Research On The Occurrence,Evolution And Function Of DNA Phosphorothioate Modification Systems

Phosphorothioate(PT)modification is a newly identified physiological modification on the DNA sugar-phosphate backbone in which the non-bridging oxygen is replaced by sulfur forming phosphorothioate diester bond.dndA,dndB,dndC,dndD and dndE are responsible for PT modification,five proteins coded by dndABCDE coordinate with each other in vivo to insert sulfur into DNA backbone in a sequence-specific and stereo-specific manner.In some of the bacteria carrying DNA PT modification,a restriction gene cluster dndFGH was found locating next to the modification cluster dndBCDE constituting a restriction-modification(R-M)system which can stabilize parasitifer's genetic material by modifying its own DNA and cut foreign DNA without PT modification.In prokaryote,some of the methyltransferases do not constitute R-M system with restriction endonuclease but establish orphan methylation system instead.Orphan methyltransferase Dam and CcrM in bacteria participate many cellular progresses including DNA replication initiation and regulation of gene expression.Epigenetics in prokaryote is involved in DNA methylation dominantly,and the frequency and distribution of methylation sites in DNA sequence could regulate the transcriptional level of genes locating downstream.This remind us of the orphan PT system without restriction gene cluster,does orphan PT system possess similar physiological signification with orphan methylation system?Although DNA PT modifications occur widely,the origination,evolution and development of PT systems across bacterial genomes is poorly understood.Here,we present the results of a comparative genomic survey of?480 PT systems which are gained by blasting DndBCD sequence of Salmonella enteric serovar Cerro 87 against various database in NCBI.More than half of PT systems are orphans without associated DndFGH.PT evolution clearly included horizontal gene transfer events,and orphan PT systems might represent a degradation of complete PT R-M pairs.Phylogenetic analysis revealed the coevolution of PT M and R counterparts.Surprisingly,few hybrid functional PT systems assembled from distantly related M and R components of different organisms were observed.Pseudomonas fluorescens pf0-1,carrying orphan PT system,was selected as object.First,we conducted whole genome PT sites analysis with the wild type by single molecular real time(SMRT)sequencing technology and mapped all the PT sites across its genome followed by classification and analysis of PT sites in ORFs,RNA genes,noncoding region and promoter region.Second,RNA-seq and metabolome analysis between wild type and PT modification deleted mutant(TT-5)were conducted.Only ten genes,including dndBCDE,were detected with change in transcription level by RNA-seq,however,none of them contain PT site in their promoter region.Genomic PT mapping along with the identification of global transcriptional change by RNA-seq suggest that orphan PT systems seem not directly involved in the regulation of global gene expression.Global metabolome analysis between wild type and TT-5 revealed level change of many metabolites related to oxidation stress resistance as the consequence of PT loss,which demonstrate that DNA PT modification acts as one of the important antioxidants to help its host adapt oxidation environment.