Mikrobiologie/Membranphysiologie1 and Organische Chemie2, Universität Tübingen,D-72076 Tübingen, Germany
Author for correspondence: Helmut Killmann. Tel: +49 7071 78849. Fax: +49 7071 295843. e-mail: hki{at}mikrobio.uni-tuebingen.de
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ABSTRACT |
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Keywords: ferrichrome transport, TonB peptides, Pantoea agglomerans
a The GenBank accession number for the sequence reported in this paper is Y15319.
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INTRODUCTION |
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Ferrichrome binding does not open the ß-barrel channel. It is thought that ß-barrel opening requires energy, which is provided by the electrochemical potential of the cytoplasmic membrane (Hancock & Braun, 1976 ; Bradbeer, 1993
; Postle, 1993
). Energy is transmitted to the outer membrane by the Ton system, which consists of the proteins TonB, ExbB and ExbD, inserted in the cytoplasmic membrane (Braun, 1995
). The interaction site of FhuA with TonB is thought to be the FhuA TonB box (residues 711; DTITV). The mutations I9P (isoleucine replaced by proline) and V11D abolish all FhuA activities except infection by phage T5, which does not require TonB. The TonB mutations Q160L, Q160K and R158L each partially restore the activity of the FhuA(I9P) mutant (Schöffler & Braun, 1989
; Günter & Braun, 1990
). Overexpressed TonB, which is degraded, is stabilized by overexpressed wild-type FhuA. The extent of stabilization of FhuA TonB box mutants depends on the degree of suppression by TonB mutants (Günter & Braun, 1990
). Analogous TonB box mutants inactivate the BtuB transport protein for vitamin B12 (Heller et al., 1988
) and the Cir transport protein for linear ferric catecholates (Bell et al., 1990
), and are suppressed by TonB Q160L and Q160K mutants. Recently, it has been shown that a random mutagenesis of the ferric enterobactin transport protein FepA also results in inactive TonB box mutants (Barnard et al., 2001
). The conclusion of an interaction of the TonB box of outer-membrane transport proteins with the region around residue 160 of TonB (in the following designated region 160) is supported by in vivo disulfide formation between cysteine residues introduced into the TonB box of BtuB and into TonB (Cadieux & Kadner, 1999
; Cadieux et al., 2000
). Addition of vitamin B12 changes the cross-linking pattern and strongly increases the flexibility of the TonB box (Merianos et al., 2000
). The cross-linking pattern is altered by amino acid substitutions in the TonB box that cause a TonB-uncoupled phenotype. Taken together, these data strongly support the concept that TonB interacts with the TonB box and that the interaction is enhanced when the substrate is bound to the transport protein.
In the light of the strong evidence in favour of a functionally important interaction between the TonB box of outer-membrane transport proteins and TonB, it was unexpected to find that the FhuA deletion mutant FhuA5160, which lacks the globular domain including the TonB box, actively transports ferrichrome at approximately 40% of the rate of wild-type FhuA and shows a TonB-dependent sensitivity to albomycin, colicin M, and the phages T1 and
80 (Braun et al., 1999
). These results have been corroborated by similar properties of FhuA
5160 mutants of Salmonella paratyphi and Salmonella enterica serovar Typhimurium, which display TonB-dependent activity for their ligands (Killmann et al., 2001
). In addition, FepA mutants with deletions analogous to the deletion in FhuA
5160 are less active than FhuA
5160, but clearly display TonB-dependent activities (Scott et al., 2001). These data point to sites of interaction between FhuA and TonB located in the FhuA ß-barrel and presumably outside region 160 of TonB (Braun et al., 1999
).
To determine whether region 160 of TonB is confined to interactions with the FhuA TonB box or interacts also with the ß-barrel, we tested whether TonB-dependent FhuA5160 activities can be inhibited by synthetic peptides identical in sequence to region 160 of TonB. In addition, we checked whether TonB mutant proteins and TonB proteins of different Enterobacteriaceae show identical or variant activities with FhuA and FhuA
5160, respectively. Furthermore, the tonB gene of Pantoea agglomerans was sequenced to obtain structural data that might explain the low activity of this encoded TonB protein in an E. coli tonB mutant.
