Barcoded overexpression screens in gut Bacteroidales identify genes with new roles in carbon utilization and stress resistance

Abstract

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 gene discovery, their applications to study gut anaerobes have been limited. For instance, most gain-of-function screens of gut bacterial genes have been performed in an aerobic host and included a small number of conditions. To address these challenges, we developed a strategy to barcode expression libraries for high-throughput interrogation of gene functions in competitive fitness assays. We demonstrate the power of this approach to uncover novel phenotypes for uncharacterized genes using pooled libraries constructed from a diverse set of gut Bacteroidales expressed in Bacteroides thetaiotaomicron. We identified new roles in carbohydrate metabolism for nine proteins, including enzymes, transporters, a regulator, and hypothetical proteins from mobile genetic elements. This approach can be readily applied to other organisms and additional phenotypic assays.

A Targeted Genome-scale Overexpression Platform for Proteobacteria

“…In this scenario, Dub-seq or promoter recombineering strategies would be advantageous, as Dub-seq expresses genes in trans and recombineering can be used to introduce more subtle cis mutations that could avoid perturbing upstream genes. Dub-seq can also introduce heterologous DNA from other species for phenotyping 14 , something CRISPRtOE is not designed to accomplish.…”

“…For instance, trackable multiplex recombineering (TRMR) is a targeted approach that uses recombinaseaided DNA insertion (recombineering) to deliver barcoded promoters or ribosome binding site variants to systematically alter gene expression 11,12 ; however, this method is restricted to bacteria with efficient recombineering systems. An innovative new OE technique, "Dub-seq," uses random genomic fragments cloned into multi-copy plasmids that can be quantified by barcode sequencing 13,14 , reducing upfront barriers to library design, but complicating downstream analysis and sharing some downsides with other plasmid-based approaches.…”

“…For instance, trackable multiplex recombineering (TRMR) is a targeted approach that uses recombinase-aided DNA insertion (recombineering) to deliver barcoded promoters or ribosome binding site variants to systematically alter gene expression 11,12 ; however, this method is restricted to bacteria with efficient recombineering systems. An innovative new OE technique, “Dub-seq,” uses random genomic fragments cloned into multi-copy plasmids that can be quantified by barcode sequencing 13,14 , reducing upfront barriers to library design, but complicating downstream analysis and sharing some downsides with other plasmid-based approaches. Moreover, the length of cloned genomic fragments dictates Dub-seq library screening results; smaller fragments may miss genes that only have phenotypes when co-expressed in operons (e.g., protein complex subunits) while longer fragments make it more difficult to pinpoint individual genes responsible for the selected phenotype.…”

Shaping human gut community assembly and butyrate production by controlling the arginine dihydrolase pathway

“…Bottom-up assembly enables a deeper understanding of the contributions of monoculture growth and inter-species interactions in response to environmental factors. While we focused on a single metabolic pathway, higherthroughput genetic approaches could also be used to discover and rank the influence of diverse molecular pathways on target community functions [59][60][61] . Our strategy could be applied to the pathway hits identified via high-throughput genetic pipelines to understand their quantitative effects and develop the capability to tune these pathways to precisely control community behaviors.…”

Many Families of Lids for TonB-dependent Transporters inBacteroides

“…Members of the SusC family work together with an accessory lipoprotein from the SusD family that is on the outer side of the outer membrane. In most cases, SusD proteins help to bind the substrate and are required for the receptors’ activity (Pollet et al 2021), but there are exceptions (Phansopa et al 2014; Huang et al 2022; Tauzin et al 2022). Structural studies show that SusD proteins bind to the outer face of the corresponding TBDT to enclose a large cavity, like a lid (Glenwright et al 2017; Madej et al 2020; Gray et al 2021).…”