Expression and purification of MalE-TonB69C. E. coli KDO23/pSTonB69C was grown to mid-log in LB broth, IPTG was added to 0.5 mM, and the cells were shaken at 37°C for 3 more hours. (A) Aliquots of 10⁸ cells were collected before (lane 1) and 1 h (lane 2) and 2 hours (lane 3) after the addition of IPTG. The bacteria were lysed and subjected to SDS-PAGE on a 10% gel. The overexpression of MalE-TonB69C (arrow) appears in lane 3. The positions of molecular size markers (m) (in kilodaltons), Bio-Rad Precision Plus protein standards, are shown to the left of the gel. (B) After lysis by passage through a French pressure cell, the lysate was clarified by centrifugation at 100,000 × g for 30 min and applied to an amylose column. MalE-TonB69C was eluted with maltose (lane 1), and TonB69C (arrow) was released by digestion with factor X_a (lane 2) (16% gel). (C) The digestion mixture was chromatographed on Sephacryl S-100 in TBS plus 0.05% SDS and analyzed on a 16% gel. The arrow indicates the position of TonB69C, which eluted last from the column (lanes 12 to 16).

Spectroscopic properties of TonB69C. (A) Guanidine HCl titration. Fluorescence emission spectra of a single tryptophan (W213) was monitored in the presence of the denaturant guanidine hydrochloride. The red shift of its emission maximum and decrease in fluorescence with increasing concentrations of guanidinium infer the unfolding of the domain and the increasing exposure of the residue to the solvent. The inset plots show emission maximum (E_max) (○) and F/F₀ (▵) versus the concentration of guanidinium-HCl. (B to D) Susceptibility of TonB69C to quenching. Purified TonB69C, either native (•) or denatured (○), was incubated with increasing concentrations of acrylamide (B), KI (C), and CsCl (D), and its fluorescence emissions were recorded and then analyzed by the Stern-Volmer equation, assuming collisional quenching by the following equation: F_o/F = 1 + K_sv [Q], where F_o and F are the fluorescence intensities in the absence or presence, respectively, of the quencher, K_sv is the Stern-Volmer constant, and [Q] is the concentration of the quencher. The data were analyzed by linear regression to determine K_sv values for each quencher (see the text). (E) Circular dichroism. The CD spectrum of purified TonB69C was recorded in TBS and showed extrema characteristic of α-helical content at 209 nm and other secondary structure at higher wavelengths. The CD data excluded the possibility that TonB69C exists in solution as a random coil form and were consistent with the form of a folded alpha-beta protein.

Affinity chromatography of MalE-TonB69C on agarose modified with proteins. (A) Purified MalE and MalETonB69C were tested for their ability to adsorb to Sepharose 6B that was derivatized with individual purified proteins. The sections of panel A show different chromatographic experiments (labeled to the right with the test protein/immobilized protein). The top three sections are control experiments: MalE-TonB69C chromatographed on unmodified Sepharose 6B, and MalE chromatographed on Sepharose 6B modified with FepA or ColB. MalE did not adsorb to any of the modified agaroses; for brevity, only the results with FepA-Sepharose and ColB-Sepharose are shown. The remaining sections show MalE-TonB69C passed over Sepharose modified with the indicated proteins. In each experiment, the test protein starting material (MalE or MalE-TonB69C; lane 1) was loaded on the column, which was washed with 10 column volumes of TBS (pH 7.5) (lanes 2 to 7), followed by a linear salt gradient (0 to 0.5 M NaCl in TBS [pH 7.5]; lanes 8 to 19). Fractions (1 ml) were collected and resolved on SDS-polyacrylamide gels that were transferred to nitrocellulose and developed with mouse monoclonal anti-MalE serum (93). HEWL, hen egg white lysozyme. (B) Purified proteins. The SDS-polyacrylamide gel (stained with Coomassie blue) shows the purified proteins that were conjugated to Sepharose: FepA, ColB, hen egg white lysozyme, OmpA, FepAΔ13-17, in lanes 1 to 5, respectively, and purified MalE-TonB69C, which was passed over the modified agaroses (lane 6). The positions of molecular size markers (m) (in kilodaltons), the BenchMark prestained protein ladder (Invitrogen), are shown to the left of the gel.

