Transport of iron-siderophores and vitamin B12 across the outer membrane (OM) of Gram-negative bacteria requires energy from the proton motive force delivered by the TonB/ExbB/ExbD complex. TonB-dependent OM receptors such as FhuA, FepA, FecA and BtuB possess a Ton box: a conserved motif located proximal to their N-terminus that has been shown to interact with TonB. However, other sites on OM receptors have been proposed to participate in interactions with TonB. To identify novel sites of interactions with TonB, we selected TonB-binding peptides from a random library of peptides displayed on phages. Fifteen peptides displayed sequence similarities to known TonB-dependent OM receptors. Of the fifteen, eight were mapped to regions of FhuA, FepA and FecA for which crystal structures are available. DNA sequences for selected peptides were fused to malE and displayed at the N-terminus of the E. coli maltose binding protein (MBP) for further characterization. Surface plasmon resonance experiments revealed that when the peptides were monovalently displayed on MBP, they retained TonB-binding activity thereby permitting assessments of their binding characteristics. In vitro and in vivo assays demonstrated that only FhuA-mapping peptides could disrupt TonB-FhuA interactions, indicating that TonB selectively binds to multiple regions distinct for each OM receptor.

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