A system for transferring large genetic loci in Bacteroides enables hemicellulose utilization in Bacteroides thetaiotaomicron and characterization of a locus from an uncultivated strain

Journal article, 2026

Bacteria from the Bacteroidota phylum are well-known for their exceptional carbohydrate degradation abilities, which are closely associated with their use of polysaccharide utilization loci (PULs). The native exchange of PULs between different Bacteroidota species via horizontal gene transfer has been confirmed experimentally, and imitating such transfers in vitro can enable detailed studies of PULs from unstudied species in readily tractable hosts and the creation of novel strains. Here, we present a new set of tools and workflow to transfer large genetic loci, such as PULs, into genetically tractable Bacteroides strains. Our method uses a newly designed pICKUP plasmid and derivatives that allow site-specific genome integration and subsequent removal of the plasmid backbone. Thus, an unlimited number of loci may be transferred using the same resistance markers. We validated the pICKUP-based workflow by transfer of a previously characterized mixed-linkage beta-glucan utilization locus (MLGUL) into the genome of Bacteroides thetaiotaomicron, which enabled metabolism of mixed-linkage beta-glucan by this strain. Subsequent transfer of a PUL from an uncultivated bacterium from the bovine rumen confers the ability to metabolize certain cellooligosaccharides and allows further scrutiny of various genes in the PUL and their involvement in cellooligosaccharide metabolism. The pICKUP-based method presented here expands the toolkit for investigation of PULs and other large loci, including those from intractable or uncharacterized members of the Bacteroidota. A robust method to transfer whole PULs between species also forms the basis for creating custom-tailored PULs and resulting strains, with potential applications in improved gut health and biomass valorization processes.IMPORTANCEThe gut environment is a highly competitive niche, where dietary fiber is a major source of carbon, and polysaccharide utilization loci (PULs) encoded by Bacteroidota species are linked to their dominance in this environment. Our proposed method to study such PULs, especially from non-cultivated or unculturable strains, by transferring them into genetically tractable hosts, represents a valuable alternative to typical studies of isolated PUL-derived proteins. By transferring PULs into culturable hosts, we enable both a better understanding of the PULs' biological roles and modification of the individual encoded genes. Our results show how a single PUL transfer can confer hemicellulose-degrading ability to Bacteroides thetaiotaomicron, which is lacking in the wild-type strain. Transfer of a PUL from a distantly related strain, speculated to confer cellulose-degrading capacity, showcases that this annotation may be incorrect and indicates that the utilization of foreign genetic material depends on species relatedness in the same phylum.