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METHODS |
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Plasmid pIM91 (wild-type E. coli tonB gene) (Killmann & Braun, 1994 ) was digested with EcoRI and HindIII and ligated to EcoRI/HindIII-digested vector pBCKS, resulting in plasmid pHK703. Plasmid pHK703 (wild-type E. coli tonB gene) was digested with EcoRI and ClaI and ligated to EcoRI/ClaI-digested pWSK29, resulting in plasmid pCH321. Plasmids pIM6, pIM53, pIM82, pIM86, pIM182 and pIM620 (Traub et al., 1993
), carrying the cloned mutated E. coli tonB genes, were digested with EcoRI and HindIII and ligated to EcoRI/HindIII-digested low-copy-number vector pWSK29, resulting in plasmids pCHK6, pCHK53, pCHK82, pCHK86, pCHK182 and pCHK620, respectively.
Plasmids pHK763, pBK7 and p76Pa were digested with HindIII/EcoRI and ligated to HindIII/EcoRI-digested pHSG576, resulting in plasmids pHK570, pBK570 and pHSGPa, respectively. Plasmids p76Sp and p76St were digested with HindIII/BamHI and ligated to HindIII/BamHI-digested pHSG576, resulting in plasmids pHSGSp and pHSGSt, respectively.
Plasmid pDM237, encoding FhuA5160
335355, was generously provided by M. Braun (University of Tübingen, Germany).
Recombinant DNA techniques.
Isolation of plasmids, use of restriction enzymes, ligation, agarose gel electrophoresis and transformation followed standard techniques (Sambrook et al., 1989 ). All genetic constructions were examined by DNA sequencing using the dideoxy chain-termination method with fluorescence-labelled or unlabelled nucleotides (Auto Read Sequencing Kit, Pharmacia) and the ALF sequencer (Pharmacia).
Protein analytical methods.
Cultures of E. coli BL21 cells freshly transformed with various plasmids were harvested at an OD578 of 0·5 by centrifugation and resuspended in 1 ml M9 salts (Miller, 1972 ) supplemented with 0·4% glucose, 0·01% methionine assay medium, 0·01% thiamin and 1 mM IPTG to induce T7 RNA polymerase synthesis. After shaking the cells for 1 h at 37 °C, rifampicin (10 µl of 5 mg ml-1 in methanol) was added and incubation continued at 37 °C for 30 min. [35S]Methionine was added, and the suspension was incubated for an additional 10 min. At suitable times a surplus of non-radiolabelled methionine was added as a chase. The cells were collected by centrifugation and suspended in sample buffer. The radioactively labelled proteins were separated by SDS-PAGE as described previously (Killmann & Braun, 1992
).
To determine the protein amounts in cells during the growth-stimulation and transport assays the strains to be tested were grown in NB medium at 37 °C to an OD578 of 1·0. The cells were harvested by centrifugation and the outer membranes were prepared by lysing the cells with lysozyme/EDTA, followed by solubilization of the cytoplasmic membrane with 0·2% Triton X-100 and differential centrifugation (Killmann & Braun, 1992 ). The proteins were separated by SDS-PAGE and stained with Serva blue.
Phenotype assays.
All phenotype assays were carried out with freshly transformed E. coli K-12 strains 41/2 aroB fhuA and HK99 aroB fhuA tonB. Sensitivity of cells to the FhuA ligands (phages T1, T5 and 80, colicin M, microcin J25, rifamycin CGP4832 and albomycin) was tested by spotting threefold- or tenfold-diluted solutions (4 µl) on TY agar plates overlaid with 3 ml TY soft agar containing 108 cells of the strain to be tested. The colicin M solution was a crude extract of a strain that carried the plasmid pTO4 cma cmi (Ölschläger et al., 1984
). The microcin J25 solution was the culture supernatant after centrifugation of a strain carrying the plasmid pTUC203 mcjABCD (Solbiati et al., 1996
) after incubation in brain heart infusion medium (37 g l-1; Difco) at 37 °C.
