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...

The cytoplasmic membrane proteins ExbB and ExbD support TonB-dependent active transport of iron siderophores and vitamin B12 across the essentially unenergized outer membrane ofEscherichia coli. In this study, in vivo formaldehyde cross-linking analysis was used to investigate the interactions of T7 epitope-tagged ExbB or ExbD proteins. ExbB and ExbD each formed two unique cross-linked complexes which were not dependent on the presence of TonB, the outer membrane receptor protein FepA, or the other Exb protein. Cross-linking analysis of ExbB- and ExbD-derived size variants demonstrated instead that these ExbB and ExbD complexes were homodimers and homotrimers and suggested that ExbB also interacted with an unidentified protein(s). Cross-linking analysis of epitope-tagged ExbB and ExbD proteins with TonB antisera afforded detection of a previously unrecognized TonB-ExbD cross-linked complex and confirmed the composition of the TonB-ExbB cross-linked complex. The implications of these findings for the mechanism of TonB-dependent energy transduction are discussed.

Leukotriene (LT) C4 synthase, the enzyme that catalyzes the conjugation ofLTA4 with reduced glutathione to form LTC4, was purified to homogeneity from the KG-1 myeloid cell line after solubilization of the microsomes utilizing a combination of 0.4% sodium deoxycholate and 0.4% Triton X-102. The solubilized enzyme was then applied to an S-hexylglutathione-agarose column that was eluted by the use of7.5 mM probenecid. After removal of the probenecid by sequential concentration and dilution in an Amicon concentrator, the enzyme was additionally purified and concentrated by binding to and elution from -75 mg ofS-hexyl-glutathione-agarose. The enzyme was further resolved by electrophoresis with a nondenaturing Tris-glycine gel, and the LTC4 synthase activity was localized to slices 3 and 4. When the remainder ofthe eluate from the nondenaturing gel was precipitated by acetone and analyzed by 14% SDS/PAGE with silver staining, a single protein band of 18 kDa was associated with LTC4 synthase activity and was not present in the eluates of slices lacking activity. The overall recovery was 12.5%. In a separate preliminary purification, in which the yield was only -1%, the eluates of the nondenaturing gel had also revealed a single protein of 18 kDa by SDS/PAGE, which was present only in the eluates with LTC4 synthase activity. These data identify LTC4 synthase as a protein of 18 kDa, a size consistent with its membership in the microsomal glutathione S-transferase family.