Eur J Biochem 269, 4948–4959 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03195.x Diffusion through channel derivatives of the Escherichia coli FhuA transport protein Michael Braun1, Helmut Killmann1, Elke Maier2, Roland Benz2 and Volkmar Braun1 Mikrobiologie/Membranphysiologie, Universitaăt Tuăbingen, Germany; 2Lehrstuhl fuăr Biotechnologie, Theodor-Boveri-Institut (Biozentrum), Universitaăt Wuărzburg, Germany FhuA is a multifunctional protein in the outer membrane of Escherichia coli that actively transports [Fe3+]ferrichrome, the antibiotics albomycin and rifamycin CGP 4832, and mediates sensitivity of cells to the unrelated phages T5, T1, /80 and UC-1, and to colicin M and microcin J25 The energy source of active transport is the proton motive force of the cytoplasmic membrane that is required for all FhuA functions except for infection by phage T5 The FhuA crystal structure reveals 22 antiparallel transmembrane b-strands that form a b-barrel which is closed by a globular N-terminal domain FhuA still displays active transport and sensitivity to all ligands except microcin J25 when the globular domain (residues 5–160) is excised and supports weakly unspecific diffusion of substrates across the outer membrane Here it is shown that isolated FhuAD5–160 supported diffusion of ions through artificial planar lipid bilayer membranes but did not form stable channels The double mutant FhuAD5– 160 D322–336 lacking in addition to the globular domain most of the large surface loop which partially constricts the channel entrance, displayed an increased single-channel conductance but formed no stable channels It transported in vivo [Fe3+]ferrichrome with 45% of the rate of wild-type FhuA and did not increase sensitivity of cells to antibiotics In contrast, a second FhuA double mutant derivative which in addition to the globular domain contained a deletion of residues 335–355 comprising one-third of surface loop and half of the transmembrane b-strand formed stable channels in lipid bilayers with a large single-channel conductance of 2.5 nS in M KCl Cells that synthesized FhuAD5–160 D335–355 showed an increased sensitivity to antibiotics and supported diffusion of maltodextrins, SDS and ferrichrome across the outer membrane FhuAD5–160 D335–355 showed no FhuA specific functions such as active transport of [Fe3+]ferrichrome or sensitivity to the other FhuA ligands It is concluded that FhuAD5–160 D335–355 assumes a conformation that is incompatible with any of the FhuA functions The outer membrane of Escherichia coli forms a permeability barrier for hydrophilic substrates larger than 600 D [1] Smaller substrates diffuse through water-filled pores formed by the porins Most of the siderophores secreted by bacteria and fungi to solubilize Fe3+ are larger than 600 Da [2] In addition, the concentration of the Fe3+ siderophores is very low so that diffusion does not provide sufficient iron for growth, which is in the order of 105 ions per E coli cell per generation Therefore, Fe3+ siderophores are actively taken up by an energy-requiring transport process The proton motive force of the cytoplasmic membrane drives their transport across the outer membrane [3] and ATP energizes uptake across the cytoplasmic membrane [4] FhuA transports ferrichrome, the structurally related antibiotic albomycin, the unrelated antibiotic rifamycin CGP 4832, and serves as receptor for the phages T5, T1, /80 and UC-1, for the toxic colicin M protein and the toxic microcin J25 peptide [4] The crystal structure of FhuA reveals 22 antiparallel transmembrane b-strands that form a b-barrel which is closed by a globular domain, also called cork [5] or plug [6] It is thought that energy input from the cytoplasmic membrane opens the channel of the b-barrel in that the globular domain somehow moves and ferrichrome dissociates from its binding site which is formed by 10 amino acid residues [5] The energy transfer from the cytoplasmic membrane to FhuA in the outer membrane is mediated by the TonB protein that is located in the periplasm and anchored by the N-proximal end in the cytoplasmic membrane [7] Two additional proteins, ExbB and ExbD, are associated with TonB and are required for TonB activity The subcellular localization of ExbD is similar to TonB [8], whereas ExbB is anchored with three transmembrane segments in the cytoplasmic membrane with most of the protein in the cytoplasm [9] FhuA changes its conformation upon binding of ferrichrome The crystal structure shows a small movement ˚ (1 A) of the globular domain towards the bound ferri˚ chrome but a large movement (17 A) of residues 19 and 22 of the globular domain that are exposed to the periplasm Binding of ferrichrome enhances interaction of FhuA with TonB [10], which may be facilitated by the structural ˚ transition of 17 A Residues 1–18 are not seen in the crystal and therefore are thought to be flexible This segment contains the TonB box (residues 7–11) which has been Correspondence to V Braun, Mikrobiologie/Membranphysiologie, Universitat Tubingen, Auf der Morgenstelle 28, D-72076 Tubingen, ¨ ¨ ¨ Germany Fax: + 49 7071 295843, Tel.: + 49 7071 2972096, E-mail: volkmar.braun@mikrobio.uni-tuebingen.de Abbreviations: LDAO, N,N-dimethyldodecylamine-N-oxide (Received June 2002, revised August 2002, accepted 21 August 2002) Keywords: channel; Escherichia coli; FhuA transport protein Ó FEBS 2002 named according to the finding that the amino acid replacements I9P (isoleucine in position replaced by proline) and V11D abolished the TonB-dependent FhuA activities [11] and reduced interaction of FhuA with TonB [12] The Q160L and Q160K replacements in TonB partially restored the activities of the FhuA TonB box mutants These results supported the notion that FhuA interacts through the TonB box with region 160 of TonB As in FhuA the TonB box is contained near the N terminus of all active outer membrane transport proteins and the group B colicins which also require TonB to kill cells Further support of the TonB box concept comes from the spontaneous disulfide formation between cysteine residues introduced into the TonB box of the BtuB protein and cysteine residues introduced into region 160 of TonB BtuB is similar to FhuA and actively transports vitamin B12 across the outer membrane [13] Site-directed spin labelling and electron paramagnetic resonance studies revealed that binding of vitamin B12 to BtuB alters the conformation and the dynamics of the TonB box segment [14] At the time when the crystal structure of FhuA was not yet available we arrived at an early tentative transmembrane model of FhuA that proposed a prominent loop at the cell surface from residue 316 to residue 355 [15] The model