Bacterial resistance to antimicrobials is among the biggest challenges affecting human health. Manyresistance mechanisms developed by bacteria involve adaptation of cell wall components that lead todecreased interaction with, or permeability to antimicrobial compounds. One such mechanism uses aminoacids (aa), carried by tRNAs, to aminoacylate phosphatidylglycerol (PG), one of the main constituents of thebacterial membrane. Since the initial discovery of this pathway, studies have shown that lipid aminoacylationenzymes (more generally referred to as aminoacyl-phosphatidylglycerol synthases; aaPGSs) are surprisinglydiverse, and have several aa in their repertoire for altering the chemical makeup of bacterial membranes.Numerous studies have demonstrated that PG aminoacylation enhances bacterial resistance to antibiotics thattarget the membrane (such as cationic antimicrobial peptides, CAMPs), as well as the virulence of pathogensfrom multiple phyla (i.e., Firmicutes and Proteobacteria). However, lipid modification systems have beenlargely ignored in the Actinobacteria, an important bacterial phylum that includes pathogens of majorimportance to human health, such as the etiological agents of tuberculosis, diphtheria, nocardiosis, andactinomycosis. An extensive genome analysis showed that bacteria in this phylum frequently harbor multiple(up to six) aaPGS homologs, suggesting that Actinobacteria display a high level of functional diversity in theirlipid modification systems. Our preliminary studies validated this hypothesis, leading to discovery of severalnovel lipid modifications systems in corynebacteria that are important for antimicrobial resistance andvirulence. A multitude of other lipid-modifying enzymes in Actinobacteria are yet to be described, and wehypothesize that some of these proteins support novel functions as well. These studies are important becauseaaPGSs represent general factors for membrane adaptation and are expressed in species of extremeimportance to human health. Identification of novel lipid modifications will not only increase our understandingof fundamental bacterial defense mechanisms, but will reveal new targets for development of antimicrobialcompounds. Our long-term goal is to define the repertoire of lipid modifications across bacterial species and tocharacterize their role in cellular physiology, antimicrobial resistance, and pathogenesis. The aims of thisproposal, which will lay the groundwork for future studies, are to i) explore aaPGS homolog diversity inActinobacteria and characterize the biochemical functions of representative enzymes from human pathogensin the genera Mycobacterium, Nocardia, Rhodococcus, and Streptomyces; and ii) determine the role of theseproteins in resistance to CAMPs of the human immune system and other antimicrobial agents.