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The DNA phosphorothioate (PT) modification existing in many prokaryotes, including bacterial pathogens and commensals, confers multiple characteristics, including restricting gene transfer, influencing the global transcriptional response, and reducing fitness during exposure to chemical mediators of inflammation. While PT-containing bacteria have been investigated in a variety of environments, they have not been studied in the human microbiome. Here, we investigated the distribution of PT-harboring strains and verified their existence in the human microbiome. We found over 2000 PT gene-containing strains distributed in different body sites, especially in the gastrointestinal tract. PT-modifying genes are preferentially distributed within several genera, including Pseudomonas, Clostridioides, and Escherichia, with phylogenic diversities. We also assessed the PT modification patterns and found six new PT-linked dinucleotides (CpsG, CpsT, ApsG, TpsG, GpsC, ApsT) in human fecal DNA. To further investigate the PT in the human gut microbiome, we analyzed the abundance of PT-modifying genes and quantified the PT-linked dinucleotides in the fecal DNA. These results confirmed that human microbiome is a rich reservoir for PT-containing microbes and contains a wide variety of PT modification patterns. 

Abstract DNA damage and epigenetic marks are well established to have profound influences on genome stability and cell phenotype, yet there are few technologies to obtain high-resolution genomic maps of the many types of chemical modifications of DNA. Here we present Nick-seq for quantitative, sensitive, and accurate mapping of DNA modifications at single-nucleotide resolution across genomes. Pre-existing breaks are first blocked and DNA modifications are then converted enzymatically or chemically to strand-breaks for both 3′-extension by nick-translation to produce nuclease-resistant oligonucleotides and 3′-terminal transferase tailing. Following library preparation and next generation sequencing, the complementary datasets are mined with a custom workflow to increase sensitivity, specificity and accuracy of the map. The utility of Nick-seq is demonstrated with genomic maps of site-specific endonuclease strand-breaks in purified DNA from Eschericia coli, phosphorothioate epigenetics in Salmonella enterica Cerro 87, and oxidation-induced abasic sites in DNA from E. coli treated with a sublethal dose of hydrogen peroxide. Nick-seq applicability is demonstrated with strategies for >25 types of DNA modification and damage.