There are now dozens of enzymatically-installed DNA modifications – the epigenome – in all forms of life, with the greatest diversity occurring in prokaryotes and bacteriophage. While bacterial DNA modifications are best known from restriction-modification systems, they are now known to regulate gene expression. Phosphorothioation (PT) of DNA, in which a sulfur atom replaces a non-bridging oxygen in the sugar-phosphate backbone, is the only known naturally occurring DNA backbone modification and is inserted by coordinated action of 4-5 enzymes encoded by the ssp and dnd families of modification and restriction genes. Using chromatography-coupled mass spectrometry (LC-MS), we recently found that 5-10% of the bacteria in the human gut microbiome possess PTs. Here we describe efforts to define the landscape of PT-possessing gut organisms using a combination of LC-MS, new next-generation sequencing (NGS) technologies, and metagenomic analysis. Nuclease digestion and LC-MS analysis of fecal DNA isolated from 11 healthy human donors revealed 8 of the 16 possible dinucleotide contexts, with each donor possessing an average of 2-3 PT dinucleotide species and wide variation among donors. A year-long time course in one donor revealed striking changes in PT levels over time. To identify organisms possessing PTs, fecal DNA was treated with iodine to convert PTs to strand-breaks and the breaks mapped by poly-T tailing NGS and bacteria identified by metagenomic analysis referenced to sequenced genomes of &gt;9000 bacterial isolates from the gut microbiome. Ten PT sequence motifs from Bacteroidales, Clostridiales, and Enterobacterales were identified in the sequencing analysis of 3 donors. Although more studies are needed to define the determinants of the PT-containing gut microbiome, the redox-active and nucleophilic PT sulfur is known to affect bacterial fitness in the face of oxidative stress. Future studies will address the effects of inflammatory bowel disease on PT epigenetics in the human gut microbiome.

Uncovering the landscape of phosphorothioates in the human gut microbiome

ACS Fall 2021

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