MalE- and GFP-TonB fusion proteins. The illustration depicts some of the experimentally defined elements of TonB structure: the hydrophobic helix (residues 1 to 32; black rectangles in the schematic representations of fusion proteins shown below the sequence), the proline-rich rod domain (residues 63 to 105; cyan and green rectangles), and the C-terminal ββαβ domain (residues 169 to 236; yellow and magenta rectangles). Our constructions fused the complete malE or ssgfp structural genes to the E. coli K-12 tonB structural gene at different locations. We engineered MalE-TonB hybrid proteins that retained all the elements of TonB structure (MalE-TonB) or the rigid rod and the C terminus (MalE-TonBC135), or only the C-terminal ββαβ domain (MalE-TonBC69). We also designed constructs that encoded ssgfp downstream of wild-type tonB (TonB-GFP) and its derivatives with altered N termini (TatTonB-GFP and Δ4-32TonB-GFP). Last, we created plasmids that encoded ssgfp upstream of tonB, under the control of the tonB promoter, either alone (TonBp-GFP) or fused to wild-type TonB (GFP-TonB and GFP-L-TonB) or without the C-terminal ββαβ region (GFP-TonBΔ69C). The small red box denotes a designed factor Xa cleavage site, and the orange rectangle represents a designed, helical, 5-amino-acid linker region.

(A to C) Fluorescence microscopic and electrophoretic analyses of GFP-TonB expression and localization. E. coli OKN3/pTpG (A), OKN3/pGT (B), and OKN3/pGLT (C) were grown in MOPS minimal medium and observed by fluorescence microscopy. Note that E. coli OKN3/pTpG, which expresses cytoplasmic GFP, showed diffuse, uniformly distributed fluorescence throughout the cells, whereas when GFP was fused upstream of TonB in E. coli OKN3/pGT and OKN3/pGLT, its fluorescence associated with the cell envelope, presumably from the association of TonB with the IM. (D and E) Expression of GFP-TonB hybrid proteins. A total of 10⁸ cells of E. coli strains OKN1 (ΔtonB), BN1071 (tonB⁺), OKN3 (ΔfepA), OKN1/pT23 (tonB⁺), OKN1/pGT (ssgfp-tonB), and OKN1/pGLT (ssgfp-L-tonB), shown in lanes 1 to 6, respectively, were grown in MOPS minimal medium, lysed in sample buffer, and subjected to SDS-PAGE and Western immunoblotting with mouse anti-FepA monoclonal antibody 45 (α-FepA) (88) (D) or polyclonal rabbit anti-TonB (α-TonB) (E). The molecular size markers (m) (sizes in kilodaltons) were the BenchMark prestained protein ladder (Invitrogen). Panel D shows comparable expression of FepA in all fepA⁺ strains; panel E shows the expression of wild-type TonB (lanes 2 to 4), GFP-TonB (lane 5), and GFP-L-TonB (lane 6) at comparable levels and the expected molecular sizes. (F and G) Fractionation of cell envelopes by French pressure cell lysis and sucrose gradient centrifugation. Bacteria were grown in MOPS minimal medium, collected by centrifugation, and lysed in a French pressure cell at 14,000 lb/in², and their membranes were resolved on sucrose step gradients and subjected to SDS-PAGE (F). IM samples from strains BN1071, OKN1, OKN1/pT23, OKN1/pGT, and OKN1/pGLT appear in lanes 1, 3, 5, 7, and 9, respectively; OM samples from the same strains appear in lanes 2, 4, 6, 8 and 10, respectively. In panel G, an identical gel was transferred to nitrocellulose and subjected to Western immunoblotting with rabbit anti-TonB sera.

