Functional screens of barcoded expression libraries uncover new gene functions in carbon utilization among gut Bactereroidales
A mechanistic understanding of host-microbe interactions in the gut microbiome is hindered by poorly annotated bacterial genomes. While functional genomics can generate large gene-to- phenotype datasets to accelerate functional discovery, their applications to study gut anaerobes have been limited. For instance, most gain-of-function screens of gut-derived genes have been performed in Escherichia coli and assayed in a small number of conditions. To address these challenges, we developed Barcoded Overexpression BActerial shotgun library sequencing (Boba-seq). We demonstrate the power of this approach by assaying genes from diverse gut Bacteroidales overexpressed in Bacteroides thetaiotaomicron. From hundreds of experiments, we identified new functions and novel phenotypes for 29 genes involved in carbohydrate metabolism or tolerance to antibiotics or bile salts. Highlights include the discovery of a D- glucosamine kinase, a raffinose transporter, and several routes that increase tolerance to bile salts through lipid biosynthesis. This approach can be readily applied to develop screens in other strains and additional phenotypic assay types.