TonB dependent transport processes through Escherichia coli FepA.

One of the previously proposed mechanisms of TonB is called the "shuttle hypothesis" which states that TonB shuttles from the inner membrane to the outer membrane. The data presented here demonstrates that TonB does not need to leave the inner membrane to accomplish its function, thereby presenting evidence against this hypothesis.Gram negative bacteria use elaborate transport systems to acquire nutrients from their environment. One type of system is the ligand gated porin (LGP) transport system. All LGP's are TonB dependent and require energy in the form of proton motive force (PMF) to transport their substrates. LGP's are beta-barrels with globular N-domains that reside in the barrel blocking the passage of molecules. Therefore in order for transport to occur the N-domain must change in conformation. This change in conformation is dependent on TonB, PMF and the presence of Ferric Enterobactin (FeEnt).The tail protein of H8 that binds to FepA was compared to the tail proteins of the other bacteriophage T5 and BF23 that are also in the Siphovirida family. Relative to these other tail proteins H8's tail protein contains acidic and aromatic residues that are unique to it and are probably involved in the binding of the phage to FepA. Other than these residues this protein is similar to T5's tail protein suggesting a similar poreforming mechanism through the cell envelope. Of further interest is a region on the H8 tail forming protein that contains a TonB box sequence. This suggests a link between this sequence and TonB dependent processes.H8 is a bacteriophage that was recently discovered to be FepA dependent for its infection of E. coli and the binding of H8 to E. coli is inhibited (50% inhibition concentration, 98 nM) by FeEnt the native siderophore of FepA. H8 did not require the other ferric catecholate receptors (Fui, Cir, or Iron) for infection. H8 has morphology and genomic structure of similarity to bacteriophage T5, a member of the family Siphoviridae. The DNA sequence of H8 showed that H8 has a 104.4 kb genome with a total of 143 open reading frames (ORFS). Of the 143 ORFS 120 of them are homologous to ORFS found in T5.TonB is a 239 residue protein anchored in the inner membrane, spanning into the periplasm of E. coli. It is required by all LGP's for their transport of siderophores and their susceptibility to group B bacteriocins and certain bacteriophage. The mechanism by which TonB accomplishes this is not yet understood. Several fusion proteins were constructed consisting of the green fluorescent protein (GFP) from Aequaria victoria either attached to the C-terminus or the N-terminus of TonB. All of the fusion proteins when expressed in E. coli were active with respect to the transport of FeEnt and the susceptibility to colicin B and D.One of E. coli's LGP transport systems is the FepA system. The FepA system is used for the acquisition of iron. Located in the outer membrane of E. coli it binds and transports ferric enterobactin (FeEnt) a siderophore. In order for transport to occur through FepA the globular N-domain needs to either leave the barrel completely or structurally change inside the barrel to allow for transport through the barrel. To study the transport mechanism of FepA, the susceptibility of 25 cysteine mutants at different locations in FepA to alkylation by fluorecein maleimide (FM) was studied in live cells. The reactivity of those cysteine residues changed in response to the addition of FeEnt. This reactivity was also dependent on the presence or absence of TonB and energy. Of particular note is the reactivity of a residue located deep inside the barrel and the dependence of this labeling on the presence of TonB and energy. Also demonstrated is the functional complementation between the N-terminus and C-terminal barrel expressed separately. The data collected offer support for the ball-and-chain theory for LGP transport

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

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 (FepAΔ3-17, 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