Identification of TonB homologs in the family Enterobacteriaceae and evidence for conservation of TonB-dependent energy transduction complexes

Abstract

The transport of Fe(III)-siderophore complexes and vitamin B 12 across the outer membrane of Escherichia coli requires the TonB-dependent energy transduction system. A set of murine monoclonal antibodies (MAbs) was generated against an E. coli TrpC-TonB fusion protein to facilitate structure and function studies. In the present study, the epitopes recognized by these MAbs were mapped, and their distribution in gram-negative organisms was examined. Cross-species reactivity patterns obtained against TonB homologs of known sequence were used to refine epitope mapping, with some epitopes ultimately confirmed by inhibition experiments using synthetic polypeptides. Epitopes recognized by this set of MAbs were conserved in TonB homologs for 9 of 12 species in the family Enterobacteriaceae (including E. coli), including previously unidentified TonB homologs in Shigella, Citrobacter, Proteus, and Kluyvera species. These homologs were also detected by a polyclonal ␣-TrpC-TonB serum that additionally recognized the known Yersinia enterocolitica TonB homolog and a putative TonB homolog in Edwardsiella tarda. These antibody preparations failed to detect the known TonB homologs of either Pseudomonas putida or Haemophilus influenzae but did identify potential TonB homologs in several other nonenteric gram-negative species. In vivo chemical cross-linking experiments demonstrated that in addition to TonB, auxiliary components of the TonB-dependent energy transduction system are broadly conserved in members of the family Enterobacteriaceae, suggesting that the TonB system represents a common system for high-affinity active transport across the gram-negative outer membrane.The outer membrane of gram-negative bacteria is a diffusion barrier that excludes a variety of toxic agents from the cell proper. This barrier is circumvented by small hydrophilic nutrients, which can enter the periplasmic space by simple diffusion through nonspecific aqueous channels created by porin proteins, and by certain larger nutrients, which can enter the periplasm by facilitated diffusion through stereospecific pores like LamB. Conversely, a third group of nutrients, Fe(III)-complexed siderophores and vitamin B 12 , are dependent on active transport via high-affinity outer membrane receptors to enter the periplasmic space.The active transport of Fe(III)-bearing siderophores and vitamin B 12 (as well as certain colicins and bacteriophage that exploit this process) across the outer membrane is complicated by the absence of a local energy source. Free diffusion of protons through porins renders the outer membrane unable to sustain an electrochemical potential sufficient to energize active transport. In addition, periplasmic phosphatases preclude the use of high-energy phosphate carriers as an energy source. Early experiments with 80 and T5 found that the electrochemical potential of the cytoplasmic membrane was required for irreversible adsorption of these phage, suggesting that the energy for this outer membrane phenomenon originates at the cytoplasmic me...

TonB protein appears to transduce energy by shuttling between the cytoplasmic membrane and the outer membrane in Escherichia coli

“…We have demonstrated, using the zero-length cross-linker formaldehyde, that TonB can cross-link in vivo to ExbB, to the outer membrane receptor FepA, and to at least two other proteins whose identities are currently under investigation (Skare et al, 1993). Furthermore, these cross-linking interactions are conserved amongst several other Gramnegative bacteria, suggesting that they represent meaningful interactions (Larsen et al, 1996). It has been clearly demonstrated that TonB-dependent energy transduction requires, either directly or indirectly, the cytoplasmic membrane pmf (Bradbeer, 1993).…”

“…In Escherichia coli, and presumably other Gram-negative bacteria (Larsen et al, 1996), TonB protein couples the cytoplasmic membrane pmf to active transport of ironsiderophore complexes and vitamin B12 across the outer membrane and into the periplasmic space. In doing so, it requires two cytoplasmic membrane proteins, ExbB and ExbD, as well as the outer membrane receptors through which those nutrients are transported.…”

“…Chemicals and reagents ␣-TonB monoclonal antibodies (mAbs) were prepared as described previously (Larsen et al, 1996). ␣-FhuA mAbs were the generous gift of Dr James Coulton.…”

1H, 13C and 15N resonance assignments of the C-terminal domain of HasB, a specific TonB like protein, from Serratia marcescens

“…Consequently, specific surface receptors promote the translocation of these various substrates by an energized mechanism. This energy-dependent transport is mediated by a cytoplasmic membrane complex consisting of three proteins: TonB, ExbB and ExbD (Postle 1993;Larsen et al 1996). Current models consider TonB to function as the energy transducer that couples the proton motive force of the cytoplasmic membrane to drive ligand translocation through the outer membrane receptors.…”

Cell Envelope Signaling in Escherichia coli

“…In addition, complexes between TonB or its homologues and other proteins (ExbB, ExbD, and as yet unidentified proteins) were detected with anti-E. coli TonB monoclonal antibodies (mAbs 1 ; Ref. 16). These findings demonstrate that the TonB-dependent energy transduction system is shared among many Gram-negative bacteria and suggest that high affinity energy-dependent iron uptake in Gram-negative aerobes is accomplished using TonB and its accessory proteins (16).…”