PROJECT SUMMARY/ABSTRACT The CDC recently released a report detailing antibiotic resistant threats in the US. Of particularemphasis in the CDC report is the increased prevalence of multidrug-resistant, Gram-negative bacteria (MDR-GNB) and the need to develop the next generation of antibiotics to combat them. All Gram-negative bacteriarely on a set of homologous, yet highly-specific, outer membrane TonB-dependent transporters (TBDTs) toimport critical nutrients from their environment, especially metals like iron, which are bound by high-affinity,metal chelating compounds called siderophores. Recent antibiotic developments have shown thatsiderophore-antibiotic conjugates can be selectively targeted to specific bacteria, and that this deliverymechanism overcomes several key antibiotic resistance mechanisms. A significant limitation of this deliverysystem is the low expression levels of the TBDTs. However, a subset of these TBDTs controls their ownexpression through a cell-surface signaling (CSS) process that up-regulates their own expression. The long-term objective of this research is to understand the CSS regulatory process and manipulate TBDT expressionto enhance siderophore-antibiotic conjugate therapy for treatment of MDR-GNB infections. Research outlinedin this proposal will help elucidate the structural basis for CSS by a sigma-regulator. As a model system, thepseudobactin BN7/8 transport system of Psuedomonas putida, which consists of the TBDT, PupB, the innermembrane ?-regulator, PupR, and the cytoplasmic ?-factor, PupI, is being used. To accomplish thisproposal's objective the following three specific aims will be pursued: 1) establish that PupR anti-?-factordomain dimerization influences transcriptional activation by PupI, 2) identify the structural determinants anddelineate the role of the PupR:PupB periplasmic interactions on the stability of the PupR periplasmic C-terminal CSS domain (CCSSD), and 3) determine changes in the full-length PupB:PupR CCSSD complex inthe presence and absence of its cognate siderophore, pseudobactin BN7/8. These aims will be accomplishedusing a multidisciplinary approach; including X-ray crystallography, small-angle X-ray scattering, molecularbiology, cellular assays, and biophysical techniques such as isothermal titration calorimetry and circulardichroism spectroscopy. This research will provide critical structural information about a ?-regulator; explainhow it interacts with a ?-factor at the inner membrane, and the extent to which periplasmic conformationalchanges between the TBDT and ?-regulator lead to proteolytic degradation that is important for controllingtranscriptional activation.