was experimentally derived from the proteolytic cleavage of peptides of up to 16 amino acids which had been inserted into FhuA, and by computer-based prediction programs In support of this notion we showed that this region serves as the binding site for the phages T1, T5 and /80, because synthetic peptides covering this region inhibited infection by the phages [16] Under the assumption that the surface loop might also control a putative channel of FhuA we excised residues 322–355 which indeed converted FhuA into an open channel that exhibited stable single-channel conductance in artificial lipid bilayer membranes [17] Excision of residues 322–336 and 335–355 resulted in no stable singlechannel conductance [18] The crystal structure later revealed that the largest surface loop (L4), indeed extends from residues 318–339 and that residues 340–355 are located above the outer membrane lipid bilayer and form half of the b-sheet number The cork domain revealed by the crystal structure could not be predicted by the methods used To understand the role of the cork domain in channel formation of FhuA we have constructed FhuAD5–160 based on the crystal structure under the assumption that excision of the entire globular domain would convert FhuA into an open channel similar to the channel formed by FhuAD322–355 [17] and abolish all TonB-dependent FhuA activities However, FhuAD5–160 still displayed all TonBrelated activities between 40 and 100% of wild-type FhuA activity, depending on the function tested, and the permeability of the outer membrane for substrates and antibiotics increased only slightly [19] These findings were supported by a study using FhuAD5–160 derivatives of Salmonella paratyphi B and Salmonella enterica serovar Typhimurium which in addition showed that hybrid proteins consisting of the b-barrel of one strain and the globular domain of another strain were functional [20] An investigation with an E coli deletion derivative of FepA, the outer membrane transport protein for ferric enterobactin, in which the cork domain was removed and comparison with FhuAD5–160 supported the TonB dependent activities of the b-barrels [21] FhuA transport protein of E coli (Eur J Biochem 269) 4949 To gain further insights into the mode of action of FhuAD5–160, and in particular to the transport activity of the b-barrel, we determined in this report single-channel conductance of isolated FhuAD5–160 incorporated into artificial bilayer membranes We wanted to relate the in vitro activities of FhuAD5–160 with the in vivo activities Since FhuAD5–160 formed no stable channels in vitro we deleted loop which constricts half the channel entrance of FhuA to about half the area of the total cross-section [6] As no stable channels were formed by FhuAD5–160 D322–336, we combined deletion D5–160 with deletion D335–355 which as a single deletion did not display stable singlechannel conductance [18] FhuAD5–160 D335–355 formed large stable channels which were consistent with the in vivo unspecific increase of the outer membrane permeability FhuAD5–160 D335–355 did not actively transport [Fe3+]ferrichrome, in contrast with FhuAD5–160 D322–336 which displayed TonB-dependent transport activity MATERIALS AND METHODS Bacterial strains, plasmids and growth conditions The E coli strains and plasmids used are listed in Table Cells were grown in TY medium [10 gỈL)1 bactotryptone (Difco Laboratories), gỈL)1 yeast extract, gỈL)1 NaCl] or NB medium (8 gỈL)1 nutrient broth, gỈL)1 NaCl, 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 antibiotic ampicillin (40 gỈmL)1) was added when required Plasmids pHK234 and pHK237 were digested with MluI and SalI and ligated into MluI/SalI-cleaved plasmid pHK763 resulting in plasmids p7634 and p7637, respectively Plasmids p7634 and p7637 were digested with BspEI and EcoRI and ligated into BspEI/EcoRI-cleaved plasmid pBK7 resulting in plasmids pDM234 and pDM237, respectively Plasmid pSKF405-04 was digested with MluI and BstEII and ligated into MluI/BstEII-cleaved plasmid pHK763 resulting in plasmid p76405 To introduce a His6-tag 10 lg of the primers His1 5¢-GATCATCACCATCACCATCAC3¢ and His2 5¢-GATCGTGATGGTGATGGTGAT-3¢ were mixed and incubated for 30 at 83 °C The mixture was then allowed to cool down slowly to 25 °C during 30 followed by an incubation for at room temperature The annealed His1 and His2 primers were ligated into BglII-cleaved plasmid p76405 resulting in plasmid pHK763H Plasmid pHK763H was digested with SalI and EcoRI and ligated into SalI/EcoRI-cleaved plasmids pBK7, p7634, pDM234, p7637, and pDM237 resulting in plasmids pBK7H, p7634H, pDM234H, p7637H, and pDM237H, respectively Recombinant DNA techniques Isolation of plasmids, use of restriction enzymes, ligation, agarose gel electrophoresis, and transformation followed standard techniques [22] All genetic constructions were examined by DNA sequencing using the dideoxy chain-termination method with fluorescence- Ó FEBS 2002 4950 M Braun et al (Eur J Biochem 269) Table E coli strains and plasmids used in this study Strain or plasmid Strains AB2847 HK99 CH1857 HK97 BL21 (DE3) omp8 CH21 KB419 Plasmids pHK763 pHK763H pAB pBK7 pBK7H pHK234 p7634 p7634H pDM234 pDM234H pHK237 p7637 p7637H pDM237 pDM237H pSKF405-04 p76405 pTO4 pTUC203 pT7-6 Genotype or phenotype Reference or source aroB tsx malT thi AB2847 tonB fhuA AB2847 DfhuACDB tonB F– araD139 lacU169 rpsL150 relA1 flbB5301 deoC1 ptsF25 rbsR aroB thi fhuE::kplac Mu53fhuA F– hsdSB B(rB– mB–) gal ompT dcm (DE3) DlamB ompF::Tn5 DompA T7 polymerase under lacUV5 control BL21 (DE3) omp8 fhuA lamB [34] [17] [19] [23] [20] Krieger-Brauer pT7-6 fhuA wild-type pT7-6 fhuA (P405 PDH6DLA V406) pT7-6 fhuA with BamHI E159D pT7-6 fhuA D5–160 E3D pT7-6 fhuA D5–160 E3D (P405 PDH6DLA V406) pBluescript SK + fhuAD322–336 (P321 PDL K337) pT7-6 fhuA D322–336 (P321 PDL K337) pT7-6 fhuA D322–336 (P321 PDL K337) (P405 PDH6DLA V406) pT7-6 fhuA D5–160 D322–336 E3D (P321 PDL K337) pT7-6 fhuA D5–160 D322–336 E3D (P321 PDL K337) (P405 PDH6DLA V406) pBluescript SK + fhuAD335–355 (P334 DL S356) pT7-6 fhuA D335–355 (P334 DL S356) pT7-6 fhuA D335–355 (P334 DL S356) (P405 PDH6DLA V406) pT7-6 fhuA D5–160 D335–355 E3D (P334 DL S356) pT7-6 fhuA D5–160 D335–355 E3D (P334 DL S356) (P405 PDH6DLA V406) pBluescript SK + fhuA (P405 PDLA V406) pT7-6 fhuA (P405 PDLA V406) pBR322 cma cmi pACYC184 mcjABCD Ampr [17] This [36] [19] This [18] This This This This [18] This This This This [15] This [24] [25] [37] labelled or unlabelled nucleotides (Auto Read Sequencing Kit, Pharmacia Biotech) and the A.L.F sequencer (Pharmacia) Protein analytical methods To show the FhuA proteins in cells that were grown under the same conditions as the phenotype