Protein-Protein Interactions Involved in Siderophore Biosynthesis and Secretion

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Most metabolic processes that support life require protein-protein interactions. Furthermore, metabolic enzymes that participate in such processes are often clustered into large complexes known as metabolons. Organization of enzymes into metabolons can enhance the efficiency of metabolic processes and can facilitate membrane localization. My laboratory studies the role of protein-protein interactions in siderophore-mediated bacterial iron uptake. Siderophores are small-molecule iron chelators that bacteria synthesize and secrete to obtain scarce iron from extracellular environment. Little is currently known about how the enzymes involved in siderophore biosynthesis are organized within a bacterial cell. We now have evidence that the enzymes involved in the biosynthesis of the E. coli siderophore enterobactin are organized as a metabolon in E. coli cells. We propose that interactions between proteins involved in enterobactin biosynthesis and secretion (i) enhance metabolic flux via substrate channeling and (ii) anchor the enterobactin metabolon to the E. coli inner membrane to facilitate secretion via direct interaction between the metabolon and an efflux transporter. We will build on our successful efforts to fully characterize the nature of interactions between enterobactin biosynthetic (Ent) enzymes using biophysical and structural approaches. We will also investigate how such protein interactions may enhance metabolic flux via substrate channeling. Knowledge gained from these experiments will allow us to identify molecular targets to disrupt Ent protein interactions and substrate channeling processes. We will study the effects of such disruptions on bacterial growth and siderophore secretion. Some E. coli strains glycosylate enterobactin prior to its secretion in order to evade host immune systems. We will study IroB, the enterobactin glycosyltransferase, to understand how its functions may be coordinated with the Ent biosynthetic machinery. In addition to our biophysical and structural approaches, we will investigate how the Ent biosynthetic machinery is localized within E. coli cells. An in vivo crosslinking approach will be used to identify protein partners in contact with the efflux transporter EntS, and to identify higher-order Ent protein complexes. In collaboration with microscopy experts, we will also use super-resolution microscopy and FRET microscopy to determine co-localization of Ent proteins in intact E. coli cells. The research proposed in this application will identify targets for either disruption or enhancement of siderophore-mediated iron uptake in E. coli cells. Small-molecule disruptors of Ent-Ent and Ent-IroB protein interactions may lead to novel antibiotics to protect poultry and livestock. Enhancement of these processes could lead to the engineering of novel bacterial strains for sustainable biomining of iron and other metals.