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 (FepA⌬3-17, 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.Iron is one target of gram-negative bacterial cell envelope transport systems, and microbes elaborate high-affinity siderophores that complex extracellular iron (70). However, ferric siderophores, like ferric enterobactin (FeEnt), are too large (716 Da) to pass through general porins in the outer membrane (OM), necessitating a different type of transporter to acquire them. On the basis of their 22-stranded transmembrane -barrels, OM metal transporters like FepA belong to the porin superfamily (89). Ligand binding to such receptors initiates the transport reaction through their transmembrane channels, which led to their designation as ligand-gated porins (LGP) (88), by analogy to the family of eukaryotic ligand-gated ion channels. It is noteworthy that LGP are mechanistically distinct from general, diffusive porins because they bind metal complexes with high affinity and actively transport them against a concentration gradient into the cell. Once in the periplasm, binding proteins adsorb ferric siderophores and del...