assays were performed, E coli HK97 fhuA was transformed with fhuA wild-type and fhuA mutant genes and grown overnight in NB medium The overnight cultures were used to inoculate fresh NB medium and the cultures were grown at 37 °C to a D578 of 1.0 before the outer membrane fractions were isolated by lysing cells with lysozyme–EDTA, followed by solubilization of the cytoplasmic membrane in 0.2% Triton X-100 and differential centrifugation The proteins of the undissolved outer membrane fraction were dissolved by heating in sample buffer, separated by SDS/PAGE and stained with Serva blue [23] Phenotype assays All phenotype assays were carried out with freshly transformed E coli K-12 strains HK97 aroB fhuA fhuE and HK99 aroB fhuA tonB These strains carry the same four amino acid replacements and an amino acid deletion in fhuA and contain the mutated FhuA protein in the outer [35] study study study study study study study study study study study membrane [23] The plasmid-encoded fhuA genes in the transformants were transcribed from the fhuA promoter Sensitivity of cells to the FhuA ligands was tested by spotting 10-fold (phages T1, T5, /80, and colicin M) or 3-fold (microcin J25, rifamycin CGP 4832, and albomycin) diluted solutions (3 lL) on TY agar plates overlaid with mL TY soft agar containing 108 cells of the strain to be tested The colicin M solution was a crude extract of a strain carrying plasmid pTO4 cma cmi [24] The microcin J25 solution was a supernatant of E coli MC4100 carrying the plasmid pTUC203 mcjABCD [25] after growth of the transformants in brain heart infusion medium (37 gỈL)1; Difco Laboratories) at 37 °C Growth inhibition by SDS and various antibiotics was determined by placing filter paper discs supplemented with 10 lL of the agents in concentrations as indicated on TY agar plates overlaid with mL TY soft agar containing 108 cells of the strain to be tested The tests were performed in parallel with TY agar plates and TY soft agar both containing ferrichrome at a final concentration of lM Growth promotion by siderophores was tested by placing filter paper discs containing 10 lL of a siderophore solution of different concentrations on NBD agar plates overlaid with mL NB soft agar containing 108 cells of the strain to be tested After overnight incubation, the diameter and the growth density around the filter paper discs were determined Ó FEBS 2002 Growth promotion by maltodextrins was tested with the lamB strain KB419 (as a control) and with strain KB419 transformed with plasmids encoding various FhuA derivatives Overnight cultures were washed twice with M9 medium and adjusted to a D578 of 1.0 in M9 minimal medium The test was performed by placing filter paper discs supplemented with 10 lL of a 40% solution of maltodextrins (maltose to maltohexaose) on M9 minimal agar plates that contained no other carbon source overlaid with M9 minimal top agar containing 100 lL of the strain to be tested After incubation overnight at 37 °C the diameter and the growth density around the filter discs were determined Ferrichrome uptake assays E coli K-12 strains HK97 aroB fhuA fhuE, and HK99 aroB fhuA tonB, freshly transformed with the plasmids to be tested were grown overnight on TY plates Cells were washed and suspended in transport medium (M9 salts [26], 0.4% glucose), and the cell density was adjusted to a D578 of 0.5 Free iron ions were removed by adding 25 lL 10 mM nitrilotriacetate, pH 7.0 to mL cells After incubation for at 37 °C, time-dependent ferrichrome uptake was started by adding [55Fe3+]ferrichrome to a final concentration of lM in the case of transport assays and 10 lM when diffusion of ferrichrome into cells was tested In the latter case, E coli HK99 aroB fhuA tonB was used as the test strain and a 150-fold surplus of nonradioactive ferrichrome was added as a chase after 17 to remove adsorbed ferrichrome molecules The concentration-dependent uptake assays were started by adding [55Fe3+]ferrichrome to final concentrations of 1, 3, 6, or 10 lM Samples of 50 lL or 100 lL were withdrawn and added to 10 mL 0.1 M LiCl Cells were harvested on cellulose nitrate filters (pore size 0.45 lm; Sartorius AG) and washed twice with mL 0.1 M LiCl The filters were dried, and the radioactivity was determined by liquid scintillation counting Ferrichrome binding assay E coli CH1857 aroB DfhuACDB tonB, freshly transformed with the plasmids to be tested were grown overnight on TY plates Thirty lL of a 50% NaI solution (density 1.5 gỈcm)3) was overlaid with 80 lL silicone oil PN200 (density 1.03 gỈcm)3 in a 400-lL microtest tube) The binding assay was started by adding [55Fe3+]ferrichrome to a final concentration of lM to cells prepared as described in ferrichrome uptake assays At the times indicated, samples of 50 or 100 lL were withdrawn and applied onto the silicone oil layer in the microtest tube The tubes were centrifuged immediately for 90 s in a Beckman Microfuge E The cells passed the silicone oil layer according to their density (1.2–1.3 gỈcm)3), and accumulated on top of the NaI layer The residual radiolabelled ferrichrome in the binding medium remained on top of the silicone layer After the centrifugation step, cells were stored in the test tube at room temperature until the uptake assay was completed (H Killmann and G Gestwa, unpublished data) At the end of the assay, the microtest tubes were cut with a scalpel in the middle of the silicone oil layer and the lower part of the test tube containing the cells was placed upside down in a fresh test tube and centrifuged for 10 at FhuA transport protein of E coli (Eur J Biochem 269) 4951 10 000 g The empty tube was removed, and the mixture of NaI, silicone oil and cells was suspended in 900 lL H2O and completely transferred to a 20-mL polyethylene vial After adding 10 mL liquid scintillation counting cocktail, the radioactivity was determined by liquid scintillation counting Purification of the FhuA proteins The E coli K-12 strain CH21 freshly transformed with the plasmids encoding the FhuA derivatives carrying a His6-tag at residue 405 were grown in 250 mL TY medium to a D578 of 0.5 before T7 RNA polymerase synthesis was induced by adding isopropyl-b-D-thiogalactopyranoside to a final concentration of mM After shaking the cultures for additional h at 37 °C the cells were harvested by centrifugation After suspending in 15 mL 0.2 M Tris/HCl (pH 8.0) that contained mg deoxyribonuclease and two tablets of COMPLETE (Roche), cells were disrupted with a French press The disrupted cells were mixed with 15 mL extraction buffer (50 mM Tris/HCl pH 8.0, 10 mM MgCl2, 2% Triton X-100) and incubated for 10 at room temperature The outer membrane fractions were isolated by