FeEnt uptake by bacteria expressing GFP-TonB hybrid proteins. (Left) Siderophore nutrition tests showed the relative abilities of bacteria expressing hybrid TonB proteins to transport FeEnt (50 μM). When ssgfp was fused downstream of tonB (pTG and pTatTG), the function of FepA was compromised (large, faint growth halos); deletion of the TonB signal sequence (pΔ4-32TG) abrogated function. When GFP was fused upstream of TonB (pGT and pGLT), FeEnt uptake was comparable to that of strains expressing wild-type TonB (BN1071 and OKN1/pT23). Strain OKN3 (ΔfepA) gave the same result (data not shown) as the negative-control OKN1 (ΔtonB). (Right) The binding (top) and transport (bottom) abilities of strains expressing GFP-TonB and GFP-L-TonB were evaluated in ⁵⁹FeEnt uptake experiments. The binding and transport values are shown in picomoles/10⁹ cells and in picomoles/10⁹ cells/min, respectively. Strain OKN1 (□) harboring pT23 (♦), pGT (▴), or pGLT (▾) was compared to BN1071 (○) and its FepA-deficient derivative OKN3 (⋄). GFP-TonB and GFP-L-TonB facilitated uptake with efficiencies similar to that of the wild-type TonB (see text for further explanation).

Homology between the LysM domain and TonB. (A) CLUSTALW analysis of YcfS homologs. CLUSTALW was used to align the sequence of the LysM motif from the E. coli membrane-bound lytic murein transglycosylase D (MltD), which is structurally solved as an αβ domain (7), to the E. coli proteins YcfS, YbiS, YnhG, ErfK, and to the TonB C terminus. The figure shows the alignment of the relevant regions of the four proteins, and the consensus sequence (Cons) from the analysis. Hydrophilic residues are colored green, acidic residues are blue, basic residues are magenta, and hydrophobic residues are red. The consensus is also aligned to the sequence of the crystallized LysM domain of MltD. LysM alignment to TonB identified a sequence relationship at the C terminus of TonB. Asp 11 in LysM (red), which denotes the PG-binding surface in its structure (7), aligns with TonB residue Asp 189. (B) LSQMAN analysis of LysM and TonB. Using the crystallographic coordinates of LysM (7) and the TonB C terminus (26), LSQMAN found a second site of structural homology between the two polypeptides. The program identified a site in TonB (green), distinct from the region noted in panel A, to which LysM (yellow) superimposes. Nearly identical disposition and projection of residues occur in the two structures. The image on the right shows a 90° rotation of the image on the left. (C) On the left, the site identified by CLUSTALW in panel A was manually superimposed to the appropriate region of TonB structure, using α-carbon backbone representation. The regions of LysM that align with TonB (including D11 in stick format) are colored cyan; D189 in TonB, also in stick format, is colored red. The image on the right is a cartoon representation of LysM structure. (D) Four LysM motifs in the TonB dimer. Two LysM polypeptides were aligned to the right lobe of the dimeric TonB C-terminal domain (residues 165 to 239). The first (cyan polypeptide, cyan outline) was found by CLUSTALW; the second (yellow polypeptide, yellow outline) was found by LSQMAN; D11 (stick format, CPK color scheme) aligns with TonB residue D189 (stick format, red) in the first site and with E205 (stick format, red) in the second site. The reflection of these two sites in the left lobe of the structure (yellow and cyan outlines) creates four potential PG-binding sites in the TonB C-domain.

