Phosphorothioate DNA modification by BREX Type 4 systems in the human gut microbiome

Among dozens of microbial DNA modifications regulating gene expression and host defense, phosphorothioation (PT) is the only known backbone modification, with sulfur inserted at a non-bridging oxygen by dnd and ssp gene families. Here we explored the distribution of PT genes in 13,663 human gut microbiome genomes, finding that 6.3% possessed dnd or ssp genes predominantly in Bacillota, Bacteroidota, and Pseudomonadota. This analysis uncovered several putative new PT synthesis systems, including Type 4 Bacteriophage Exclusion (BREX) brx genes, which were genetically validated in Bacteroides salyersiae. Mass spectrometric analysis of DNA from 226 gut microbiome isolates possessing dnd, ssp, and brx genes revealed 8 PT dinucleotide settings confirmed in 6 consensus sequences by PT-specific DNA sequencing. Genomic analysis showed PT enrichment in rRNA genes and depletion at gene boundaries. These results illustrate the power of the microbiome for discovering prokaryotic epigenetics and the widespread distribution of oxidation-sensitive PTs in gut microbes.