Antibiotic resistance among bacterial pathogens remains one of the great challenges confronting public health in the world today. The widespread use of antibiotics has facilitated the rise of multi-drug resistant pathogens that threaten to undermine the remarkable success of modern medicine. The Centers for Disease Control and Prevention have identified multi-drug resistant enterococci as a “Serious Threat” requiring prompt and sustained activity to limit proliferation. Daptomycin is a frontline antibiotic with efficacy against Gram positive organisms and is used with increasing frequency against multi-drug resistant enterococci such as vancomycin-resistant enterococci (VREs). The goal of this proposal is to comprehensively map the evolutionary trajectories leading to DAP resistance in Enterococcus faecium and elucidate how the identified changes in protein structure-function establish the physicochemical basis for the observed resistance phenotypes. We use quantitative experimental evolution in a novel, continuous culture bioreactor system to identify and rank the most important evolutionary trajectories leading to resistance. Based upon these results, we then characterize the most relevant proteins and pathways to daptomycin resistance using a combination of biochemical and structural approaches that link the change in biophysical properties to resistance. Techniques include X-ray crystallography, enzyme activity, ligand affinity, protein stability studies, RNAseq, qPCR, and others. This approach seeks to determine, not only the biochemical basis for resistance, but also those candidate proteins and pathways that would be well suited for the development of a new class of co-drugs that would target and delay the development of resistance. Our studies can also provide valuable molecular indicators of emerging resistance. Using our expertise in experimental evolution, we have also developed a new approach to harness both the power of evolution and the largely unexplored biochemical diversity and killing strategies of one of Nature's best antibiotic producing organisms: Streptomyces. We use experimental evolution within micro-emulsion droplets to produce selection conditions to identify variants of S. roseosporus (the “Predator”) that have improved their ability to kill a VRE strain (the “Prey”). Within each micro-droplet, we trap the two populations (Predator and a Prey). If the Predator can adapt to kill the Prey, the adapted Predator has a significant resource advantage, and increased reproductive success, over the un-adapted Predator. Taken together, this project takes a multi-pronged approach to uncovering the mechanisms and physicochemical basis for the evolution of antibiotic resistance and extends experimental evolution to include a novel method for discovering new antimicrobials.