Project Summary New approaches to antibiotic development are needed to combat multidrug-resistant bacteria, and especially Gram-negative pathogens. The Gram-negative outer membrane prevents most molecules from entering the cell and is one of the greatest hurdles to new antibiotic development. A remarkable strategy to overcome this permeability barrier is shown by the new clinical antibiotic cefiderocol, which binds outer membrane siderophore receptors and uses active transport systems to pull itself into the periplasm. Using energy-coupled import allows otherwise exclude molecules, like the antibacterial component of cefiderocol, to enter bacteria. Unfortunately, few molecules capable of this translocation have been identified, and we have been largely limited to using siderophore conjugates to develop these innovative Trojan horse antibiotics. Our work on the understudied class of bacteriocins called microcins provides a rare opportunity to study a broader class of antibacterials with this membrane translocation ability. Microcins are small antibacterial proteins (<10kDa) that selectively bind Gram-negative outer membrane proteins and hijack active transport processes to enter the periplasm. Microcins have are effective at controlling pathogen growth in vivo and have many characteristics that could make them attractive antibiotic scaffolds. Despite their potential value, advances in microcin biology have been impeded by the challenges of their identification and the limited characterization of the only 15 known examples. To overcome the discovery bottleneck, we developed an approach for systematic identification and validation of new microcins. We have focused our research on class II microcins due to their prevalence among Gram-negative bacteria. Coupling an in silico pipeline with a new method for microcin activity screening, we are identifying microcins across phylogenetically diverse bacteria, including phylogroups that have never been examined for microcin activity. We have already validated over 50 novel class II microcins, which is 5X what has been discovered in the past 40 years. Our new appreciation for class II microcin diversity has made plain the critical need to develop detailed knowledge of their sequence-activity relationships to empower their future use in antibiotic development. Foremost among our gaps in knowledge is our lack of understanding of microcin cell entry and the diversity of receptors that microcins can target. These factors dictate the initial step required for microcins to cross the outer membrane and are central to controlling microcin spectrum of activity. To pursue these critical gaps in knowledge, we will take advantage of the large exclusive repertoire of unrecognized class II microcins we have discovered and will (Aim 1) provide the first in-depth microcin sequence- activity study to uncover postions import for receptor binding, cell entry, and antibacterial activity, (Aim 2) reveal how sequence variations a...