DNA Phosphorothioate Modification Expands Microbial Growth Range Under Multiple Stresses

The discovery of DNA phosphorothioation?PT?modification supplements the classical theory that DNA is only composed of carbon,hydrogen,oxygen,carbon,phosphorus.DNA PT modification is a sulfur modification on the backbone of DNA introduced by five proteins DndA-E.Bioinformatic and phenotypic analysis showed that DNA PT modifications were widespread among bacteria.Furthermore,DNA PT modification was related to bacterial restriction modification,DNA methylation modification system and antioxidant function.This study focused on the mechanisms of anti-oxidation and anti-extreme environmental conditions of DNA PT modification,and explored these scientific questions:?1?Whether the antioxidation property of PT modification caused by promoting the activity of other anti-oxidation enzymes,and whether DNA PT modification system functions as an anti-oxidation system by itself;?2?Whether PT modification strains have higher tolerance to other extreme environmental conditions;?3?Could the DNA PT modification system protect DNA and protein against the oxidative damage;?4?Mechanism of the reaction of PT modification DNA with reactive oxygen.It has been found that Salmonella enterica with DNA PT modification has a survival advantage under hydrogen peroxide stress,and phosphorothioate DNA can react with oxidants such as hydrogen peroxide and peroxyacetic acid.To elucidate whether DNA PT modification is an anti-oxidation system by itself,the plasmids containing the full-length sulfur modification gene cluster?pJTU1238?or the inframe-deletion fragment of dptC-E?pJTU3527,pJTU3528,pJTU3529?were introduced into the E.coli Hpx^-and DH5?.Under hydrogen peroxide treatment,growth curves showed that the Hpx^-/pJTU1238and DH5?/pJTU1238 grew faster than the dnd^-mutants.Indicating that the DNA PT modification system functions as an anti-oxidation system,rather than regulating the recombinant enzyme or catalase/peroxidase genes.To test whether sulfur modification strains have stronger tolerance to extreme environmental conditions,E.coli wild-type strain MG1655,Hpx^-/Dnd⁺and its dnd^-mutants?dptC-E mutants?were treated with X-ray and UV radiation,acidic condition,EDTA,antibiotics or metal ions.?1?The survival rate of Dnd⁺strain is remarkably different from the dnd^-mutants in response to X-ray and UV,suggesting that DNA PT modification confers advantages under these stress conditions.?2?The growth curves showed that the sulfur modified strains had stronger tolerance to EDTA.?3?DNA PT modification bacteria was also deteced to have stronger tolerance to acidic conditions.?4?However,it was found that Dnd⁺strains have no significant tolerance to high concentrations of metal ions or antibiotics.In this study,we investigated the mechanism of antioixdation function of sulfur modification system by detecting mutation rate,clearance rate and enzyme activity.It was shown that DNA sulfur modification system can scavenge the reactive oxygen species to protect the DNA and protein.Supplementation of 0.5 mM H₂O₂ resulted in a 2-fold increased thyA mutation rate of Dnd⁺;And the thyA mutation rate of dndC^-,dndD^-and dndE^-enhanced by 20,4 and 6 folds respectively,indicated that DNA PT modification can significantly reduce the mutation rate of bacteria.To monitored the oxidative damage,the genomic DNA was isolated and purification followed with PCR analysis.It was found that the DNA damage of Dnd⁺strain was less than dnd^-mutants.Threonine dehydrogenase?TDH?activity assays showed that dnd^-strains lose 85%of the total activity while the Dnd⁺strain remains 75%of the activity,suggesting that DNA phosphorothioate modification can protect both DNA and proteins from oxidative damage.Moreover,in solution,Fenton reagent and ultraviolet radiation can lead to the generation of hydroxyl radicals,which are easily trapped in vitro and analyzed using EPR.It was detected that2.5 mM dG_SA can scavenge about 100?M of the free radicals generated by Fenton reaction,whereas dG_OA didn't have the scavenge capability under the same concentration.Hydroxyl radical in bacterial cells can also be trapped and analyzed by EPR.It was observed that a notable increase in adduct formation in dnd^-cells with the supplementation of 50?M and 75?M H₂O₂.But for the Dnd⁺strain,only extremely weak signals were detected.These suggested that DNA sulfur modification system is an active oxygen scavenging system and has a strong reactive oxygen species scavenging capability.It was used HPLC/MS to analyze the reaction of the phosphorothioate bond?dG_SA?with H₂O₂,X-ray and UV,and the reaction production were dG_OA and dG_HA.When H₂O and H₂O₂ in reaction system were replaced by H₂¹⁸O₂ and H₂¹⁸O,it was found that the oxygen atoms in the dG_OA were mainly derived from water.Based on these results,the chemical mechanism of the reaction between sulfur-modified DNA with free radicals was proposed.Overall,DNA phosphorothioate modification system functions as an antioxidation system by itself rather than by regulating other genes.Sulfur modification system can enhance the tolerance of E.coli to a variety of extreme environmental conditions.Phosphorothioation DNA can protect DNA and proteins from oxidative damage by scavenging reactive oxygen.Our findings might shed light on the physiological functions of DNA phosphorothioation system.