The active transport of ferric-siderophores across the outer membrane of Gram-negative bacteria is coupled to the proton motive force of the cytoplasmic membrane by the TonB energy transduction system. The cytoplasmic membrane proteins that comprise this complex system include TonB, ExbB, and ExbD. Import of the scarce ferric iron from the environment is an integral activity for these organisms and is a determinant of virulence in many bacteria. A complete understanding of the mechanistic details of this complex is the ultimate goal of our studies.

TonB appears to be energized by a heteromultimeric complex at the cytoplasmic membrane composed of ExbB and ExbD. Currently it is not known how the complex couples the energy of the proton motive force to TonB, although it is proposed to form a proton-conducting channel in the cytoplasmic membrane. Consistent with that idea, TonB responds conformationally to the proton motive force only in the presence of ExbB/ExbD. TonB and ExbD are topologically similar; each has a single N-terminal transmembrane domain with the majority of the protein exposed to the periplasm. ExbB on the other hand contains three transmembrane domains with the majority of the protein exposed to the cytoplasm. Since ExbD and TonB have short cytoplasmic domains, the major region through which they can interact with ExbB is through their transmembrane domains. Mutagenic studies of ExbB have identified a few key residues in two of its three transmembrane domains. However, the majority of ExbB consists of two soluble cytoplasmic domains including a ∼90 amino acid cytoplasmic loop between the first and second transmembrane domains and a ∼50 amino acid C-terminal tail. Nothing is known about the cytoplasmic domains of ExbB. In this study, a deletion scanning analysis was applied to the cytoplasmic loop of ExbB to determine its role in ExbB activity if any. The studies in this dissertation show that the ExbB cytoplasmic loop is essential for its role in energizing siderophore-mediated iron transport across the outer membrane. The majority of the loop deletions appear to collapse the proton motive force, which is the energy source for iron transport. Because the cytoplasmic loop domain of ExbB is also required for trimerization of ExbD in the periplasm and dissociation of the TonB C terminus from its outer membrane association, we hypothesize that under wild type circumstances signal transduction occurs from the cytoplasm to the periplasm and outer membrane through the transmembrane domains of ExbB. Each of the inactive ExbB loop deletions also form a previously undetected crosslinked complex. In summary, in this dissertation, the role of ExbB as a proton translocator is established and more specifically, evidence is presented that the cytoplasmic loop plays a role in regulating translocation of protons through ExbB.