Growth promotion by siderophores was tested by placing filter-paper discs containing 10 µl of a siderophore solution concentrated as indicated on NBD agar plates overlaid with 3 ml NB soft agar containing 108 cells of the strain to be tested. The diameter and the growth density around the filter-paper disc were determined after incubation overnight.
Transport 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 collected, washed, and suspended in transport medium [M9 salts (Miller, 1972 ), 0·4% glucose] before cell density was adjusted to an OD578 of 0·5.
To test the inhibitory activity of synthetic TonB peptides, E. coli AB2847 aroB and HK97 aroB fhuA, freshly transformed with p7626, which encodes FhuA322355 (Killmann et al., 1993
), and, in the case of strain HK97, with the fhuA plasmid to be tested, were grown overnight on TY agar plates. Cells were collected, washed, and suspended in transport medium to an OD578 of 5. Peptides were transferred into cells by incubating 200 µl TonB nonapeptides (1 mg ml-1), 40 µl 10x M9 salts, 4 µl 40% glucose, 116 µl water, and 40 µl cells to adjust the OD578 to 0·5. The culture was shaken gently for 15 min at 37 °C. Then the free iron ions were removed by adding 10 µl 10 mM nitrilotriacetate, pH 7·0. After incubation for 5 min at 37 °C, transport was started by adding 4 µl 100 µM [55Fe3+]ferrichrome. Samples of 50 µl were withdrawn and added to 10 ml 0·1 M LiCl. Cells were harvested on cellulose nitrate filters (pore size 0·45 µm; Sartorius) and washed twice with 5 ml 0·1 M LiCl. The filters were dried, and the radioactivity was determined by liquid scintillation counting.
Peptide synthesis.
Peptide amides were synthesized on a simultaneous multiple peptide synthesizer (Beck-Sickinger & Jung, 1996 ) (SMPS 350, Zinsser Analytic, Frankfurt; Software Syro, MultiSynTech, Bochum) using the fluorenylmethoxycarbonyl (Fmoc) tertiary-butyl (tBu) strategy. The resin employed (30 mg per peptide, 0·55 mmol g-1) was 4-(2',4' - dimethoxyphenyl - Fmoc - aminomethyl) - phenoxyacetamidonorleucyl-MBHA resin (Rink MBHA resin). Fmoc-protected amino acids were activated with diisopropylcarbodiimide/1-hydroxybenzotriazole in N,N-dimethylformamide (DMF) and coupled in tenfold excess amino acid with respect to resin loading for 90 min. The N-terminal protecting group was cleaved with piperidine/dimethylformamide (1:1, v/v). The peptides were cleaved from the resin using reagent K (1 ml) containing 82·5% trifluoroacetic acid, 5% (w/v) phenol, 5% (w/v) thioanisole, 2·5% (w/v) ethanedithiol and 5% (w/v) water, for 3 h at room temperature. After precipitation and washing with cold diethyl ether (three cycles), the compounds were dissolved in tert-butyl alcohol (4:1, v/v) and were then lyophilized.
The identity of the synthetic peptides was proven by pneumatically assisted electrospray mass spectrometry on an API III triple-quadrupole mass spectrometer equipped with an IonSpray source (Sciex).
Computer-assisted sequence analysis.
Sequences were analysed using the program package PC.GENE and the BLAST homology search (Altschul et al., 1990 ).
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RESULTS |
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The four TonB nonapeptides covering residues 150 to 161 inhibited ferrichrome transport to rates between 17 and 27% of that of untreated E. coli AB2847 cells taken as 100% (Table 2). Peptide 158166 reduced the rate to 42%. The other peptides reduced the ferrichrome transport to a lesser extent (Table 2
).
