Abstract Lipoproteins (LPs) play a substantial role in orchestrating symbiosis between commensal gut Bacteroides and their host and dictate survival in the intestine, yet little is known about LP synthesis or acylation state in these bacteria. Bacteroides are abundant in the human gut and communicate with the host via cell surface and outer membrane vesicle (OMV)-packaged LPs. LPs are critical for the construction and integrity of the cell envelope, and act as virulence factors in extraintestinal pathogens like Bacteroides fragilis, and critical players in nutrient uptake in symbionts like Bacteroides thetaiotaomicron (B. theta). The long-term goal of our research is to understand fundamental aspects of Bacteroides physiology that facilitate adaptation to the host. Bacteroides synthesize 2-3x as many LPs as many bacteria yet are missing recognizable homologs of key enzymes required for LP maturation in other bacteria. Via genetic screen we identified a gene conserved within gut Bacteroides that encodes a domain of unknown function (DUF4105) at its N-terminus and transmembrane domain at its C- terminus and performs N-acylation of diacylated LPs. This Bacteroides Lipoprotein N-acyltransferase (BLnt) complements lnt depletion in E. coli. BLnt is encoded downstream of a conserved gene encoding homology to the alkaline phosphatase (AP) superfamily. B. theta ∆Blnt/AP mutants produce diacylated LP and display growth deficiencies, altered cell morphology, and decreased OMV production suggesting this is a key fitness factor. Our objective is to determine how BLnt performs N-acylation, and if deficiencies in BLnt/AP affect LP presentation, cell morphology and interactions with the immune system. In Aim 1 we will determine the substrate specificity, protein structure and localization of BLnt. Our working hypothesis is that BLnt performs N- acylation of diacylated lipoproteins at the inner membrane. We will determine BLnt location and potential protein partners, such as AP, via fractionation and co-immunoprecipitation. Mass spectrometry will be employed to confirm the acylation state of endogenous B. theta proteins in wild type (WT), ∆BLnt and ∆BLnt/AP cells. We will assay BLnt activity to identify its phospholipid substrate and determine its crystal structure. In Aim 2 we will measure how BLnt influences cell shape, LP presentation, and immune activation. Our working hypothesis is that loss of BLnt results in defects at the cell surface that influence growth and morphology. Using high-content quantitative microscopy, we will measure cell shape and the abundance of surface proteins in WT, ∆BLnt and ∆BLnt/AP strains. We will measure the interaction of WT, mutant cells and their OMVs with macrophages and HEK cells expressing various Toll-like receptors. The successful completion of these aims will reveal fundamental details about Bacteroides LP maturation via a novel N-acyltransferase and provide a comprehensive picture of how LP synthesis influences interacti...