centrifugation for h with 30 000 g To remove the added Triton X-100 the outer membranes were washed four times with 10 mL H2O The outer membranes were suspended in 12 mL solubilization buffer containing 50 mM Tris/HCl pH 8.0, mM EDTA, 1% N,N-dimethyldodecylamine-N-oxide (LDAO) To solubilize the FhuA proteins samples of mL were shaken overnight at 15 °C, then centrifuged and mL of the pooled supernatant fractions dialysed for h at room temperature in 500 vols binding buffer containing 50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, 0.1% LDAO (pH 8.0) The protein solutions were concentrated to mL by ultrafiltration (Centricon YM30, Millipore) before loading onto a Ni2+–NTA agarose column equilibrated with binding buffer Chromatography was performed as described by the manufacturer (Qiagen) with the exception that all buffers contained 0.1% LDAO The elution buffer containing the purified proteins was replaced by a buffer containing 0.2 M Tris/HCl pH 8.0, 0.2% LDAO using PD10 columns (Pharmacia) Black lipid bilayer membrane experiments Membranes were formed from a 1% (w/v) solution of diphytanoyl PtdCho (Avanti Polar Lipids) in n-decane in a Teflon cell consisting of two aqueous compartments connected by a circular hole with an area of approximately 0.4 mm2 [27,28] The aqueous salt solutions (analytical grade; Merck) were used unbuffered and had a pH of approximately The temperature was kept at 20 °C throughout the experiments The single-channel measurements were performed with a pair of Ag/AgCl electrodes (with salt bridges) switched in series with a voltage source and a current amplifier (Keithley 427) The amplified signal was monitored with a storage oscilloscope and recorded with a strip chart recorder Stock solutions containing the FhuA deletion derivatives were added after the lipid membrane turned optically black to reflected light For determination of zero-current membrane potentials the membranes were formed in a 50-mM KCl solution and 4952 M Braun et al (Eur J Biochem 269) Ó FEBS 2002 insertion of pores was observed until a conductance of at least 0.1 nS was reached corresponding to the formation of a sufficient number of channels Then the instrumentation was switched to the measurement of the zero-current potentials and a KCl gradient was established by adding M KCl solution to one side of the membrane while stirring The zero-current membrane voltage reached its stationary value about 2–5 after addition of the concentrated KCl-solution and was analysed using the Goldman–Hodgkin–Katz equation [29] RESULTS FhuAD5–160 does not form stable channels in lipid bilayers The FhuA b-barrel lacking the central N-terminal globular domain was incorporated into lipid bilayer membranes to determine the increase in conductance To purify the protein, the fhuAD5–160 gene was cloned into plasmid pBK7H (Table 1) downstream of the phage T7 gene 10 promoter and specifically transcribed by the T7 RNA polymerase FhuAD5–160 was labelled with a His6-tag at residue 405 [30] which is exposed at the cell surface [5,6] FhuAD5–160 was contained in the outer membrane fraction in lower amounts than wild-type FhuA (Fig 1A) It was purified by affinity chromatography using an Ni2+–NTA agarose column (Fig 1B) Control purifications were performed with strain CH21 transformed with the pT7-6 expression vector FhuAD5–160 was added to the aqueous phase on one or both sides bathing a black lipid bilayer membrane formed from diphytanoyl PtdCho/n-decane across a small circular hole FhuAD5–160 increased the membrane conductance as demonstrated by the current recording in M KCl (Fig 2) The conductance increase did not occur in a step-wise fashion as found with porins of Gram-negative bacteria [27,31] This means that FhuAD5–160 failed to form stable channels The current recordings revealed a high degree of current noise The most frequently observed conductance step was about 0.5 nS in M KCl No conductance increase was recorded when purified wild-type FhuA was added to the lipid bilayer membranes This was also the case in the Fig Single-channel recording of a diphytanoyl PtdCho/n-decane membrane in the presence of the FhuAD5–160 mutant The aqueous phase contained M KCl (pH 6) and  50 ng FhuAD5–160ỈmL)1 The applied membrane potential was 20 mV; T ¼ 20 °C Note that the current did not increase in a step-wise fashion but showed a high current noise indicating rapid fluctuations of the channel-forming unit control recordings with purified samples of the control strain CH21 transformed with pT7-6 Control measurements were carried out with a 100-fold concentrated protein solution compared to the measurements of the different FhuA deletion derivatives (data not shown) FhuAD5–160 D322–336 increased the permeability of lipid bilayer membranes The loop L4 reduces the entrance of the surface cavity of FhuA to about half its diameter [6] To see whether loop restricts the permeability of the open channel that was formed by removal of the globular domain, we constructed Fig (A) Stained proteins after SDS/PAGE of outer membrane fractions of E coli HK97 fhuA transformed with plasmids (listed in Table 1) encoding the FhuA proteins indicated in the figure Arrows denote wild-type FhuA and the various FhuA deletion derivatives The molecular masses of standard proteins in kDa are indicated (B) His-tagged proteins obtained by chromatography on Ni–agarose columns as they were used for the lipid bilayer experiments Ten-lL samples of a 0.5 mgỈmL)1 protein solution were applied per lane Ó FEBS 2002 FhuA transport protein of E coli (Eur J Biochem 269) 4953 much higher amplitude The conductance of FhuAD5–160 D322–336 was higher than that of FhuAD5–160 (Table 2) which indicated that removal of half of the L4 loop increased the conductance of the b-barrel FhuAD5–160 D335–355 forms stable channels Fig Single-channel recording of a diphytanoyl PtdCho/n-decane membrane in the presence of FhuAD5–160 D322–336 The aqueous phase contained M KCl (pH 6) and  50 ng FhuAD5–160 D322– 336ỈmL)1 The applied membrane potential was 20 mV; T ¼ 20 °C Note that the time resolution of the current recording is higher than that of Figs and His6-tagged FhuAD5–160 D322–336 The purified deletion derivative (Fig 1) increased the conductance of lipid bilayer membranes but did not show uniform single-channel conductance or a step-wise increase (Fig 3) Higher time resolution of the current than shown in Fig revealed frequent and rapid opening and closing of channels which was also observed with FhuAD5–160 (Fig 2) but had a Because FhuAD5–160 did not form stable channels in the reconstitution experiments we attempted to combine the D5–160 excision with the 322–355 deletion which we have