Project Summary Pathogenic bacteria require iron for their survival and virulence. The opportunistic pathogen Pseudomonas aeruginosa has many mechanisms by which it can acquire iron, including ferric and ferrous iron uptake systems. However, within the host P. aeruginosa adapts to utilize heme as an iron source via the heme assimilation (has) and Pseudomonas heme utilization (phu) systems. The has operon encodes an extracellular hemophore, HasAp that scavenges heme and transfers it to the outer membrane (OM) receptor, HasR which acts as the cell surface signaling (CSS) system. The hemophore and receptor genes are downstream of an Extra Cytoplasmic Function (ECF) sigma and anti-sigma factor (HasI and S, respectively) associated with signal transduction and regulation of heme uptake. We have recently shown that in contrast to the bis-His coordination of HasR, the OM receptor PhuR has a unique His-Tyr coordination, an emerging motif in high affinity heme acquisition systems. 13C-heme isotopic labeling studies in combination with bacterial genetics suggested the PhuR receptor is the high capacity uptake receptor, with the HasR receptor acts primarily as a sensor and regulator of heme utilization. The goal of my proposal will be to determine the contributions of the Has and Phu systems to heme sensing and transport through 1) identifying the downstream targets of the HasAp-HasR cell surface signaling system (CSS) and its extra-cytoplasmic function (ECF) sigma factor HasI; and 2) determine the contributions of the HasR and PhuR receptors to heme uptake and utilization. We will utilize a combination of in vitro biochemical and spectroscopic techniques to characterize the molecular mechanism of heme regulation and uptake in combination with in vivo techniques including bacterial genetics and 13C-heme isotopic labeling coupled to LC-MS/MS to determine heme uptake in knockout and complemented strains. At the conclusion of this project I will have determined the regulatory cascade of the Has heme sensing system and the contributions of the non-redundant Has and Phu systems in heme utilization by P. aeruginosa. This study will provide a platform for the identification of novel antimicrobial therapies.