REGIONS OF ESCHERICHIA-COLI TONB AND FEPA PROTEINS ESSENTIAL FOR IN-VIVO PHYSICAL INTERACTIONS

The transport of Fe(III)-siderophore complexes and vitamin B-12 across the outer membrane of Escherichia coli is an active transport process requiring a cognate outer membrane receptor, cytoplasmic membrane-der ived proton motive force, and an energy-transducing protein anchored i n the cytoplasmic membrane, TonB, This process requires direct physica l contact between the outer membrane receptor and TonB, Previous studi es have identified an amino-terminally located region (termed the TonB box) conserved in all known TonB-dependent outer membrane receptors a s being essential for productive energy transduction, In the present s tudy, a mutation in the TonB box of the ferric enterochelin receptor F epA resulted in the loss of detectable in vivo chemical cross-linking between FepA and TonB, Protease susceptibility studies indicated this effect was due to an alteration of conformation rather than the direct disruption of a specific site of physical contact, This suggested tha t TonB residue 160, implicated in previous studies as a site of allele -specific suppression of TonB box mutants, also made a conformational rather than a direct contribution to the physical interaction between TonB and the outer membrane receptors, This possibility was supported by the finding that TonB carboxyl-terminal truncations that retained G ln-160 were unable to participate in TonB-FepA complex formation, indi cating that this site alone was not sufficient to support the physical interactions involved in energy transduction, These studies indicated that the final 48 residues of TonB were essential to this physical in teraction, This region contains a putative amphipathic helix which cou ld facilitate TonB-outer membrane interaction, Amino acid replacements at one site in this region were found to affect energy transduction b ut did not appear to greatly alter TonB conformation or the formation of a TonB-FepA complex, The effects of amino acid substitutions at sev eral other TonB sites were also examined.