previously shown converts FhuA into a stable channel [17] We failed to observe transformants which expressed FhuAD5–160 D322–355, presumably because the protein was toxic to cells We then constructed FhuAD5–160 D335– 355 As reported previously [18] FhuAD335–355 increased the membrane conductance only slightly and did not form stable channels in lipid bilayer membranes SDS/PAGE of isolated outer membrane fractions identified the FhuAD5– 160 D335–355 protein The amounts were lower than those of wild-type FhuA or FhuAD5–160 (Fig 1A) FhuAD5–160 D335–355 purified as His6-tagged derivative on nickel agarose (Fig 1B) inserted readily into planar lipid bilayers and produced discrete step-wise current increase at a transmembrane potential of 20 mV (Fig 4) If each step corresponded to a single channel, the unitary conductance was  2.5 nS in M KCl The conductance steps were fairly homogeneous as shown by the histogram (Fig 5) Only a small number of small steps were observed, which probably represent smaller substates of the open channel (see Table for a summary of the results of the lipid bilayer experiments) The conductance of the FhuAD5–160 D335–355 mutant was smaller than that of FhuAD322–355 (3 nS) determined previously under otherwise identical conditions [17] Single-channel analysis of the FhuA deletion mutants Table shows the average single-channel conductance G of the FhuA mutant proteins as a function of the KCl concentration in the aqueous phase Measurements were performed from 0.1 to M KCl and with M LiCl and M KAc Only FhuAD5–160 D335–355 displayed a linear relationship between single-channel conductance and KCl Table Single-channel properties of the various FhuA deletion mutants in different salt solutions The membranes were formed of diphytanoyl PtdCho dissolved in n-decane The aqueous solutions were used unbuffered and had a pH of  unless otherwise indicated The applied voltage was 20 mV, and the temperature was 20 °C The average single-channel conductance, G, was calculated from at least 80 single events The selectivity of the different mutants in KCl was derived from zero-current membrane potential measurements ND, not determined FhuA mutants FhuAD5–160 FhuAD5–160 D322–336 Salt Concentration c [M] Single-channel conductance G [nS] KCl 0.1 0.3 1.0 3.0 1.0 1.0 1.0 0.2 0.3 0.5 0.5 0.5 0.8 0.75 11 FhuAD5–160 D335–355 LiCl KAc (pH 7) KCl + 0.1% SDS Selectivity PK/PCl 0.4 0.9 1.3 1.5 0.5 ND 2.5 0.3 0.7 2.5 6.5 1.3 1.5 ND 3.3 Ó FEBS 2002 4954 M Braun et al (Eur J Biochem 269) concentration, which is expected for wide water-filled channels similar to those formed by Gram-negative bacterial porins [32,33] The single-channel conductance showed a minor restriction as it increased 21-fold while the KCl concentration was increased 30-fold FhuAD5–160 D335– 355 showed a higher conductance in KAc as compared with LiCl which suggested some preference for cations over anions For the other mutant proteins no clear dependence on the aqueous salt concentrations was recorded which may be caused by the rapid transition of the FhuA deletion channels between different conductance states as Figs and indicate Furthermore, with the latter mutant proteins no clear selectivity for ions was observed Selectivity of the FhuA mutant proteins Fig Single-channel recording of a diphytanoyl PtdCho/n-decane membrane in the presence of FhuAD5–160 D335–355 The aqueous phase contained M KCl (pH 6) and  50 ng FhuAD5–160 D335– 355ỈmL)1 The applied membrane potential was 20 mV, T ¼ 20 °C Zero-current membrane potential measurements were performed to further determine the selectivity of the FhuA mutant proteins After the incorporation of 100–1000 channels into the membranes, the KCl concentration on one side of the membranes was raised from 0.1 to 0.5 M by the addition of concentrated KCl The more dilute side of the membrane (0.1 M) became positive which indicated preferential movement of potassium ions through the mutant channels These data demonstrate selectivity for cations and support the data obtained from the singlechannel experiments (Table 2) The zero-current membrane potentials for KCl were on average between 14 and 32 mV at a fivefold KCl gradient across the membranes Analysis of the potential using the Goldman–Hodgkin–Katz equation [29] suggested that anions also move through the channel because the ratio of the permeabilities PK+ divided by PCl- was between 2.5 and 11 (Table 2) The ion selectivity of the mutant proteins with higher single-channel conductance was lower than that of FhuAD5–160 with a smaller conductance Active transport of ferrichrome by FhuA deletion derivatives Fig Histogram of the probability P(G) of the occurrence of a given conductivity unit observed with membranes formed of diphytanoyl PtdCho/n-decane in the presence of FhuAD5–160 D335–355 mutant P(G) is the probability that a given conductance increment G is observed in the single-channel experiments It was calculated by dividing the number of fluctuations similar to those of Fig with a given conductance increment by the total number of conductance fluctuations The aqueous phase contained M KCl (pH 6) and  50 ng FhuAD5–160 D335–355ỈmL)1 The applied membrane potential was 20 mV; T ¼ 20 °C The average single-channel conductance was 2.5 nS for 94 single-channel events (right-hand maximum) Previously, we have shown that FhuAD5–160 exhibits all TonB-dependent FhuA activities [19] except uptake of microcin J25 [20] Therefore, we tested whether FhuAD5– 160 retained ferrichrome transport activities when additional deletions were introduced E coli HK97 with a chromosomal fhuA mutation was transformed with the plasmids carrying fhuA deletion genes The amounts of the FhuA mutant proteins present in cells used to determine the properties of the mutants are shown in Fig 1A Cells containing FhuAD5–160 transported [55Fe3+]ferrichrome with a rate that amounted to 59% of wild-type FhuA This comparison has to take into account that the cells contained less FhuAD5–160 than wild-type FhuA There is no linear increase of the ferrichrome transport rates with increasing concentrations of FhuA above a certain FhuA concentration The ferrichrome transport rate of FhuAD5– 160 D322–336 amounted to 45% and that of FhuAD322– 336 to 75% of the rate of wild-type FhuA (Fig 6A and Table 3) In contrast, FhuAD5–160 D335–355 did not transport ferrichrome (Fig 6A and Table 3) Since FhuAD335–355 was also transport inactive, removal of the globular domain did not convert FhuAD335–355 into an active transporter Ó FEBS 2002 FhuA transport protein of E coli (Eur J Biochem 269) 4955 Increase of outer membrane permeability by the FhuA deletion derivatives Fig (A) Time-dependent transport of [55Fe3+]ferrichrome (1 lM) into E coli HK97, (B) Time-dependent uptake of [55Fe3+]ferrichrome (10 lM) into E coli HK99 After 21 a 150-fold surplus of nonradioactive ferrichrome was added as a chase The E coli strains HK97 fhuA aroB and HK99 fhuA tonB aroB were transformed with plasmids (listed in Table 1) encoding the FhuA deletion derivatives indicated in the figure As FhuAD5–160 D335–355 formed , in lipid bilayer membranes, stable open diffusion channels for ions such as K+ and Cl–, we examined whether FhuAD5–160 D335–355 also formed open channels in the outer membrane of E coli cells To study diffusion of ferrichrome into the periplasm, we used the fhuA tonB double mutant HK99 that was devoid of active transport across the outer membrane but actively transported ferrichrome via the FhuBCD proteins from the periplasm across the cytoplasmic membrane into the cytoplasm [55Fe3+]Ferrichrome remains in the cytoplasm when cells are washed on filters to remove excess radioactivity, whereas [55Fe3+]ferrichrome that has entered only the periplasm is washed away As HK99 is devoid of FhuA activity, [55Fe3+]ferrichrome can only pass the outer membrane via the FhuA deletion derivatives synthesized after transformation of HK99 with plasmids carrying the fhuA deletion genes To measure diffusion we increased the concentration of [55Fe3+]ferrichrome from lM used in the transport assay to 10 lM Of the FhuA deletion derivatives tested, only FhuAD5–160 D335–355 supported uptake of [55Fe3+]ferrichrome via diffusion across the outer membrane and subsequent active transport across the cytoplasmic membrane (Fig 6B) Addition of excess nonradioactive ferrichrome (1.5 mM) after 21 of transport released a small portion (10%) of the [55Fe3+]ferrichrome which was probably bound to periplasmic FhuD and some may have been unspecifically bound to cells and the filters Ninety per cent of [55Fe3+]ferrichrome has been taken up from the periplasm into the cytoplasm from where it was no longer released during the chase Wild-type FhuA and the FhuA deletion mutants other than FhuAD5–160 D335–355 did not support diffusion of [55Fe3+]ferrichrome into the periplasm of the HK99 transformants (Fig 6B) [55Fe3+]Ferrichrome bound to HK99 synthesizing plasmid-encoded wild-type FhuA remained constant during the 21 incubation period [55Fe3+] Ferrichrome was released by the chase with nonradioactive ferrichrome which is considered to be the amount of [55Fe3+]ferrichrome that is bound to FhuA HK99 Table Ferrichrome binding and transport rates of wild-type FhuA and various FhuA deletion derivatives The E coli strains CH1857 fhuACDB tonB and HK97 fhuA were transformed with the plasmids listed in Table that encoded the FhuA proteins listed in the left panel FhuA proteins Fc transport rates per into HK97a (% wild-type) Fc binding to CH1857 Iron ions/cellb (% wild-type) FhuA wild-type FhuAD5–160 FhuAD322–336 FhuAD5–160 D322–336 FhuAD335–355 FhuAD5–160 D335–355c 9960 5905 7456 4492 – 10198 (100%) 3108 (30%) 8972 (88%) 1171 (11%) 2146 (21%) – a 55 (100%) (59%) (75%) (45%) [ Fe3+]Ferrichrome transport rates per minute were calculated from the linear region between and 13 of Fig 6A The rate was related to the transport rate of wild-type FhuA (100%) b The mean values of [55Fe3+]ferrichrome (Fc) bound to the FhuA derivatives minus the mean values after addition of 150 lM nonradioactive ferrichrome (chase) was taken as the fraction that is bound to FhuA The percentage is related to ferrichrome bound to wild-type FhuA (100%) c FhuAD5–160 D335–355 did not take up ferrichrome by active transport but by TonB independent diffusion The high concentration of iron ions measured during the binding assay (17597 iron ions per cell) was due to the diffusion of ferrichrome into the periplasm and does not reflect the binding capacitiy of FhuAD5–160 D335–355 for ferrichrome 4956 M Braun et al (Eur J Biochem 269) FhuAD322–336 bound similar amounts of [55Fe3+]ferrichrome as HK99 wild-type FhuA Cells of the other FhuA deletion derivatives contained only very small amounts of bound [55Fe3+]ferrichrome (Fig 6B) To support the conclusion of a diffusive entry of [55Fe3+]ferrichrome across the outer membrane of HK99 FhuAD5–160 D335–355, we determined the concentrationdependent uptake rate of [55Fe3+]ferrichrome and compared it with the uptake into HK99 transformed with plasmids that encoded wild-type FhuA and the other FhuA deletion derivatives Only HK99 FhuAD5–160 D335–355 showed a linear increase of uptake with increasing concentrations of [55Fe3+]ferrichrome from to lM (Fig 7) From to 10 lM [55Fe3+]ferrichrome the uptake into HK99 FhuAD5–160 D335–355 was no longer linear, presumably because transport across the cytoplasmic membrane became rate limiting The FhuA deletion mutants showed only a comparatively small increase of [55Fe3+]ferrichrome associated with the cells which in the case of HK99 FhuAD322– 336 and FhuA wild-type reflected mostly binding to FhuA At higher concentrations [55Fe3+]ferrichrome diffused somewhat through the other FhuA deletion derivatives into the periplasm (Fig 7) Fig not only reveals transport of [55Fe3+]ferrichrome but also binding of [55Fe3+]ferrichrome For cells that synthesized wild-type FhuA and FhuAD322–336 transport into HK97 (Fig 6A) correlated qualitatively with binding to HK99 (Fig 6B) However, although HK97 FhuAD5– 160 and HK97 FhuAD5–160 D322–336 transported [55Fe3+]ferrichrome, binding of [55Fe3+]ferrichrome was Ó FEBS 2002 very low Because during the transport assay cells on filters were washed twice with mL 0.1 M LiCl, weakly bound [55Fe3+]ferrichrome could have been released from cells and washed through the filter Therefore, binding was determined by a recently devised method in which cells are centrifuged through silicone oil and collected above a layer of a NaI solution During this procedure cells remain viable (H Killmann and G Gestwa, unpublished data) This method was used for the determination of [55Fe3+]ferrichrome binding to FhuA and the FhuA deletion derivatives The used E coli CH1857 is a tonB mutant deficient in ferrichrome transport across the outer membrane and it lacks the fhuABCD genes for transport across the cytoplasmic membrane The only [55Fe3+]ferrichrome binding site left is that of plasmid encoded FhuA and its derivatives In fact, cells that synthesized FhuAD5–160 bound [55Fe3+] ferrichrome to 30% of the level of the wild-type FhuA Reduction of [55Fe3+]ferrichrome binding probably resulted from the loss of four binding sites delivered by the cork domain out of a total of 10 ferrichrome binding sites in FhuA This result demonstrated that centrifugation through silicone oil was a much milder procedure than washing of cells on