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Since reduction of the transport rates by the TonB peptides also depended on the expression level of FhuA, the amounts of FhuA in the outer membrane were determined. E. coli AB2847 and HK97 transformed with pHK570 or pBK570 were grown in NB medium under similar conditions as used for the transport assays. Compared to wild-type FhuA (pHK570), FhuA5160 (pBK570) was present in lower amounts, which, however, were higher than the amounts produced by chromosomally encoded FhuA (Fig. 1
). Therefore, reduction of the ferrichrome transport by FhuA
5160 should rather be compared with the results obtained with chromosomally encoded FhuA (AB2847) than with the results obtained with plasmid-encoded wild-type FhuA. This comparison shows that the reduction of ferrichrome transport by FhuA
5160 caused by the TonB peptides spanning residues 150161 was weaker than with chromosomally encoded wild-type FhuA, probably because the TonB peptides only interfered with wild-type TonB binding to the ß-barrel of FhuA
5160 missing the TonB box.
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As found previously (Braun et al., 1999 ), FhuA
5160 transported ferrichrome, but at a rate lower than that of wild-type FhuA (Fig. 3
). All of the tonB mutations impaired ferrichrome transport activity of FhuA
5160, and the reduction was stronger than the reduction of wild-type FhuA activity (Fig. 3
, compare a with c, and b with d). The results showed that the mutations in the TonB proteins also affected FhuA
5160 activity, but they did not reveal specific interactions between TonB and wild-type FhuA or FhuA
5160.
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Synthetic E. coli TonB peptides inhibit ferrichrome transport by FhuA proteins from various species
To study the specificity of the interaction between TonB and FhuA further, inhibition of ferrichrome transport by synthetic E. coli (Ec) TonB peptides was examined with FhuA proteins of Salmonella paratyphi (Sp), Salmonella enterica serovar Typhimurium (St) and P. agglomerans (Pa), whose fhuA genes have been sequenced (Killmann et al., 1998 ). E. coli HK97 fhuA aroB carrying plasmid p7626 (FhuA
322355) was transformed with plasmids encoding the fhuA genes of these organisms. All four FhuA proteins were expressed in comparable amounts, as was shown previously (Killmann et al., 2001
). Three TonB peptides that inhibited ferrichrome transport into E. coli AB2847 and the inactive TonB peptide 149157 were used in the assays. TonB 152160 reduced ferrichrome transport by FhuA (Sp), FhuA (St) and FhuA (Pa) to rates observed for FhuA (Ec) (Table 4
). Compared to FhuA (Ec) the transport rates by FhuA (Sp) and FhuA (St) were somewhat less inhibited by the TonB peptides TonB 150158 and TonB 151159, whereas FhuA (Pa) was inhibited the least (Table 4
). TonB 149157 only weakly reduced ferrichrome transport by all four FhuA proteins.
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The transformants formed the same size and type of growth zone around filter paper discs supplemented with 10 µl 0·03 mM ferrichrome on NBD agar plates, except for transformants producing TonB (Pa), which formed a less-dense growth zone, indicating a reduced ferrichrome uptake (data not shown).
Interaction of the TonB proteins with the ß-barrel domain of E. coli FhuA5160 was determined in E. coli HK99 transformed with plasmid pBK7 (FhuA
5160) and the TonB plasmids of the strains listed above. No differences were observed in the sensitivity to the phages T1,
80 and T5, and to colicin M and albomycin, showing that all TonB proteins tested interact with the ß-barrel domain of E. coli FhuA
5160, and the interaction was indistinguishable from that with wild-type FhuA (data not shown).
