exported across the cytoplasmic membrane, and
0.3 mM ferrichrome was used. At this concentration, cells that inserted correctly into the outer membrane. Moreover, it was
synthesized FhuA⌬5–160 of E. coli or P. agglomerans showed a of interest to determine whether FhuA hybrids consisting of
strong growth zone of 10 mm in diameter (6-mm disk diameter -barrel domains and unrelated cork domains display activity
not subtracted). At 10 mM ferrichrome, cells synthesizing E. with some or all of the ligands and whether the reconstituted
coli FhuA⌬5–160, S. paratyphi B FhuA⌬5–160, Salmonella se- FhuA proteins still respond to TonB.
rovar Typhimurium FhuA⌬5–160, and P. agglomerans The cork domains of S. paratyphi, Salmonella serovar Typhi-
FhuA⌬5–160 had growth zones of 18, 13, 18, and 22 mm, murium, and P. agglomerans were each combined with the
respectively. -barrel domain of E. coli. The derivatives showed the same
Another means to measure diffusion through the FhuA⌬5– electrophoretic mobility (Fig. 1, lanes 2 to 4) as wild-type FhuA
160 derivatives is provided by antibiotics that are too large to of E. coli (Fig. 1, lane 1). The yield of the hybrid FhuA proteins
diffuse readily through the porin channels. Growth inhibition resulting from transcription by T7 RNA polymerase was com-
around filter paper disks to which these antibiotics had been parable to the yield of wild-type FhuA cloned in the same
applied was measured. The sensitivities to erythromycin (734 vector. Similar results were obtained with each of the unrelated
Da), rifamycin (823 Da), and vancomycin (1,486 Da) of E. coli cork domains fused to the -barrel domain of S. paratyphi BVOL. 183, 2001 ENTEROBACTERIAL FhuA BARREL-CORK HYBRIDS 3483
(Fig. 1, lanes 6 to 9), Salmonella serovar Typhimurium (Fig. 1, low or no binding (Table 2), which was largely correlated with
lanes 11 to 14), and P. agglomerans (Fig. 1, lanes 16 to 19). the amounts of FhuA protein and the additional FhuA-derived
To use the same conditions as those under which the FhuA protein bands (Fig. 2). However, the ferrichrome transport
activity assays were performed, FhuA synthesis was examined rates were not strictly related to binding since FhuAPaBEcC
in transformants in which the fhuA genes were transcribed by with no binding transported better than FhuAPaBSpC with
E. coli RNA polymerase under the control of the fhuA pro- residual binding (Table 2). Furthermore, FhuASpBPaC binds
moters, and cells were grown under assay conditions. SDS- poorly (7%) and FhuAEcBPaC binds very poorly (0.8%), but
PAGE analysis revealed a somewhat heterogeneous band pat- they display high transport activities.
tern at the electrophoretic position of FhuA, especially with The degree of albomycin sensitivity of E. coli 41/2 fhuA
FhuAEcBStC and the FhuA hybrids containing the P. agglo- transformed with plasmids encoding the FhuA hybrids is in
merans -barrel (Fig. 2). However, each of the transformants agreement with the ferrichrome transport rates (Table 3). A
contained a stronger protein band at the electrophoretic posi- few minor deviations may result from the lower transport rate
tion of FhuA than E. coli contains when it synthesizes chro- of albomycin than ferrichrome and from the type of assay used,
mosomally encoded wild-type FhuA (10, 12). The amount of either the transport assay within 30 min or the growth assay
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hybrid FhuA is considered to be sufficient to confer all FhuA within 15 h.
activities. The amounts of the hybrid proteins were similar to The sensitivity of the FhuA hybrids to phages T1, T5, and
the amounts of plasmid-encoded wild-type proteins. All FhuA 80 was determined with transformants of E. coli 41/2 fhuA.
proteins, including E. coli wild-type FhuA, contained a minor The FhuA hybrids containing the -barrel of E. coli or S.
band which most likely is a degradation product. The upper paratyphi B FhuA were as phage sensitive as cells synthesizing
band close to FhuA in the samples of the FhuA⌬5–160 pro- wild-type FhuA (Table 3). Exceptions were transformants with
teins is not an FhuA product since it is not contained in Fig. 1 the -barrel of E. coli and the cork of Salmonella serovar
in which the fhuA⌬5–160 genes were specifically transcribed by Typhimurium, which were resistant to phage T1, 1,000-fold less
T7 RNA polymerase (Fig. 1). sensitive to phage 80, and 10-fold less sensitive to phage T5.
