A versatile genetic toolbox for Prevotella copri enables studying polysaccharide utilization systems.

Prevotella copri is a prevalent inhabitant of the human gut and has been associated with plant-rich diet consumption and diverse health states. The underlying genetic basis of these associations remains enigmatic due to the lack of genetic tools. Here, we developed a novel versatile genetic toolbox for rapid and efficient genetic insertion and allelic exchange applicable to P. copri strains from multiple clades. Enabled by the genetic platform, we systematically investigated the specificity of polysaccharide utilization loci (PULs) and identified four highly conserved PULs for utilizing arabinan, pectic galactan, arabinoxylan, and inulin, respectively. Further genetic and functional analysis of arabinan utilization systems illustrate that P. copri has evolved two distinct types of arabinan-processing PULs (PULAra ) and that the type-II PULAra is significantly enriched in individuals consuming a vegan diet compared to other diets. In summary, this genetic toolbox will enable functional genetic studies for P. copri in future.

Anaerobic Respiration of NOX1-Derived Hydrogen Peroxide Licenses Bacterial Growth at the Colonic Surface.

The colonic microbiota exhibits cross-sectional heterogeneity, but the mechanisms that govern its spatial organization remain incompletely understood. Here we used Citrobacter rodentium, a pathogen that colonizes the colonic surface, to identify microbial traits that license growth and survival in this spatial niche. Previous work showed that during colonic crypt hyperplasia, type III secretion system (T3SS)-mediated intimate epithelial attachment provides C. rodentium with oxygen for aerobic respiration. However, we find that prior to the development of colonic crypt hyperplasia, T3SS-mediated intimate attachment is not required for aerobic respiration but for hydrogen peroxide (H2O2) respiration using cytochrome c peroxidase (Ccp). The epithelial NADPH oxidase NOX1 is the primary source of luminal H2O2 early after C. rodentium infection and is required for Ccp-dependent growth. Our results suggest that NOX1-derived H2O2 is a resource that governs bacterial growth and survival in close proximity to the mucosal surface during gut homeostasis.