Reduced activity of P. agglomerans TonB coupled to E. coli FhuA
Growth on ferrichrome of E. coli HK99 synthesizing FhuA (Ec) and TonB (Pa) was reduced. Therefore, E. coli and P. agglomerans wild-type strains were transformed with pHK763, encoding FhuA (Ec), or p76Pa, encoding FhuA (Pa), and ferrichrome transport rates measured to determine whether the transport rate increased through the synthesis of a high number of plasmid-encoded heterologous FhuA proteins in addition to the chromosomally encoded homologous FhuA proteins. All combinations showed a two- to threefold increase in ferrichrome transport with the exception of P. agglomerans expressing FhuA (Ec), which displayed nearly the same transport rate as untransformed P. agglomerans, suggesting that the interaction of FhuA (Ec) with TonB (Pa) is somewhat disturbed (data not shown).
Transport of ferrichrome was also determined quantitatively using E. coli HK99 fhuA tonB transformed with pHK763, encoding wild-type FhuA (Ec), or p76Pa, encoding wild-type FhuA (Pa), and tonB plasmids of E. coli or P. agglomerans cloned on the low-copy-number plasmid pHSG576. The strains expressing FhuA (Pa) and TonB (Ec) or TonB (Pa) showed similar transport rates, which amounted to 80% compared to a strain expressing FhuA (Ec) and TonB (Ec) taken as 100% (Fig. 5). In contrast, the strain expressing FhuA (Ec) and TonB (Pa) showed only a transport rate of 40%, supporting the assumption that the interaction of FhuA (Ec) with TonB (Pa) is somewhat disturbed (Fig. 5
). Since the amounts of FhuA (Ec) and FhuA (Pa) were the same (Killmann et al., 2001
) we also wanted to rule out that different expression levels of TonB (Ec) and TonB (Pa) were the reason for the reduced transport rates. A pulsechase experiment showed comparable amounts of [35S]methionine-labelled TonB (Ec) and TonB (Pa); however, the TonB (Pa) was somewhat more stable than TonB (Ec) (data not shown).
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Nucleotide sequence of the P. agglomerans tonB gene and derived amino acid sequence
To obtain structural information that might explain the low activity of TonB (Pa) in E. coli, the tonB gene of P. agglomerans was cloned. E. coli BR158 tonB aroB was transformed with DNA fragments of 13 kb from the P. agglomerans chromosome. Transformants that had acquired the tonB gene were selected by growth on NBD agar plates supplemented with 4 µM ferrichrome as sole iron source. The recombinant plasmid carrying the P. agglomerans tonB gene, pPaTonB, carried a 1·7 kb PstI fragment. Both strands of the insert were sequenced (GenBank accession number Y15319); an open reading frame of 756 bp with 64·5% identity to the tonB gene of E. coli was identified. Upstream of the tonB gene are a ribosome-binding site and typical -35 and -10 promoter regions, with 29 out of 31 nucleotides identical to the tonB-specific promoter consensus sequence (Gaisser & Braun, 1991 ). In addition, a region with a high level of identity to the binding site of the Fe2+ Fur repressor was identified, with 14 out of 19 nucleotides identical to the Fur consensus sequence (de Lorenzo et al., 1987
). The P. agglomerans tonB gene encodes a protein of 251 amino acids with a molecular mass of 27·4 kDa and 61·5% sequence identity to the TonB protein of E. coli. Only two copies each of the repetitive sequence motifs Glu-Pro and Lys-Pro, which are typical for TonB proteins, are found in the P. agglomerans TonB protein, whereas TonB of E. coli has four copies of Glu-Pro and six copies of Lys-Pro. The amino acids R158, Q160, Q162 and Y163, which have been shown to be involved in the interaction of E. coli TonB with the TonB box of various transport proteins and colicins, were substituted in P. agglomerans TonB by V170, K172, G174 and Y175 (Fig. 6
).
