Master of Science / Department of Biochemistry and Molecular Biophysics / Phillip Klebba / Iron acquisition is a component of Gram-negative bacteria pathogenesis, therefore as a form of 'nutritional immunity' host organisms sequester iron. To obtain iron bacteria secrete siderophores that scavenge iron. The E. coli outer membrane protein FepA actively transports the siderophore ferric enterobactin into the periplasm. We observe this uptake reaction by fluorescently labeling FepA in live bacteria, monitoring quenching that occurs upon binding of FeEnt, and then fluorescence recovery during transport. Energy poisons azide, arsenate, and 2,4-dinitrophenol were evaluated to determine sensitivity to known transport inhibitors. We developed and optimized methods to screen for iron transport inhibitors using a cell-based high-throughput screening platform. These inhibitors may have broad spectrum bacteriostatic antibiotic properties.

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Phillip E. Klebba / Siderophore uptake systems are one the most prominent methods of Fe³+-iron acquisition in Gram negative bacteria. The catecholate siderophore enterobactin is synthesized and utilized by many members of Enterobacteriaceae as well as several of the ESKAPE pathogens. The outer membrane (OM) transporter of ferric enterobactin (FeEnt), FepA is a ligand-gated porin (LGP) that requires interaction with the inner membrane (IM) protein TonB in order to accomplish active transport. TonB is thought to transduce the electrochemical energy created by the proton gradient across the IM to LGPs like FepA in the OM, to promote siderophore transport through their occluded channels. However, we do not yet have a clear picture of either how TonB transfers energy to FepA, or what kind of conformational changes occur in the occluding domain of FepA to allow ligand passage. The experiments described herein investigate these two questions, building on previously outlined models and observations. Using fluorescence labeling of strategically substituted cysteines in the surface loops of FepA, we unraveled a hierarchy of loop motion during binding of FeEnt to FepA. Additionally, by rendering parts of the FepA protein immobile as a result of engineered disulfide bonds, I identified residues or regions within its occluding domain that may normally unfold to open a size-specific channel for FeEnt. I also elucidated the role of the peptidoglycan polymer beneath the OM a framework for protein-protein interactions between IM and OM proteins. This includes the proposed interaction between a rotating TonB and FepA, or other LGPs, that may transfer kinetic energy to the OM transporter. The role of iron in microbial survival and pathogenesis makes iron-uptake pathways an attractive target for therapeutic intervention. Using the FeEnt-FepA uptake system as a model, we used a fluorescence based high-throughput screening method to identify novel small molecule inhibitors of TonB action in E. coli. The approach used can be potentially adopted to screen bigger chemical libraries as well as used to find inhibitors of ESKAPE pathogens that use FeEnt such as, Acinetobacter baumannii, Klebsiella pneumoniae or Pseudomonas aeruginosa. Finally, we discoverd a TonB-dependent OM transporter of heme/hemoglobin called HutA in the oligotrophic bacterium Caulobacter crescentus.

Doctor of Philosophy / Biochemistry and Molecular Biophysics / Phillip E. Klebba / The goal of the research included in this dissertation is to provide a more complete model of the role of TonB, an energy transducing protein that resides in the inner membrane and is an essential component of the iron transport of Escherichia coli under iron-starved conditions. Using fluorescent hybrid proteins, the anisotropy of TonB in the cytoplasmic membrane (CM) of Escherichia coli was determined. With the aim of understanding the bioenergetics of outer membrane (OM) iron transport, the dependence of TonB motion on the electrochemical gradient and the effect of CM proteins ExbB and ExbD on this phenomenon was monitored and analyzed. The native E. coli siderophore, enterobactin chelates Fe⁺³ in the environment and ferric enterobactin (FeEnt) enters the cell by energy- and TonB-dependent uptake through FepA, its OM transporter. The TonB-ExbBD complex in the CM is hypothesized to transfer energy to OM transporters such as FepA. We observed the polarization of GFPTonB hybrid proteins and used metabolic inhibitors (CCCP, azide and dinitrophenol) and chromosomal deletions of exbBD to study these questions. The results showed higher anisotropy (R) values for GFP-TonB in energy-depleted cells, and lower R-values in bacteria lacking ExbBD. Metabolic inhibitors did not change the anisotropy of GFP-TonB in ΔexbBD cells. These findings suggest that TonB undergoes constant, energized motion in the bacterial CM, and that ExbBD mediates its coupling to the electrochemical gradient. By spectroscopic analyses of extrinsic fluorophore labeled site-directed Cys residues in 7 surface loops of Escherichia coli FepA, binding and transport of ferric enterobactin (FeEnt) was characterized. Changes in fluorescence emissions reflected conformational motion of loops that altered the environment of the fluorophore, and we observed these dynamics as quenching phenomena during FeEnt binding and transport in living cells or outer membrane vesicles. Cys residues in each of the 7 surface loops (L2, L3, L4, L5, L7 L8, and L11) behaved individually and characteristically with regard to both fluorophore maleimide reactivity and conformational motion. Fluorescence measurements of FeEnt transport, by either microscopic or spectroscopic methodologies, demonstrated that ligand uptake occurs uniformly throughout the cell envelope, and susceptibility of FeEnt uptake to the proton ionophore m-chlorophenyl hydrazone (CCCP) at concentrations as low as 5 uM. The latter result recapitulates the sensitivity of inner membrane major facilitator transporters to CCCP (Kaback, 1974), providing further evidence of the electrochemical gradient as a driving force for TonB-dependent metal transport.