Affinity of the TonB C terminus for PG. (A to F) Purified E coli PG was suspended in distilled water at 1.7 mg/ml. Increasing amounts of the PG solution were mixed with 30 μg of purified proteins MalETonB69C, MalE, and FepB in a final volume of 100 μl, incubated for 30 min at room temperature, and subjected to ultracentrifugation (100,000 × g, 45 min). The pellets (A to C) and supernatants (D to F) were resolved by SDS-PAGE, and the gels were stained with Coomassie blue. The positions of molecular size markers (m) (in kilodaltons), the BenchMark prestained protein ladder (Invitrogen), are shown to the left of the gels. Panels A and D are composite pictures from two separate experiments; the amount of added PG (in micrograms) is denoted beneath each lane. (G) SDS-PAGE of purified PG. Aliquots of the solution of purified sacculi were subjected to SDS-PAGE, and the gel was stained with Coomassie blue. The positions of molecular weight markers (m) (in kilodaltons), Precision Plus protein standards (Bio-Rad), are shown to the left of the gel. (H) Quantification of MalE-TonB69C precipitation in pellets and depletion from supernatants by PG. SDS-polyacrylamide gels were photographed, and the images were quantified using ImageQuant (Molecular Dynamics). Filled symbols are derived from analysis of pellets, while open symbols are derived from analysis of supernatants: MalE-TonB69C (circles) was precipitated, while MalE (inverted triangles) and FepB (triangles) were not precipitated by PG. The quantities of protein and PG (in micrograms) are shown on the y and x axes. The graph depicts the means ± standard errors (error bars) from two experiments.

PG-associated OM protein surveillance model of TonB action. (Left) In a cross-sectional view of the gram-negative bacterial cell envelope, dimeric TonB (green and blue) associates with the IM by α-helices in its N terminus that form a complex with ExbBD (red). TonB also contains extended lengths of rigid polypeptide (depicted here as coiled helices) that span the periplasm and LysM motifs in its C terminus that associate with the PG layer (gray) underlying the OM bilayer. The TonB dimer has general affinity for PG and TonB-independent OM proteins (e.g., OmpA), which tends to localize the C terminus at the periplasmic interface of the OM bilayer. The monomeric form of the C terminus, on the other hand, has a specific affinity for accessible TonB boxes of (ligand-bound) TonB-dependent receptors, resulting in their recruitment by its β-sheet. These dual affinities allow TonB to physically survey the periplasmic surface of the OM bilayer until it encounters bound LGP (FhuA [dark green and orange]). This motion across the internal surface of the OM may derive from movement of the N terminus in the fluid IM bilayer. FepA (purple and red) and the TonB-independent OM proteins OmpF (white) and TolC (white) are also shown associated with PG. The latter protein complexes with AcrAB in the IM bilayer, which provides a reference for the distance between the IM and OM bilayers. (Right) From a periplasmic view, the dimeric form of TonB (green and blue) moves among the roughly hexagonal, 50-Å cells of the PG polymer (gray), which associate with the β-barrels of OM proteins (TolC, OmpF, and LamB [all shown in white]). LGP in the OM bilayer may be ligand-free (FepA [purple β-barrel, red N-domain, and cyan TonB box]; FhuA [dark green β-barrel, orange N-domain, and cyan TonB box]) or ligand-bound (note FhuA at top and bottom right, with TonB box relocated to the center of the channel). The TonB-C-domain remains dimeric until it encounters a ligand-bound receptor (FhuA-Fc, top right) and then dissociates into a monomeric form that recruits the TonB box region into its β-sheet. This binding reaction and additional unknown energy-dependent reactions promote the unfolding of the LGP N-domain from the barrel and concomitant internalization of its bound metal complex. Crystallographic coordinates for the following proteins were from the RCSB Protein Data Bank (http://pdbbeta.rcsb.org/pdb/home/home.do): FepA (1FEP), FhuA (1BY3 and 1BY5), OmpF (1BT9), LamB (1MAL), TolC (1EK9), TonB C terminus (1IHR and 1QXX). The N-terminal and central regions of TonB and ExbBD were drawn (at this time, there are no high-resolution structural data for these polypeptides), and the PG polymer was drawn from Meroueh et al. (65).