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DISCUSSION |
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Four overlapping TonB peptides covering residues 150161 inhibited FhuA transport activity most strongly. The specificity of the inhibition was demonstrated by peptides flanking this region which were inactive or much less active. Peptide 158166 also interfered with TonB-dependent FhuA activities. The peptides from residues 150161 and 158166 cover the region in which the TonB mutations R158L, Q160L and Q160K are located; these mutant TonB proteins suppress the mutations I9P and V11D in the TonB box of FhuA (Schöffler & Braun, 1989 ; Günter & Braun, 1990
). It was expected that peptide 149157 would be inactive but the inactive peptides covering positions 154168 still contained the residues of the suppressor mutations. The inactive TonB peptides might not be able to adapt to the conformation of the FhuA TonB box (and the unknown interaction site(s) of the ß-barrel). A detailed analysis of mutations in the TonB box of the BtuB protein by Cadieux et al. (2000)
revealed that the suppressor mutations did not identify interacting amino acid residues but rather changed the conformation of the TonB region such that it was able to interact with the mutated TonB box sequence, leading to partial restoration of the BtuB activity.
The same set of peptides that inhibited FhuA also inhibited FhuA5160 transport activity. Therefore, interaction of the TonB peptides cannot be only confined to the TonB box of FhuA, but must also affect other regions of FhuA. It is even possible that the peptides did not inhibit the interaction of TonB with the TonB box, but only interactions with the FhuA ß-barrel.
Recently the crystal structure of a TonB fragment (residues 164239) has been determined; the crystal structure reveals a dimer that forms an antiparallel ß-sheet composed of six ß-strands (Chang et al., 2001 ). The TonB peptides used in our study could have interfered with TonB dimer formation, thereby inhibiting TonB activity. We consider this unlikely for the following reasons. Overexpression of FhuA relieved inhibition by the TonB peptides, which has also been observed previously with TonB peptides (residues 33239, 103239 and 122239) synthesized in vivo and secreted into the periplasm via a cleavable signal peptide (Howard et al., 2001
). The frequency of binding of TonB to FhuA increases when FhuA is overexpressed, which decreases the chance of the competing peptides to interfere. TonB (residues 33239) was isolated from the cytoplasm without the use of denaturing compounds and was mainly a monomer (Moeck & Letellier, 2001
), chemical cross-linking of TonB with FhuA and FepA results predominantly in dimers that contain a single polypeptide of each of the proteins (Larsen et al., 1997
; Moeck et al., 1997
), and disulfide-linked BtuB with TonB also consists of a mixed dimer (Cadieux & Kadner, 1999
). These data with complete TonB and TonB fragments larger than those used for crystallization argue in favour of a monomeric complete TonB. Moreover, TonB is anchored to the cytoplasmic membrane by the N-proximal end (residues 132) and interacts within the cytoplasmic membrane with transmembrane regions of ExbB (Traub et al., 1993
; Postle, 1993
). The polypeptides in the crystallized dimer fragment have opposite polarities, which implies that the identical TonB monomers interact differently with FhuA. It is difficult to envisage how the TonB polypeptides in the dimer run in opposite directions in the periplasm when they start identically in the cytoplasmic membrane. Moreover, TonB-mediated energy transfer from the cytoplasmic membrane to the outer membrane is a vectorial process, and it is highly likely that this is achieved by TonB polypeptides that assume the same orientation. Even with a model that proposes rotation of TonB to bind to and dissociate from FhuA (Chang et al., 2001
), the asymmetry of the interaction of the two TonB polypeptides with FhuA is not resolved.
Another explanation for interference of TonB activity by TonB peptides is inhibition of the interaction of TonB with ExbB. This possibility does not seem to play a major role, as has been demonstrated with the in vivo-synthesized periplasmic TonB fragments mentioned above (Howard et al., 2001 ). Since the periplasmic nonapeptides are within these larger TonB peptides, it is unlikely, but not completely ruled out, that they interfere with the interaction of TonB with ExbB.
