Mapping PT modifications in bacteria

One microbial organism always contains multiple DNA modification systems as defense strategies or epigenetic controls. Here, we describe crosstalk between two distinct DNA modifications with different target sequence motif: DNA phosphorothioation (PT, GPSAAC/GPSTTC by DndACDE proteins) and N6-methyl-adenine methylation (m6A, G6mATC by Dam protein). By using a newly developed DNA modification sequencing method PT nick-seq, we found that half of PT modifications change from GAAC/GTTC to GATC sites after bacteria have lost Dam. Map the PT modification sites in bacteria and ∆dam mutant To further confirm this result and get a full understanding of where the new modifications are, we exploited PT-nick-seq method to get the single-base resolution of PT modification map of E.coli BW25113 and ∆dam mutant harboring S. enterica DndBCDE proteins.With PT-nick-seq technology, PT modifications on DNA were turned into strand breaks after iodine cleavage and were inferred through the higher read coverage ratio compared to their neighbor nucleotides and negative controls.To further get the PT modification site information in the same ChIP-seq samples, PT-nick-seq was adopted to sequence the genomic DNA of the same strain used for ChIP-seq experiment.