The activities of the FhuA hybrids were determined by mea- Cells that synthesized wild-type FhuA of Salmonella serovar
suring the ferrichrome transport rate of E. coli HK97 aroB Typhimurium or P. agglomerans and FhuA hybrids composed
fhuA fhuE transformed with plasmids encoding each of the of the -barrel of Salmonella serovar Typhimurium or P. ag-
FhuA hybrids. As shown in Fig. 3 and summarized in Table 2, glomerans were equally resistant to the phages (Table 3). None
half of the FhuA hybrids displayed transport activities as high of the FhuA hybrids conferred sensitivity to phage ES18, which
or nearly as high as the wild-type FhuA proteins. For example, indicates that infection by phage ES18 requires both the cork
the -barrel of E. coli FhuA fused to the cork of S. paratyphi or and the -barrel of Salmonella serovar Typhimurium FhuA.
P. agglomerans showed 100 and 84% of the transport rate of E. E. coli 41/2 fhuA transformants that synthesized FhuA hy-
coli wild-type FhuA (Table 2). In contrast, the E. coli -barrel brids containing the E. coli or S. paratyphi B -barrel were
fused to the Salmonella serovar Typhimurium cork displayed sensitive to colicin M to somewhat variable degrees (Table 3).
only 17% of the wild-type activity, which may be explained by Transformants that expressed wild-type FhuA of Salmonella
the lower amounts of the mutant FhuA protein (Fig. 2). The serovar Typhimurium or P. agglomerans and FhuA hybrids
same cork fused to the -barrel of S. paratyphi showed 74% of containing the -barrel of Salmonella serovar Typhimurium or
the wild-type activity, which agrees with the high amount of the P. agglomerans were resistant to colicin M. Colicin M sensitivity
mutant FhuA protein (Fig. 2). The cork of P. agglomerans conferred by the FhuA hybrids was TonB dependent, as trans-
fused to the -barrel of E. coli or S. paratyphi B was highly formants of E. coli HK99 tonB were resistant to colicin M (data
active (84 and 67% of the wild-type activity) but showed no not shown).
activity when combined with the -barrel of Salmonella serovar The -barrel and the cork of E. coli or S. paratyphi B were
Typhimurium, despite high levels of protein (Fig. 2). The required to render cells sensitive to microcin J25. However, the
-barrel of P. agglomerans displayed the lowest tolerance to cork of P. agglomerans did not reconstitute the activity of
unrelated cork domains. FhuAPaBPaC, FhuAPaBPaSp, and FhuA⌬5–160 of E. coli or S. paratyphi B (Table 3).
FhuAPaBStC displayed only 37, 18, and 20% of the FhuAPa Of all the FhuA hybrids examined here, only those that
wild-type activity, and the amount of unaltered reconstituted synthesized the E. coli -barrel fused to the S. paratyphi B cork
FhuA hybrid proteins was the lowest of all the hybrid proteins and the -barrel of S. paratyphi B fused to the cork of E. coli
(Fig. 2). or Salmonella serovar Typhimurium conferred TonB-depen-
To see whether the transport rates are related to fer- dent sensitivity to CGP 4832 that was higher than the sensitiv-
richrome binding activities, binding of radioactive ferrichrome ity to rifamycin (Table 3).
was measured in cells of CH1857 ⌬fhuABCD tonB expressing There is no preference for the TonB protein related to the
the FhuA hybrid proteins. As shown in Table 2, FhuAEcBSpC, FhuA cork or -barrel. We first determined the transport
FhuASpBEcC, FhuASpBStC, FhuAStBEcC, and FhuAStB- activities of all FhuA derivatives in E. coli, which means in
SpC bound ferrichrome approximately to the same extent as combination with the E. coli TonB protein. We then wanted to
the wild-type FhuA proteins. FhuAEcBStC displayed only find out whether it makes a difference in FhuA activity when
25% of these binding activities, which was correlated with the the FhuA hybrids are combined with the TonB proteins of the
heterogeneous FhuA protein profile (Fig. 2) and the low fer- same strains from which the FhuA hybrids were derived. In
richrome transport rate (Fig. 3A; Table 2). Binding of fer- addition, since TonB apparently interacts with the cork and the
richrome to FhuAPa was lower than to the wild-type FhuA -barrel it was of interest to determine whether the cork or the
proteins of the other strains, and the hybrid proteins showed -barrel should be from the same strain as the TonB protein.3484 KILLMANN ET AL. J. BACTERIOL.