filters, and indicates weak binding of [55Fe3+]ferrichrome to FhuAD5–160 Cells synthesizing FhuAD5–160 D322–336 bound 11% [55Fe3+]ferrichrome, FhuAD322–336 88% and FhuAD335-335 showed 21% binding compared to cells with wild-type FhuA (Table 3) Binding to FhuAD5–160 D335–355 could not be determined as ferrichrome diffused into the periplasm and remained there during centrifugation through silicone oil (determined value was 17 597 iron ions per cell) Sensitivity of cells synthesizing FhuA deletion derivatives to antibiotics Fig Concentration-dependent uptake of [55Fe3+]ferrichrome into E coli HK99 fhuA tonB aroB expressing the plasmid-encoded FhuA proteins indicated in the figure Another approach to identify water-filled protein channels in the outer membrane is the determination of the sensitivity of cells to antibiotics which are prevented from entering cells by the permeability barrier of the outer membrane Novobiocin, erythromycin, rifamycin and vancomycin are antibiotics which are too large to diffuse rapidly through the pores formed by the porins (Table 4) Cells of HK97 wildtype FhuA were resistant to the indicated antibiotics except rifamycin (Table 4) All FhuA deletion derivatives increased sensitivity to the antibiotics The highest sensitivity to the antibiotics was conferred by FhuAD335–355 and FhuAD5– 160 D335–355 Sensitivity mediated by FhuAD5–160 was increased by the additional deletion 335–355, whereas the sensitivity of FhuAD335–355 against SDS and Novobiocin was increased only slightly when the cork domain had been removed Another indicator for outer membrane permeability is sensitivity to SDS to which E coli K-12 is resistant as long as the outer membrane barrier is intact Only FhuAD335–355 and FhuAD5–160 D335–355 rendered cells sensitive to SDS Sensitivity to the antibiotics was also determined in the presence of lM ferrichrome to test whether binding of ferrichrome and the concomitant structural changes in FhuA affected the permeability of the FhuA deletion derivatives We observed only slight effects if any (data not shown) which might be caused by the low binding of ferrichrome to the FhuA deletion derivatives This conclusion was supported by the decrease of the sensitivity of HK97 FhuAD322–336 to rifamycin by ferrichrome which binds to FhuAD322–336 (Table 3) Maltotetraose 667 Da mg – – – 14 Maltotriose 504 Da mg 14 10 16 – – – – Growth of FhuA deletion mutants on maltodextrins LamB is the maltoporin through which maltodextrins diffuse across the outer membrane into the periplasm of E coli [31] If lamB is deleted maltodextrins larger than maltotriose diffuse too slowly into the periplasm to support growth on maltodextrins as the sole carbon source We used E coli KB419 which is a lamB mutant with no polar effect on transport genes required for maltose transport across the cytoplasmic membrane Among the FhuA mutants tested E coli KB419 FhuAD5–160 D335–355 formed the largest growth zone on maltotetraose and could grow on maltopentaose (Table 4) In contrast, the growth zone of E coli KB419 FhuAD335–355 on maltotetraose was smaller and no growth occurred on maltopentaose The other FhuA deletion derivatives did not support growth on maltotetraose and maltopentaose except FhuAD5–160 (Table 4) Deletion 322–336 in FhuAD5–160 D322–336 did not increase but somewhat reduced growth of KB419 on the maltodextrins, as compared with strains expressing only FhuAD5–160 Rifamycin 823 Da lg 9 – – 10 10 – – FhuA activities of the FhuA deletion mutants Erythromycin 734 Da 15 lg Vancomycin 1485 Da 20 lg Growth of E coli KB419 lamB on Maltopentaose 829 Da mg FhuA transport protein of E coli (Eur J Biochem 269) 4957 FhuAD5–160 confers to E coli HK97 devoid of wild-type FhuA sensitivity to the FhuA-specific phages T1, T5, /80, to colicin M and albomycin to the same or similar degree as wild-type FhuA We examined sensitivities of HK97 that synthesized the various FhuA deletion derivatives HK97 FhuAD5–160 D335–355 was resistant to all FhuA ligands as was HK97 FhuAD335–355 In contrast, HK97 FhuAD5– 160 D322–335 displayed sensitivity to T5, colicin M and albomycin which, however, was lower than the sensitivity of HK97 FhuAD322–335 The sensitivity to T5 was reduced 100-fold and the growth inhibition zones caused by colicin M and albomycin were turbid indicating partial inhibition, whereas those of HK97 FhuAD322–335 were clear (data not shown) – – – – – – FhuA wild-type FhuAD5–160 FhuAD322–336 FhuAD5–160 D322–336 FhuAD335–355 FhuAD5–160 D335–355 Novobiocin 634 Da 30 lg DISCUSSION SDS 288 Da 750 lg Growth inhibition of E coli HK97 fhuA by Table Growth inhibition of E coli HK97 fhuA by SDS and antibtiotics and growth promotion of E coli KB419 lamB by maltodextrins Sensitivity of E coli HK97 fhuA transformants and growth promotion of E coli KB419 lamB transformants expressing various plasmid-encoded FhuA deletion derivatives as indicated in the table The molar masses and the absolute amounts of the inhibitors and the maltodextrins added to the filter paper discs are indicated The size of the zones of growth inhibition (in mm) of E coli HK97 with SDS and the antibiotics is given with subtraction of the sensitivity of the control strain HK97 pT7-6 including the diameter of the filter paper disc (6 mm) The growth zones of E coli KB419 with the maltodextrins is given in mm with subtraction of the filter paper disc (6 mm) No sensitivity or no growth is indicated by a single line (–) Ó FEBS 2002 The globular domain of FhuA tightly closes the channel formed by the b-barrel and for this reason was designated cork [5] or plug [6] Although binding of ferrichrome causes a large structural change in the crystal structure of FhuA it does not open the channel Excision of the entire globular domain resulted in FhuAD5–160 that showed TonBdependent active ferrichrome transport The b-barrel domain alone functioned as an active transporter With higher concentrations of ferrichrome than are required for transport, tonB mutant cells grew on ferrichrome as sole iron source, indicating an open channel [19] Apparently, ferrichrome diffused through FhuAD5–160 in contrast to wildtype FhuA that failed to support growth of a tonB mutant FhuAD5–160 also somewhat increased sensitivity of cells to antibiotics that are prevented from access to their target site by the permeability barrier of the outer membrane [19] Maltodextrins that pass through the outer membrane via the LamB maltoporin entered the periplasm via FhuAD5–160 in a lamB mutant These results indicated that FhuAD5–160 increased unspecifically the permeability of the outer Ó FEBS 2002 4958 M Braun et al (Eur J Biochem 269) membrane However, cells synthesizing FhuAD5–160 remained resistant to