Voogdt, C. G. P. | bioRxiv Science

bioRxiv2025

A toolkit for transposon libraries and functional genomics in intestinal Bacteroidales

Members of the order Bacteroidales include some of the most prevalent and abundant bacterial species in the healthy human gut microbiota. Yet, most of the functions encoded in their genomes remain poorly characterized, limiting our understanding of the different roles they play in the human gut microbiome. Towards addressing this gap, we developed tools and methods for genome-wide transposon mutagenesis in Bacteroidales, including broad-range transposon vectors with several antibiotic selection markers, a dual conjugation-cloning donor strain, and protocols for convenient library generation in liquid media. We then created saturated, barcoded, insertion mutant libraries in the type strains of three key representatives of the main genera within Bacteroidales: Bacteroides uniformis (ATCC 8492), Phocaeicola vulgatus (ATCC 8482) and Parabacteroides merdae (ATCC 43184). Based on the dense transposon insertion profiles and a workflow for comparing essentialomes across species, we identified 275 core essential genes shared across the three species, and 163 species-specific essential genes, some of which could be explained by functional redundancy and alternative metabolic pathways. We further identified essential non-protein coding elements and essential protein domains with known and unknown functions. Finally, using insertion directionality bias, we could map potential toxic modalities in the three genomes, including toxin-antitoxin pairs, mobile elements encoding toxic products and enzymes leading to toxic metabolic intermediates. Overall, the tools, workflows and genome-wide resources reported here expand the experimental repertoire for characterizing genes in key bacteria of the human gut microbiome, and pave the way for the establishment of similar genetic toolkits for other gut bacteria.

microbiology↗

bioRxiv2025

Establishing an in vitro pipeline for the high-throughput quantification of epithelial permeability of gut bacterial metabolites

The epithelium of the human gastrointestinal tract is key for controlling the absorption of small molecules and for forming a tight barrier between the gut microbiota and the host, thereby maintaining metabolic homeostasis. Both the microbiota and the barrier function of the intestinal epithelium have a role in pharmacokinetic variability of medical drugs. In this study, we developed a high-throughput workflow to assess the absorption of bacterially produced (drug) metabolites by intestinal epithelial cells through the combination of anaerobic bacterial cultures and human-intestine derived Caco2 cell cultures in a transwell system. To functionally monitor the barrier integrity during the experiments, we introduced a panel of marker compounds, whose concentration kinetics on either side of the epithelial monolayer indicates barrier integrity and transport. We employed this workflow to systematically probe the effect of different gut bacterial species on the epithelial absorption of 482 drugs and their 172 bacterially produced metabolites. While we could recapitulate known bacterial drug biotransformation reactions and expected drug metabolite absorption profiles, we also identified 33 new bacteria-drug pairs for which bacterial biotransformation alters epithelial permeability. Further, we combined the developed experimental workflow with untargeted metabolomics analysis to systematically study epithelial permeability of metabolites naturally produced by gut bacteria. Tracking the absorption kinetics of 397 bacterially produced metabolites revealed that the majority (>79%) of these metabolites do not pass the Caco2 monolayer, illustrating its role as a physical and metabolic barrier. In summary, we present a highly adaptable high-throughput workflow to quantitatively study the metabolic interactions at the intestinal microbiota-host interface which can impact pharmacokinetics, toxicokinetics, and human physiology.

pharmacology and toxicology↗