We constructed combinations of tonB genes on a low-copy channel formed by the -barrel, the N-proximal peptide ap-
plasmid with the plasmid-encoded wild-type fhuA and mutated pears to strongly impair the binding of ferrichrome, which
fhuA genes in E. coli HK99 fhuA tonB, with the exception of occurs well above the cell surface. In addition to binding, the
the tonB gene of S. paratyphi B, which was unavailable. All the transport activity must also be impaired since FhuA⌬5–160
combinations were active, and the absolute transport rates also binds ferrichrome poorly but transports ferrichrome
listed as 100% in Table 2 are similar to the highest transport rather well. It should be stated that the relative amount of
rates shown in Fig. 3. No alterations of the FhuA activities FhuA⌬25–160(P24D) protein observed after SDS-PAGE was
were observed that could be related to homologous versus comparable to that of wild-type FhuA (data not shown).
heterologous FhuA-TonB combinations or to the cork or the
-barrel (Table 2). The FhuA activities of the E. coli FhuA DISCUSSION
-barrel derivatives combined with TonB of E. coli (Table 2,
HK97 and HK99 1) differed only slightly from the E. coli FhuA Our previous finding of high and specific activities of cork-
-barrel derivatives combined with the TonB protein of Sal- less FhuA of E. coli (4) are supported by the results described
monella serovar Typhimurium and P. agglomerans (HK99 2). in this paper with the corkless FhuA proteins of S. paratyphi B
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When the -barrel of S. paratyphi B was combined with TonB and Salmonella serovar Typhimurium. These corkless FhuA
of Salmonella serovar Typhimurium, the FhuA activities were derivatives exhibit TonB-dependent ferrichrome transport, al-
somewhat higher (HK99 1) than when combined with the E. though at rates lower than that of the E. coli corkless FhuA.
coli TonB (HK97). This increase may be a result of the over- The amounts of the corkless derivatives were also lower (ap-
expression of plasmid-encoded TonB, although in other cases proximately 25% that of wild-type FhuA), which may have
the FhuA -barrel derivatives showed lower FhuA activities reduced the activities. The rates of 14 and 21% in comparison
when combined with plasmid-encoded TonB (HK99) than with to the rates obtained with the complete FhuA of the same
chromosomally encoded E. coli TonB (HK97) (Table 2), which strain decreased to zero in the tonB mutant strain HK99 car-
has been observed previously (22). There was a tendency of a rying the same fhuA mutation as that of E. coli HK97 fhuA
higher FhuA activity with TonB combined with the related used for the transport experiments. To rule out complemen-
cork domain (HK99 2) than with TonB combined with the tation of the mutated E. coli HK97 FhuA protein by the E. coli
related -barrel domain (HK99 1). However, we doubt that the corkless FhuA mutant protein through formation of a func-
observed differences are large enough to suggest a stronger tional oligomer, we previously carried out experiments with E.
impact of the cork than the -barrel in the interaction of FhuA coli H1857 in which the fhuABCD genes are deleted (4). After
with TonB. transformation of E. coli H1857 with fhuA⌬5–160 and the
The -barrel domain of E. coli FhuA containing the TonB fhuBCD genes for transport across the cytoplasmic membrane,
box is less active. The results reported here and in our previous ferrichrome transport is even higher than transport into E. coli
papers (4, 9, 30) indicated that FhuA activity is mediated by HK97 since E. coli H1857 synthesizes greater amounts of plas-
TonB through interaction with the cork and the -barrel. mid-encoded FhuBCD proteins than E. coli HK97. In addition,
Therefore, we examined whether the TonB box linked to the X-ray analysis does not support the formation of an FhuA
-barrel domain affects the activity of the -barrel. We con- oligomer as the FhuA crystals consisted of a monomer (7, 20).
structed FhuA⌬25–160, in which the N-proximal 23 residues of FhuA⌬5–160 of P. agglomerans was considered inactive, as it
mature FhuA, including the TonB box, were linked to residue did not transport ferrichrome, conferred no sensitivity to albo-
161 of the -barrel domain. The genetic manipulation replaced mycin, and showed the same sensitivity to rifamycin CGP 4832
Pro24 with Asp. E. coli 41/2 fhuA synthesizing FhuA⌬25– as to rifamycin. Among the FhuA proteins studied, that of P.