If TonB interacts with the TonB box and the ß-barrel of FhuA, TonB missense mutations might affect one or the other interaction. Therefore, TonB missense mutants with impaired activities were used to identify regions in TonB that differ in their interaction with wild-type FhuA and FhuA5160. All TonB mutant proteins reduced the activities of wild-type FhuA and FhuA
5160. The weaker transport activity of FhuA
5160 for colicin M, rifamycin CGP 4832 and microcin J25 was more strongly reduced than the wild-type FhuA activity by the mutated TonB proteins. Although some minor differences were noted, the TonB mutants did not reveal TonB mutations that impaired FhuA activities as opposed to FhuA
5160 activities, which could have been ascribed to interactions with the TonB box and the ß-barrel, respectively.
TonB proteins of different species were employed to identify differences in the functional response of wild-type FhuA and FhuA5160 to TonB. No such differences were noted, which again demonstrates that the ß-barrel can convey all the FhuA activities.
The P. agglomerans TonB coupled to E. coli FhuA or FhuA5160 in the assays had low activity. Interactions of FhuA (Pa) with TonB (Pa) have structural requirements which are not completely met by FhuA (Ec) and TonB (Ec), respectively. To obtain structural information that might shed light on the interaction specificity, the tonB gene of P. agglomerans was sequenced. Residue Q160, which has been identified both genetically and biochemically as being important for interaction with the TonB box of the outer-membrane proteins FhuA, Cir, FepA and BtuB (Braun, 1995
), is in TonB (Pa) replaced by K (Fig. 6
). In addition, R158, which when replaced by L suppressed the I9P TonB box mutation in E. coli FhuA, is replaced by V. In TonB proteins from other species that fully complement an E. coli tonB mutant, Q160 is conserved or replaced by the similar amino acid N, and R158 is conserved or replaced by the similar amino acid K. The functional significance of the Q
K substitution is supported by the incomplete complementation of an E. coli tonB mutant by a TonB mutant of Y. enterocolitica with the same Q
K substitution (Koebnik et al., 1993
).
TonB of P. agglomerans showed high activities when coupled to FepA (TonB box sequence DTIVV), colicin M (ETLTV) and colicin B (DTMVV), whereas the activity in combination with FhuA (DTITV) and FhuE (ETVIV) was strongly reduced and with colicin D (HSMVV) completely abolished. The TonB box of colicin D contains histidine where the TonB box of all the other TonB-dependent colicins and transporters contain aspartate or glutamate. This deviation from the consensus sequence may contribute to the reduced colicin D sensitivity (uptake) of those E. coli cells which synthesize the heterologous TonB proteins of P. agglomerans, Y. enterocolitica, Salmonella enterica serovar Typhimurium and Serratia marcescens. Although the data suggest the importance of the interaction between region 160 of TonB and the TonB box sequence, other regions of TonB and in particular the ß-barrel of FhuA contribute to the activation of FhuA by TonB.
The most important result of this study is that competitive peptide mapping, the use of TonB missense and nonsense mutants, and TonB proteins of different strains did not discriminate between interaction of TonB with wild-type FhuA and FhuA5160. The ß-barrel is the major player in all FhuA activities, and interaction of TonB with the TonB box may only add to the release of ferrichrome, albomycin and rifamycin CGP 4832 from the common binding site to which the globular domain contributes amino acid side chains (Ferguson et al., 1998
, 2000
, 2001
) and the movement of the globular domain to open the channel formed by the ß-barrel.
In a detailed thermodynamic and kinetic analysis, Scott et al. (2001) compared ferrichrome transport into FhuA and FhuA
5160 and ferric enterobactin transport into FepA
17150 and into hybrid proteins in which the globular domains and the ß-barrels were interchanged. They arrived at the conclusion that the globular domain within the pore is dispensable to the siderophore internalization reaction and, when present, acts without specificity during substrate uptake. The data presented here, which show interaction of all TonB proteins and TonB peptides with the ß-barrel, are consistent with this concept, although we think that additional interactions of TonB with the TonB box are experimentally well supported.
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ACKNOWLEDGEMENTS |
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Received 17 June 2002;
revised 12 July 2002;
accepted 19 July 2002.