Occurrence, evolution, and functions of DNA phosphorothioate epigenetics in bacteria

Significance Phosphorothioate (PT) modification of the DNA sugar-phosphate backbone is an important microbial epigenetic modification governed by DndABCDE, which together with DndFGH, constitutes a restriction-modification system. We show that up to 45% of 1,349 identified bacterial dnd systems exhibit the form of solitary dndABCDE without the restriction counterparts of dndFGH. The combination of epigenomics, transcriptome analysis, and metabolomics suggests that in addition to providing a genetic barrier against invasive DNA, PT modification is a versatile player involved in the epigenetic control of gene expression and the maintenance of cellular redox homeostasis. This finding provides evolutionary and functional insights into this unusual epigenetic modification. Our results imply that PT systems might evolve similar to other epigenetic modification systems with multiple cellular functions. The chemical diversity of physiological DNA modifications has expanded with the identification of phosphorothioate (PT) modification in which the nonbridging oxygen in the sugar-phosphate backbone of DNA is replaced by sulfur. Together with DndFGH as cognate restriction enzymes, DNA PT modification, which is catalyzed by the DndABCDE proteins, functions as a bacterial restriction-modification (R-M) system that protects cells against invading foreign DNA. However, the occurrence of dnd systems across a large number of bacterial genomes and their functions other than R-M are poorly understood. Here, a genomic survey revealed the prevalence of bacterial dnd systems: 1,349 bacterial dnd systems were observed to occur sporadically across diverse phylogenetic groups, and nearly half of these occur in the form of a solitary dndBCDE gene cluster that lacks the dndFGH restriction counterparts. A phylogenetic analysis of 734 complete PT R-M pairs revealed the coevolution of M and R components, despite the observation that several PT R-M pairs appeared to be assembled from M and R parts acquired from distantly related organisms. Concurrent epigenomic analysis, transcriptome analysis, and metabolome characterization showed that a solitary PT modification contributed to the overall cellular redox state, the loss of which perturbed the cellular redox balance and induced Pseudomonas fluorescens to reconfigure its metabolism to fend off oxidative stress. An in vitro transcriptional assay revealed altered transcriptional efficiency in the presence of PT DNA modification, implicating its function in epigenetic regulation. These data suggest the versatility of PT in addition to its involvement in R-M protection.

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