160(P24D) was as sensitive to phage 80 as E. coli 41/2 fhuA agglomerans exhibits the least sequence similarity to E. coli
synthesizing FhuA⌬5–160 but was 10-fold less sensitive to FhuA (59%). The construction of the deletion introduced the
phages T1 and T5 and to colicin M and was resistant to albo- amino acid replacements A3D and E4P; the latter replacement
mycin and microcin J25. Since TonB-independent infection by may not affect FhuA⌬5–160 activity, since similar replace-
phage T5 was also reduced, the lower activity of FhuA⌬25– ments at the A3 site in FhuA⌬5–160 of E. coli (E3D), S.
160(P24D) cannot be ascribed to an unproductive binding of paratyphi B (Q3D), and Salmonella serovar Typhimurium
TonB to the TonB box of FhuA⌬25–160(P24D). This inter- (Q3D) did not abolish activity.
pretation is supported by the finding that phage T5 sensitivity FhuA of S. paratyphi B is the only non-E. coli FhuA that
is also reduced 10-fold in the HK99 tonB mutant. The fer- mediates sensitivities to phages T1, T5, and 80 and to colicin
richrome transport rate was near zero. After 30 min, there M and albomycin, and this specificity was retained in the S.
were 3,000 ferrichrome molecules per cell compared to paratyphi B corkless FhuA, although at 1 or 2 orders of mag-
140,000 in cells expressing wild-type FhuA and 45,000 in cells nitude lower than the sensitivity conferred by the complete
expressing FhuA⌬5–160 in experiments run in parallel. In ad- FhuA. Sensitivity to these FhuA ligands was TonB-dependent,
dition, binding of ferrichrome to FhuA⌬25–160(P24D) was except for infection by phage T5, which occurs independent of
examined. Using E. coli CH21(pBK71), binding of ferrichrome TonB. FhuA⌬5–160 of S. paratyphi, like that of E. coli, did not
amounted to about 3,000 molecules per cell compared to mediate sensitivity to microcin J25 and differed from the E. coli
20,000 molecules bound to wild-type FhuA of E. coli FhuA⌬5–160 in that it did not enhance sensitivity to rifamycin
CH21(pHK763), which after a chase with a 150-fold surplus of CGP 4832.
unlabeled ferrichrome was reduced to 3,000 molecules per cell. The -barrel of E. coli FhuA without the cork mediates all
Although the binding site (residue 161) of the N-proximal FhuA functions except uptake of microcin J25 (4). Uptake of
24-residue peptide is exposed to the periplasm outside the microcin J25 and infection of Salmonella serovar TyphimuriumVOL. 183, 2001 ENTEROBACTERIAL FhuA BARREL-CORK HYBRIDS 3485
by phage ES18 may require both the cork and the -barrel. We in processed form in amounts similar to those of the wild-type
have previously shown that the prominent loop of the FhuA FhuA proteins. The exceptions were the FhuA hybrids which
-barrel (18), which is loop 4 in the E. coli FhuA crystal contained the P. agglomerans -barrel and heterologous cork
structure (7, 20) and lies above the cell surface, serves as the domains, which formed several bands of which one was prob-
principal binding site of the phages and colicin M (13, 14, 15). ably the genuine FhuA hybrid. However, the reduced amounts
This result implies that TonB, without the help of the cork, can of these hybrids do not fully explain the low activity, as they
change the conformation of loop 4 such that binding of phages were present in higher amounts than that observed with chro-
T1 and 80 triggers DNA release from the phage head. This mosomally encoded wild-type FhuA, which confers full FhuA
conformational change is not restricted to loop 4, since release activity (10, 12). FhuAPa⌬5–160 is somewhat unstable, as the
of ferrichrome from its binding sites in the -barrel (residues band pattern demonstrates, and the hybrid proteins appear to
Y244, W246, Y313, Y315, F391, and F693) probably also re- be even less stable. Nevertheless, the hybrid proteins exhibit
quires a conformational change of the -barrel, and none of ferrichrome transport activity, while the corkless mutant does
the ferrichrome binding sites are located in loop 4. These not. Most FhuA hybrids transported ferrichrome with rates
binding sites are contained in the four corkless FhuA proteins, higher than those of the corkless FhuA proteins from which
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with the exception of Y315, which is replaced in Salmonella they were derived. FhuAEcBStC displayed a low transport rate
serovar Typhimurium and P. agglomerans by T and N, respec- (17% that of E. coli FhuA wild-type), which may have attrib-
tively, and F693, which is replaced in P. agglomerans by Y. uted to the protein’s instability (Fig. 2). In contrast, FhuASt-
Since aromatic residues play a major role in ferrichrome and BPaC is inactive despite its high amounts in the outer mem-
albomycin binding, replacement of Y315 by these nonaromatic brane fraction (Fig. 2). In this hybrid protein the cork
amino acids may well contribute to the lower transport activity apparently does not fit into the -barrel to reconstitute an
of Salmonella serovar Typhimurium FhuA⌬5–160 and the in- active FhuA protein. In all mutant FhuA proteins the degree
activity of P. agglomerans FhuA⌬5–160. However, this cannot of albomycin sensitivity correlated with the ferrichrome trans-
be the only cause since the transport activity of S. paratyphi port rates.
