Cell Envelope Signaling in Escherichia coli

Abstract

The ferrichrome-iron receptor of Escherichia coli is FhuA, an outer membrane protein that is dependent upon the energy-coupling protein TonB to enable active transport of specific hydroxamate siderophores, infection by certain phages, and cell killing by the protein antibiotics colicin M and microcin 25. In vivo cross-linking studies were performed to establish at the biochemical level the interaction between FhuA and TonB. In an E. coli strain in which both proteins were expressed from the chromosome, a high molecular mass complex was detected when the ferrichrome homologue ferricrocin was added immediately prior to addition of crosslinker. The complex included both proteins; it was absent from strains of E. coli that were devoid of either FhuA or TonB, and it was detected with anti-FhuA and anti-TonB monoclonal antibodies. These results indicate that, in vivo, the binding of ferricrocin to FhuA enhances complex formation between the receptor and TonB. An in vitro system was established with which to examine the FhuA-TonB interaction. Incubation of TonB with histidine-tagged FhuA followed by addition of Ni 2؉ -nitrilotriacetate-agarose led to the specific recovery of both TonB and FhuA. Addition of ferricrocin or colicin M to FhuA in this system greatly increased the coupling between FhuA and TonB. Conversely, a monoclonal antibody that binds near the N terminus of FhuA reduced the retention of TonB by histidine-tagged FhuA. These studies demonstrate the significance of ligand binding at the external surface of the cell to mediate signal transduction across the outer membrane.

Substrate-dependent transmembrane signaling in TonB-dependent transporters is not conserved

“…Chemical cross-linking (12,30) and recent protein crystal structures (20,31) indicate that the Ton box interacts with TonB. There also is evidence that this Ton box-TonB interaction occurs in a substrate-dependent manner (12,32); thus, some signal transduction event must initiate this interaction. Genetic studies suggest that iron siderophore and vitamin B 12 transport systems compete for TonB (33), and regulation of the transporter-TonB interaction by a substratedependent signaling event would account for this observation and optimize the use of TonB, which is stoichiometrically limiting.…”

Crystal Structure of a 92-Residue C-terminal Fragment of TonB from Escherichia coli Reveals Significant Conformational Changes Compared to Structures of Smaller TonB Fragments

“…A close apposition of the TonB box of the receptor with the Cterminal TonB region around Gln 160 has been inferred from in vivo experiments using tonB suppressor mutants (18), crosslinking data (8), disulfide formation in cysteine substitution mutants (9), and competitive binding of peptides (20). In vitro, complexes between receptors and TonB have been described that are more stable when the receptor contains bound ligand (17,10,21,22,44,45). Because of the crystallographic evidence for an allosteric conformational change at the periplasmic surface of the receptor, TonB could in principle bind there without significantly altering its conformation, but an induced fit of TonB upon binding cannot be excluded.…”

In Vivo Evidence for TonB Dimerization

“…ToxR(1-210)-PhoA(27-472) and ToxR(1-210)-MalE(27-397) were used as positive and negative controls, respectively. In addition, cells were grown in the presence and absence of 1 mM citrate, since TonB may preferentially bind to FecA loaded with ferric citrate, as has been found for ferrichrome binding to FhuA (34,35) and ferric enterobactin binding to FepA (41). Binding to ferric citrate-loaded FecA may change the conformation of TonB and affect dimer formation.…”

“…In addition, the required energy is provided by the proton motive force of the cytoplasmic membrane (2), along with an energy-transducing device in the cytoplasmic membrane which is composed of the TonB, ExbB, and ExbD proteins (3,22,37). The N terminus of TonB (residues 13 to 32) anchors it to the inner membrane, while most of the protein is located in the periplasm, where it interacts with outer membrane transporters (7,15,35,40). The topography of ExbD resembles that of TonB (23), whereas ExbB spans the cytoplasmic membrane three times, with most of the protein being located in the cytoplasm (24).…”