Exploration Of The Antioxidation Function Of DNA Sulfur Modification In Streptomyces Lividans

DNA sulfur modification is a DNA phosphate backbone modification,in which a non-bridging oxygen atom is replaced by a sulfur atom.A five-gene cluster named dnd gene cluster is responsible for DNA sulfur modification.The five genes consist of dndA,dndB,dndC,dndD and dndE.dndB-E constitute an operon.This operon and dndA transcribe divergently.dnd gene cluster is widespread in different species of bacteria,including marine bacteria,the bacteria dwelling in soil,and human pathogens.Currently,our knowledge of the physiological functions of DNA sulfur modification remains limited.It was proved to be involved in a new restrictionmodification process.This research focuses on the antioxidation function of DNA sulfur modification in Streptomyces lividans.Xie et al.found that the facultative anaerobe S.enterica strain with DNA sulfur modification is resistant to hydrogen peroxide damage.Meanwhile,sulfur modified DNA can react with peroxides such as hydrogen peroxide,and reduce them.However,these questions remains to be solved: 1,does other species of bacteria possess the antioxidation function of DNA sulfur modification? 2,does DNA sulfur modification affect gene transcription? 3,does DNA sulfur modification scavenge peroxides through activating transcription of antioxidation genes,including catalases and peroxidases ? 4,is the transcription of dnd genes responsible for DNA sulfur modification subject to oxidant regulation? 5,if transcription of dnd genes is subject to regulation of some oxidant,what is the corresponding mechanism?This research found that the survival rate of the wild type strain Streptomyces lividans 1326 was two times higher than that of the whole dnd gene cluster deletion mutant HXY6 upon treatment with 20 mM hydrogen peroxide.The survival rate of S.lividans 1326 was ten-fold higher than that of HXY6 upon treatment with 290 ?M peracetic acid(PAA)or 20 mM cumene hydroperoxide(CHP).Because S.lividans isaerobic and Gram-positive bacteria,it indicates that DNA sulfur modification is also an antioxidation system in aerobic and Gram-positive bacteria.We characterized two DNA sulfur modified sites within the dnd gene cluster in S.lividans genome,using Southern blotting combined with peracetic acid cleavage.By mutating the two modified sites and analyzing the reporter catechol-2,3-dioxygenase activity,we found that the two modification sites can not affect the expression of dndB gene.We also compared the transcriptome of 1326 and HXY6,and found that DNA sulfur modification can not activate antioxidation genes.Meanwhile,we assayed the induction level of genes responsible for scavenging peroxides to know the peroxides concentration in the cell,finding that the induction level of these genes(including 5catalases,2 alkyl hydroperoxide reductases and 1 organic hydroperoxide resistance protein)in HXY6 was higher than that in 1326.The results suggested that the peroxides concentration in HXY6 was higher than that in 1326.It also indicated that the ability of HXY6 to scavenge peroxides was weaker than that of 1326.The in vivo and in vitro experiments showed that the ability of 1326 to scavenge hydrogen peroxide and PAA was more rapidly than that of the DNA sulfur modification deficient mutant strain HXY6.These observations suggested that DNA sulfur modification is itself a novel antioxidation system in S.lividans.The transcriptional expression of most antioxidation systems is activated by the oxidant scavenged by respective antioxidant genes.Treating the wild type strain using paraquat,hydrogen peroxide,diamide,CHP and PAA,we found that diamide can up-regulate the trancription of dnd gene cluster to 2.5-fold.The transcriptional start sites of dndA and dndB genes in S.lividans 1326,and the regulatory elements in dndB promoter region were analyzed.A negative regulation sequence(r repeat)and a positive regulation sequence(R repeat)were found in the promoter region of dndB gene through deletion,mutagenesis analysis of promoter region and the assay of activity of catechol-2,3-dioxygenase encoded by the reporter gene xylE.Transcription of dnd genes was not activated by diamide afterdeletion of dndB gene.The potential underlying mechanism might be: a reduced form of DndB binds r repeat,suppressing transcription of dndB;diamide oxidizes DndB and then releases it from r repeat,allowing dnd transcription.An antioxidation system should suffice the following requirements: 1,it is resistant to some oxidant;2,it can scavenge this oxidant;3,transcription of the responsible genes is regulated by oxidant.Overall,our results support the point that DNA sulfur modification is an antioxidation system in S.lividans and pave the way for further biochemistry study on the antioxidation function of DNA sulfur modification.