FhuA⌬5–160 is rather low (after 12 min, 18,000 ions per cell Increased sensitivity to rifamycin CGP 4832, compared to
compared to 48,000 per cell with E. coli FhuA⌬5–160), despite rifamycin and sensitivity to microcin J25 were only mediated by
the identity of these residues with those of E. coli FhuA⌬5– FhuA hybrids containing the -barrel of E. coli or S. paratyphi
160. B. The binding site of CGP 4832 in FhuA, as derived from the
TonB-dependent conformational changes of the -barrel FhuA cocrystal structure (A. D. Ferguson, J. Ködding, G.
may also widen the channel to facilitate diffusion of fer- Walker, C. Bös, J. W. Coulton, K. Diederichs, V. Braun, and
richrome and albomycin once they are released from their W. Welte, unpublished data), largely overlaps with the fer-
binding sites and/or may properly position the amino acid side richrome and albomycin (8) binding site. The same amino acid
chains along which ferrichrome and albomycin diffuse through residues contribute to binding of ferrichrome, albomycin, and
FhuA. These possibilities should be considered due to the low CGP 4832 in the E. coli and S. paratyphi B FhuA proteins,
diffusion rates through the corkless FhuA proteins, as evi- except for a single, functionally equivalent E3D exchange in
denced by the small increase in sensitivity to the antibiotics S. paratyphi B. Of the total of 16 residues that bind CGP 4832
erythromycin, rifamycin, and vancomycin compared to the to E. coli FhuA, FhuA of Salmonella serovar Typhimurium and
same E. coli strain synthesizing plasmid-encoded wild-type P. agglomerans deviate by 4 and 8 residues, respectively. The
FhuA proteins. number of amino acid replacements may explain why the FhuA
If TonB interacts only with -barrel regions exposed to the proteins of Salmonella serovar Typhimurium and P. agglomer-
periplasm, the conformational change must be transmitted ans do not show increased sensitivity to CGP 4832. Two out of
across the entire FhuA molecule up to the cell surface. It is not the 10 residues that in E. coli FhuA bind ferrichrome are
known whether TonB inserts into the outer membrane. How- different in Salmonella serovar Typhimurium FhuA, and 4 out
ever, the observed shuttling of TonB between the outer mem- of 10 differ in P. agglomerans FhuA. These sites also bind
brane and the cytoplasmic membrane (19) excludes a firm albomycin and CGP 4832 in E. coli FhuA.
integration of TonB in the outer membrane. In addition to the ligand binding sites, the data indicate that
Fusions of cork domains with -barrel domains of different other regions are important for the transport activities of the
species were constructed to determine whether the corks are hybrid FhuA proteins. For example, insertion of the Salmo-
inserted into the -barrels, how they fit into the -barrels, and nella serovar Typhimurium FhuA cork decreases the activity of
whether they restore the activities to those of complete wild- the E. coli FhuA -barrel; however, the E. coli cork strongly
type homologous FhuA proteins. It was conceivable that the increases the transport activity of the Salmonella serovar Ty-
corks were not incorporated into the -barrels, that the hybrid phimurium -barrel. The Salmonella serovar Typhimurium
proteins were rapidly degraded in the cytoplasm or the cork fused to the S. paratyphi B -barrel results in a highly
periplasm, that they stayed in the cytoplasm and were not active transporter. The P. agglomerans cork increases the trans-
exported across the cytoplasmic membrane, that they re- port activities when inserted into the E. coli and S. paratyphi B
mained in the periplasm, or that they were inserted into the -barrels but fails to complement the Salmonella serovar Ty-
outer membrane in an inactive form. The heterologous corks phimurium -barrel. These results show that incorporation of
could interact with the -barrels such that structural transitions a cork into a barrel is not sufficient to restore transport activity;
in the -barrels and the corks upon binding of the ligands and rather, intimate interactions between the cork and the -barrel
TonB were blocked or aberrant. We did not have to investigate must occur in order to form an active transporter. In a previous
all these possibilities since we obtained FhuA hybrids present study, prior to the determination of the FhuA crystal structure,You can also read
