Insight from TonB hybrid proteins into the mechanism of iron transport through the outer membrane.

We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB(+) bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepADelta3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.

Single-molecule study of molecular mobility in the cytoplasm of Escherichia coli.

The cytoplasm of bacterial cells is filled with individual molecules and molecular complexes that rely on diffusion to bring them together for interaction. The mobility of molecules in the cytoplasm has been characterized by several techniques mainly using fluorescent probes and ensemble methods. In order to probe the microenvrionment inside the cytoplasm as viewed by an individual molecule, we have studied single green fluorescent proteins (GFPs) diffusing in the cytoplasm of Escherichia coli cells at observation at rates ranging from 60 to 1000 Hz. Over long times the diffusion shows confinement due to the geometry of the cells themselves. A simulation in model cells using the actual distribution of cell sizes found in the experiments describes accurately the experimental results as well as reveals a short time diffusion coefficient that agrees well with that determined by ensemble methods. Higher short time diffusion coefficients can be obtained by filling the simulated cell with small spheres modeling cytoplasmic molecules and, depending on the density of particles included in the modeled cytoplasm, can approach the diffusion coefficient of GFPs found in water. Thus, single-molecule tracking combined with analysis using simple simulation of Brownian motion is able to reveal the main contributors to the GFP mobility in the cytoplasm of E. coli.

Evidence of ball-and-chain transport of ferric enterobactin through FepA.

The Escherichia coli iron transporter, FepA, has a globular N terminus that resides within a transmembrane beta-barrel formed by its C terminus. We engineered 25 cysteine substitution mutations at different locations in FepA and modified their sulfhydryl side chains with fluorescein maleimide in live cells. The reactivity of the Cys residues changed, sometimes dramatically, during the transport of ferric enterobactin, the natural ligand of FepA. Patterns of Cys susceptibility reflected energy- and TonB-dependent motion in the receptor protein. During transport, a residue on the normally buried surface of the N-domain was labeled by fluorescein maleimide in the periplasm, providing evidence that the transport process involves expulsion of the globular domain from the beta-barrel. Porin deficiency much reduced the fluoresceination of this site, confirming the periplasmic labeling route. These data support the previously proposed, but never demonstrated, ball-and-chain theory of membrane transport. Functional complementation between a separately expressed N terminus and C-terminal beta-barrel domain confirmed the feasibility of this mechanism.

FepA- and TonB-dependent bacteriophage H8: receptor binding and genomic sequence.

H8 is derived from a collection of Salmonella enterica serotype Enteritidis bacteriophage. Its morphology and genomic structure closely resemble those of bacteriophage T5 in the family Siphoviridae. H8 infected S. enterica serotypes Enteritidis and Typhimurium and Escherichia coli by initial adsorption to the outer membrane protein FepA. Ferric enterobactin inhibited H8 binding to E. coli FepA (50% inhibition concentration, 98 nM), and other ferric catecholate receptors (Fiu, Cir, and IroN) did not participate in phage adsorption. H8 infection was TonB dependent, but exbB mutations in Salmonella or E. coli did not prevent infection; only exbB tolQ or exbB tolR double mutants were resistant to H8. Experiments with deletion and substitution mutants showed that the receptor-phage interaction first involves residues distributed over the protein's outer surface and then narrows to the same charged (R316) or aromatic (Y260) residues that participate in the binding and transport of ferric enterobactin and colicins B and D. These data rationalize the multifunctionality of FepA: toxic ligands like bacteriocins and phage penetrate the outer membrane by parasitizing residues in FepA that are adapted to the transport of the natural ligand, ferric enterobactin. DNA sequence determinations revealed the complete H8 genome of 104.4 kb. A total of 120 of its 143 predicted open reading frames (ORFS) were homologous to ORFS in T5, at a level of 84% identity and 89% similarity. As in T5, the H8 structural genes clustered on the chromosome according to their function in the phage life cycle. The T5 genome contains a large section of DNA that can be deleted and that is absent in H8: compared to T5, H8 contains a 9,000-bp deletion in the early region of its chromosome, and nine potentially unique gene products. Sequence analyses of the tail proteins of phages in the same family showed that relative to pb5 (Oad) of T5 and Hrs of BF23, the FepA-binding protein (Rbp) of H8 contains unique acidic and aromatic residues. These side chains may promote binding to basic and aromatic residues in FepA that normally function in the adsorption of ferric enterobactin. Furthermore, a predicted H8 tail protein showed extensive identity and similarity to pb2 of T5, suggesting that it also functions in pore formation through the cell envelope. The variable region of this protein contains a potential TonB box, intimating that it participates in the TonB-dependent stage of the phage infection process.