Quantitative Mapping Of DNA Phosphorothioation In Bacterial Genomes

DNA phosphorothioation(PT)is a novel DNA modofication phenomenon in which a non-bridging oxygen atom of the phosphate group on the DNA backbone is replaced by sulfur atom.DNA phosphorothioation is the new DNA modification that only has been found on the DNA backbone.DNA PT modification is widespread in prokaryotes.Although the single molecule real-time sequencing(SMRT)and the deep sequencing of iodine-induced cleavage(ICDS)have been established to determine genomic PT modified sites,methods for high resolution mapping of PT modification level are still lacking.In order to further explore the PT site and its modification characteristics,we have developed the quantitative genome sequencing methods and carried out quantitative research of phosphorylation on the Escherichia coli B7 A and Salmonella enterica serovar Cerro 87 genome.First,when the sequencing depth is increased to approximately 1000 ×,23,708 out of a total of 40,701 GAAC/GTTC sites on the E.coli B7 A genome were detected as PT modified site by using ICDS resequencing.Gradient analysis of the sequencing depth revealed that the site of DNA PT modification on the E.coli B7 A genome increased with the increase of sequencing depth.If all of these deteced GAAC/GTTC PT sites are fully modified homogenously in every cell,the abundance of PT linked dinucleotides would be ~ 12 times more than which quantified by LC-MS/MS.Secondly,we developed one approach named with iodine induced cleavage-based PT sequencing(PT-IC-seq)to quantitatively determine the PT modification percentage.Compared with the ICDS,it's omitted the ligation of the unique tag at the iodine-cleavage sites in PT-IC-seq method.Both iodine-cleaved(modified)molecules and uncleaved(unmodified)molecules are equally amplified in the PCR amplication step of PT-IC-seq method.The phosphorothioation frequency of target site can be calculated by the percentage of ended reads to the total reads of this sites.The quantitative analysis of the pBlueScript SK(+)plasmid from S.enterica serovar Cerro 87 confirmed the feasibility of the PT-IC-seq method and revealed the incomplete modification of the PT modification site on this plasmid.This incomplete modification is manifested in the presence of both modified and unmodified molecules at the same site,suggesting the heterogeneity of the modifications between the plasmid molecules.In addition,the phosphorothioate level of E.coli B7 A and S.enterica serovar Cerro 87 genomic DNA was quantified using the PT-IC-seq method.It was found that both on E.coli B7 A and S.enterica serovar Cerro 87 genomes had incomplete modification characteristics.Thus,it is revealed that PT modification at the genome level has heterogeneous among different cells.Heterogeneity increase the overall population's fitness when environmental shifts are unpredictable.PT heterogeneity may also give the population some advantage in survival.It was also found that the modification frequency of PT sites in bacterial genome was relatively low.In the E.coli B7 A genome,the site with a modification frequency greater than 5% accounted for only 25.4% of all GAAC/GTTC sites in the genome,and in the S.enterica serovar Cerro 87 genome,sites with a modification frequency greater than 5% were only 11.8% of all GAAC/GTTC sites in the genome.Similar results were obtained for both bacteria,suggesting that the heterogeneity of DNA PT modification might be ubiquitous among PT modified strains.In addition,in-depth analysis of PT sites with modification frequencies more than 5% in E.coli B7 A genome and S.enterica serovar Cerro 87 genome revealed that the high frequency modification sites were distributed relatively evenly in ORF,ncRNA and non-coding regions of the genome,and did not concentrate in a functional region.Finally,the envelope of the DndCDE protein complex was resolved as C-shaped with a radius of 4.6 nm by small angle X-ray scattering.The DNA sequence bound by the DndCDE protein complex was also explored by the method of ChIP-seq.In conclusion,an important feature of phosphorothioation,heterogeneity,is revealed this study.We describe to our knowledge the first genome-wide quantitative characterization of phosphorothioation landscape.In addition,the exploration of the envolope of the DndCDE protein complex and its combined DNA sequence laid a foundation for the subsequent research on the enzyme catalytic mechanism of DNA phosphorothioation.