Identification and Characterization of the LipoproteinN-acyltransferase inBacteroides

Abstract

AbstractMembers of the Bacteroidota compose a large portion of the human gut microbiota, contributing to overall gut health via the degradation of various polysaccharides. This process is facilitated by lipoproteins, globular proteins anchored to the cell surface by a lipidated N-terminal cysteine. Despite their importance, lipoprotein synthesis by these bacteria is understudied. InE. coli, the α-amino linked lipid of lipoproteins is added by the lipoproteinN-acyltransferase Lnt. Herein, we have identified a protein distinct from Lnt responsible for the same process inBacteroides, named lipoproteinN-acyltransferase inBacteroides(Lnb). Deletion of Lnb yields cells that synthesize diacylated lipoproteins, with impacts on cell viability and morphology, growth on polysaccharides, and protein composition of membranes and outer membrane vesicles (OMVs). Our results not only challenge the accepted paradigms of lipoprotein biosynthesis in Gram-negative bacteria, but also support the establishment of a new family of lipoproteinN-acyltransferases.SignificanceBacteroidota are key members of the human gut microbiota that influence human health by degrading polysaccharides. This degradation is achieved by a suite of lipoproteins, a class of membrane protein characterized by lipidation. Lipoprotein synthesis in Bacteroidota is understudied. Here, we used a genetic screen to identify gene(s) responsible forN-acylation, the last step in lipoprotein biosynthesis. Our screen identified the lipoproteinN-acyltransferase inBacteroides(Lnb) that performs this step. We show that deletion of Lnb negatively affects cellular growth and ability to degrade polysaccharides, deepening our understanding of Bacteroidota and lipoproteins.