DNA-utilization loci enable exogenous DNA metabolism in gut Bacteroidales

The human gut microbiome plays a central role in nutrient metabolism, yet the fate of exogenous nucleic acids within this ecosystem remains poorly understood. Here, we show that multiple Bacteroidales species efficiently metabolize exogenous DNA, with <i>Bacteroides thetaiotaomicron</i> converting it into the deaminated nucleobases uracil and xanthine. Using genetic and biochemical approaches, we identify <i>ddbABCDEF</i>, a six-gene locus encoding secreted nucleases and an outer membrane transporter, essential for exogenous DNA metabolism in <i>B. thetaiotaomicron</i>. Colonization of gnotobiotic mice with <i>ddbABCDEF</i> mutants reveals that this pathway significantly alters nucleobase pools in a gnotobiotic mouse model. Comparative genomic analyses demonstrate that <i>ddbABCDEF</i> is evolutionarily related to a natural transformation system present in Bacteroidota and has diversified into four distinct subtypes, each linked to unique DNA-processing activities in closely related gut Bacteroidales strains. These findings thus expand our understanding of DNA metabolism in the gut microbiome and reveal a distinctive pathway for nucleobase production with implications for host-microbe interactions.