Project Summary The long term goal of the application is to understand why and how bacterial lipoproteins in the medically important Firmicutes phylum undergo structural modifications. Lipoproteins are membrane associated proteins tethered to the bacterial surface through an acylated N-terminal cysteine anchor. They are ubiquitous cell envelope structures in both gram-positive and gram-negative bacteria, playing key roles in nearly every aspect of bacterial cell envelope physiology. Due to their functional importance, abundance, universal distribution, and the structurally unique acylated N-terminal cysteine, the innate immune system detects bacteria by binding the N-terminus of lipoproteins using the Toll-like receptor 2 (TLR2) family. TLR2 activation triggers a pro- inflammatory cytokine/chemokine response to clear bacteria as well as to orchestrate humoral immunity. However, there is emerging evidence that acylation patterns in lipoproteins from the Firmicutes phylum are not canonical TLR2 ligands nor are they static in structure. Firmicutes synthesize lipoprotein chemotypes varying in acyl chain number, length, and attachment position using an array of accessory lipoprotein biosynthetic genes. Gene distribution varies at both the genera and species level, and can even differ at the strain level due to circulating plasmid/transposon encoded lipoprotein remodeling genes. Lipoprotein composition is also dynamically regulated by the growth environment, including copper which has been shown to induce expression of certain N-terminal modifying genes. This project aims to uncover the physiological need(s) for distinct lipoprotein N-terminal modification systems and to characterize the enzymes involved. While select lipoprotein structural modifications confer TLR2 evading capabilities in host associated bacteria, lipoprotein N-terminal acylation/acetylation is also found in environmental lineages which suggests a broad selective pressure. Given copper co-induces both lipoprotein N-terminal modification and copper resistance genes, it has been proposed that copper directly binds to the free N-terminus and inhibits cell growth unless N-modified. This project will investigate the impact of N-terminal lipoprotein modifications on copper binding by measuring growth under copper challenge conditions requiring specific lipoprotein functions, using Tn-seq to compare chemotype-specific copper sensitization gene networks, and assaying oxidative damage to model lipopeptides isolated from both actively respiring cells and under in vitro reaction conditions. In the second aim, the recently discovered lipoprotein N-acylating enzymes LnsAB from Staphylococcus aureus will be reconstituted. Targeted mutations, acyl chain donor, and possible protein-protein complex formation will be examined using genetic and biochemical approaches. The final part of the project will examine how N-acetylated lipoproteins are made using genetic transposon screens and biochemical assay...