IDENTIFICATION OF TONB HOMOLOGS IN THE FAMILY ENTEROBACTERIACEAE AND EVIDENCE FOR CONSERVATION OF TONB-DEPENDENT ENERGY TRANSDUCTION COMPLEXES

The transport of Fe(III)-siderophore complexes and vitamin B-12 across the outer membrane of Escherichia coli requires the TonB-dependent en ergy transduction system, A set of murine monoclonal antibodies (MAbs) was generated against an E. coli TrpC-TonB fusion protein to facilita te structure and function studies, In the present study, the epitopes recognized by these MAbs were mapped, and their distribution in gram-n egative organisms was examined, Cross-species reactivity patterns obta ined against TonB homologs of known sequence were used to refine epito pe mapping, with some epitopes ultimately confirmed by inhibition expe riments using synthetic polypeptides. Epitopes recognized by this set of MAbs were conserved in TonB homologs for 9 of 12 species in the fam ily Enterobacteriaceae (including E. coli), including previously unide ntified TonB homologs in Shigella, Citrobacter, Proteus, and Kluyvera species, These homologs were also detected by a polyclonal alpha-TrpC- TonB serum that additionally recognized the known Yersinia enterocolit ica TonB homolog and a putative TonB homolog in Edwardsiella tarda, Th ese antibody preparations failed to detect the known TonB homologs of either Pseudomonas putida or Haemophilus influenzae but did identify p otential TonB homologs in several other nonenteric gram-negative speci es, In vivo chemical cross-linking experiments demonstrated that in ad dition to TonB, auxiliary components of the TonB-dependent energy tran sduction system are broadly conserved in members of the family Enterob acteriaceae, suggesting that the TonB system represents a common syste m for high-affinity active transport across the gram-negative outer me mbrane.