SDS which suggested that the FhuAD5–160 channel still restricted entry of molecules In this paper, a correlation between data obtained in vivo with those obtained in vitro was attempted Determination of the single-channel conductance of isolated FhuAD5–160 in artificial lipid bilayer membranes revealed an increase in conductance for KCl and other ions with rapidly changing amplitudes and no permanently open stable channels This finding was unexpected as the crystal structure of FhuA suggests that removal of the cork domain should result in an open channel The conductance showed rapid transitions between different states probably through rapid opening and closing of the FhuAD5–160 channel or, less likely, rapid membrane entry and exit of the protein The instability of the channels formed by FhuAD5–160 may have several causes It contains surface loop above the outer cavity of FhuA through which ferrichrome gains access to the ferrichrome binding site Loop constricts the channel entrance to about half the area of the total cross-section [6] Removal of the globular domain may increase the flexibility of loop and other surface exposed loops so that rapidly moving loops may cause the frequent transition of open and closed states of FhuAD5–160 In addition, among the more than 60 residues that are exposed to the interior of the b-barrel channel and fix the globular domain, some amino acid side chains may become flexible when the globular domain is removed, and depending on their orientation may modulate the movement of ions through the channel Furthermore, the b-barrel of FhuAD5–160 may be less rigid ˚ than is suggested by the crystallographic B-factor (52 A2) of complete FhuA [6] Removal of the globular domain may increase the flexibility of the b-strands resulting in b-barrels with changing diameters of the elliptical cross shape Although FhuAD5–160 D322–336 lacked the cork domain and most of the L4 loop, it formed no stable channels in lipid bilayer membranes, and showed no or only a marginal concentration-dependent increase in ferrichrome diffusion across the outer membrane, but increased somewhat the outer membrane permeability for antibiotics and maltodextrins Obviously, removal of the L4 loop did not subtantially increase diffusion through the b-barrel FhuAD322–336 exhibited 75% of the [55Fe3+]ferrichrome transport activity of wild-type FhuA The transport activity was reduced further when the cork domain was deleted also, probably because four ferrichrome binding sites were removed with the cork domain which resulted in only 11% binding related to wild-type FhuA FhuAD5–160 D322–336 still functioned as an active transporter and displayed all the other FhuA related functions Deletion 335–355 was within the b-barrel and caused inactivation of FhuAD335–355 and probably also of FhuAD5–160 D335–355 as transporters and as receptors for the phages and colicin M Deletion 335–355 distorted the b-barrel such that the FhuA deletion derivative was not only unable to display TonB-dependent activities but also the TonB-independent infection by phage T5 However, it formed channels through which ferrichrome diffused into the periplasm with a rate that increased linearly with the ferrichrome concentration Maltodextrins served as carbon sources for cells synthesizing FhuAD5–160 D335–355 and the deletion derivative was sensitive to SDS It is likely that these hydrophilic/amphipatic compounds diffused through FhuAD5–160 D335–355 into the periplasm We favour the same interpretation for the increase in sensitivity of the antibiotics although they are more hydrophobic and may enter cells through diffusion through the outer membrane if incorporation of FhuAD5–160 D335–355 disturbed outer membrane integrity The results obtained in vivo correlated with the results obtained in vitro The recordings of the single-channel conductance of FhuAD5–160 D335–355 revealed stable single channels of rather uniform size with high single-channel conductance Each step probably corresponded to the incorporation of one channel-forming unit into the lipid bilayer Our conclusion that the b-barrel of FhuA largely determines the transport properties including the response to TonB was confirmed by a similar study on FepA and its comparison with FhuAD5–160 [21] FepA is the outer membrane transport protein for Fe3+ enterobactin The crystal structure of FepA is similar to that of FhuA [34] Excision of the globular domain (residues 17–150) resulted in a protein that exhibited Fe3+ enterobactin transport that depended on TonB However, the Vmax of the transport rate (0.3% of wild-type FepA) was much lower than the Vmax of FhuAD5–160 (32% of wild-type FhuA) [21] Despite the low transport rate of FepAD17–150 it supports the conclusion that the b-barrel alone functions as an active transporter In wild-type FhuA the cork must be dislocated so that the channel of the b-barrel opens which may require the concerted action of TonB on the b-barrel and the cork ACKNOWLEDGEMENTS We thank K.A Brune for critical reading of the manuscript This work was supported by the Deutsche Forschungsgemeinschaft (BR330/20–1, Forschergruppe Bakterielle Zellhulle: Synthese, Funktion und Wirkort) ă and the Fonds der Chemischen Industrie REFERENCES Nikaido, H (1992) Porins and specific channels of bacterial outer membranes Mol Microbiol 6, 435–442 Drechsel, H & Winkelmann, G (1997) Iron chelation and siderophores in transition metals in microbial metabolism In Transition Metals in Microbial Metabolism (Winkelmann, G & Carrano, C.J., eds), pp 1–49 Harwood Academic Publishers, Amsterdam Bradbeer, C (1993) The proton motive force drives the outer membrane transport of cobalamin in 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Wolff, H & Braun, V (1998) Identification of a new site for ferrichrome transport by comparison of the FhuA proteins of Escherichia coli, Salmonella paratyphi, Salmonella typhimurium, and Pantoea agglomerans J Bacteriol 180, 3845–3852 37 Tabor, S & Richardson, C.C (1985) A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes Proc Natl Acad Sci USA 82, 1074–1078  ... mutated FhuA protein in the outer [35] study study study study study study study study study study study membrane [23] The plasmid-encoded fhuA genes in the transformants were transcribed from the fhuA. .. rapid opening and closing of the FhuAD5–160 channel or, less likely, rapid membrane entry and exit of the protein The instability of the channels formed by FhuAD5–160 may have several causes It... and depending on their orientation may modulate the movement of ions through the channel Furthermore, the b-barrel of FhuAD5–160 may be less rigid ˚ than is suggested by the crystallographic B-factor