Physiological Functions And Dual-modification Systems Of Genome Phosphorylation

DNA macro-molecule and its epigenetic modification,as the basic genetic material of life,have always been one of the hotspots in Biology research.DNA phosphorothioate(PT)modification,a sulfur replacing the non-bridged oxygen of phosphorus,is the exclusive known natural modification on the DNA skeleton.PT modification exists widely in prokaryotes and archaea,including human pathogenic bacteria and intestinal colonies,with Rp spatial conformation in a sequence-specific manner.At present,the physiological function and action mechanism of PT modification have not been fully elucidated.Focus on the action mechanism of PT modification-dependent restriction system,we established a method for identification of DNA damage.It was found that PT restriction muntant in DNA phosphorylation restriction system was produced more than 50,000characteristic sites with the wild type and PT double knockout strains,it also found the DNA cleavage hotspots in adenylate.In order to analyze the physiological significance of PT modification to the host,a competitive growth platform of DNA phosphorylation modification bacteria based on fluorescent protein labeling was developed.It found that:(1)Under oxidative stress,PT modified strains(PT+)showed higher sensitivity to Na Cl O(Cl O~-)with PT modified mutant(PT-)strains;(2)Under the pressure of nutrition competition,the growth of PT+strain to PT-was weak at the beginning of the two strains,but after 3 days of growth competition,PT+strain gained a new growth advantage.(3)Under temperature stress and pressure,E.coli PT+strain showed stronger tolerance to high temperature;(4)In the model of competitive colonization of intestinal microorganisms in mice,PT+strain showed weak growth activity in colonization and growth,builting intestinal ulceration and transfer between organs in host mice.In order to clarify the dynamic process of PT modification and the physiological significance of stable abundance,we mapped the Genomic PT sites of Pseudomonas putida S1.The genome DNA alignment determined that the recognition motif was 5'-Gps ATC-3',which accounted for 7.33%of the total GATC of the genome,with a single-stranded distribution and mainly occurred in the protein coding region.On the other hand,this study also introduced another type of PT modification into S1strain,and realize the construction of DNA PT double modification system.The modifications are 5'-Gps ATC-3'and 5'-Gps GCC-3',expression with the radio of 1:2.92.While the modification frequency of 5'-Gps ATC-3'in the double modification system was 59%lower than that of the single modification system in S1 strain.What's more,by analyzed the functional regions of PT modification sites,it was found that the abundance of PT modification was up-regulated in 95 CDS regions.In this study,the double modification system was also analyzed by transcriptome sequencing and physiological analysis.According to the statistics of differentially expressed genes,251 genes were up-regulated in S1 double PT modification system,which mainly related to organic acid metabolism and nucleotides/small molecule binding;while 398 genes were down-regulated,which mainly related to cell membrane composition.In terms of physiological activity,the S1 double modification system showed higher sensitivity to oxidants,which confirmed that the PT modification on the genomic DNA gave the host bacteria an oxidative disadvantage and reduced the genomic stability.In summary,this study focused on the phosphorylation of genomic DNA:(1)By constructing the detection method of DNA damage site,the damage hot spot and damage frequency of PT limiting system are identified.(2)By establishing a co-culture system,we clarified the physiological significance of PT modification to host bacteria under different conditions.(3)Studied the colonization ability and growth status of PT modified strains in the intestinal tract of mice.(4)Genomic mapping and distributed characteristics a new PT modified of Pseudomonas aeruginosa S1.(5)The double expression system of DNA PT modification was constructed.And the distribution characteristics and interaction of two different PT modifications on the genome DNA were clarified.This study updated the original understanding of the dynamic stability in PT modification frequency,and laid an experimental foundation for studying the stability of host maintaining the abundance of PT modification,while provided a new platform for genomic PT modification research.(6)The effect of double system of PT modification on host bacteria transcriptome was clarified,and the apparent regulatory network of PT modification was revealed.In this study,the physiological function,epigenetic regulation mechanism and biosynthesis pathway of PT modification were comprehensively analyzed.Several technical platforms established will lay a technical foundation and provide theoretical guidance and scientific basis for the further study of PT modification,especially for PT modification,which is widely distributed in intestinal microorganisms.