Abstract
Phosphorothioate (PT) modifications of the DNA backbone, widespread in prokaryotes, are first identified in bacterial enteropathogens Escherichia coli B7A more than a decade ago. However, methods for high resolution mapping of PT modification level are still lacking. Here, we developed the PT-IC-seq technique, based on iodine-induced selective cleavage at PT sites and high-throughput next generation sequencing, as a mean to quantitatively characterizing the genomic landscape of PT modifications. Using PT-IC-seq we foud that most PT sites are partially modified at a lower PT frequency (< 5%) in E. coli B7A and Salmonella enterica serovar Cerro 87, and both show a heterogeneity pattern of PT modification similar to those of the typical methylation modification. Combining the iodine-induced cleavage and absolute quantification by droplet digital PCR, we developed the PT-IC-ddPCR technique to further measure the PT modification level. Consistent with the PT-IC-seq measurements, PT-IC-ddPCR analysis confirmed the lower PT frequency in E. coli B7A. Our study has demonstrated the heterogeneity of PT modification in the bacterial population and we also established general tools for rigorous mapping and characterization of PT modification events at whole genome level. We describe to our knowledge the first genome-wide quantitative characterization of PT landscape and provides appropriate strategies for further functional studies of PT modification.
Highlights
Phosphorothioate (PT) modification of DNA, in which the non-bridging oxygen in the phosphate moiety of the sugar-phosphate backbone is replaced by sulfur, is widespread in prokaryotes in and R-configuration and a sequence-selective manner
Phosphorothioate (PT) modification is a novel DNA modification, previous studies showed that PT modifications in E. coli occure at GpsAAC/GpsTTC motifs, but the modification frequency at each site are not known
We introduced two methods: PT-IC-seq, which could quantitatively characterize the genomic landscape of PT modifications; and PT-IC-droplet digital PCR (ddPCR), which could measure PT modification frequency precisely
Summary
Phosphorothioate (PT) modification of DNA, in which the non-bridging oxygen in the phosphate moiety of the sugar-phosphate backbone is replaced by sulfur, is widespread in prokaryotes in and R-configuration and a sequence-selective manner. The biochemical study of PT modification revealed that PT-modifying enzymes DndACDE function as a large protein complex, with DndB actings as a negative transcriptional regulator[5,6,7]. Many bacterial PT-modifying enzymes act in concert with and are encoded in close proximity to cognate restriction endonucleases DndFGH. PT modifications catalysed by PT-modifying enzyme protects DNA from digestion by DndFGH with which it forms a restriction-modification (R-M) system[8]. More than half of all PT bacterial strains lack the dndF-H restriction system in spite of containing dndA-E and PT. The fact that many strains of bacteria lack the restriction enzyme components of a typical R-M system is consistent with the idea that PT modifications and dndA-E genes provide functions other than R-M, such as control of gene expression[10]
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