Studies On The DNA Phosphorothioate Modification Systems In Extremely Halophilic Archaea

DNA phosphorothioate(PT)modification is the first reported physiological modification in the DNA double helix backbone,in which the non-bridging oxygen in the sugar-phosphate linkage is replaced by sulfur.DNA with this unusual DNA modification resulted in Tris-dependent DNA degradation(Dnd)during electrophoresis.After decades of research,PT modification is found to be governed by the five-member dndABCDE gene cluster and occurs in sequence-selective and Rp stereo-specific manner.Besides dndABCDE gene cluster,there are also another three-member cluster,dndFGH,which locates downstream of dndBCDE in many kinds of bacterial stains.According to the results of a transformation assay,dndFGH provide a genetic barrier to foreign plasmids without PT.That is,dndABCDE and dndFGH gene clusters consitute a special PT modification-based restriction-modification(R-M)system with similarity to DNA methylation-based R-M system.In addition,DNA with PT modification shows better resistance to oxidation.DNA PT is widely distributed in prokaryotes and are taxonomically unrelated with each other,represent genera of variable origins and diverse habitats.Therefore,it is hypothesized that dnd gene clusters may be transmitted by horizontal gene transfer(HGT).As a significant domain of cellular lives,Archaea is regarded as the bridge between Bacteria and Eukarya.After running BLAST search by dnd clusters sequence,we also found dnd cluster widely distributing in archaea,indicating that DNA PT modification occurs in archaea as well.Archaea is thought to be the most slowly evolving cellular life on earth,it is important for us to study the relationship between DNA PT and the evolution of prokaryotes.According to previous research,we used LC-MS/MS to detect PT modification in three stain,including salt-tolerant terrestrial archaea Haloterrigena jeotgali A29,haloalkaliphilic archaea Natronorubrum bangense JCM 10635 and an acidiphilic methanogen Methanoregula boonei 6A8.The results confirm the presence of PT modification in archaea.In this study,we also used LC-MS/MS to detect and quantify PT modification in salt-tolerant archaea Halapricum salinum JCM 19729,and we found H.salinum JCM 19729 possess d(GpsA)RP· At meantime,we applied the latest DNA sequencing technology,Single Molecule Real Time DNA sequencing(SMRT)to map PTs across the genome of Htg.jeotgali A29 andNrr.bangense JCM 10635.With the assistance of SMRT and newly designed Mass spectrometric analysis of DNA PT-Methylation double modification after Methylation by dam Methyltransferase in vitro,we confirmed that the motifs of PT modification in Htg.jeotgali A29 and Nrr.bangense JCM 10635 are both 5,-GpsATC-3,.We also focus on the horizontal gene transfer of dnd clusters among different archaea,By PEG-mediated method,we transformed recombinant plasmid which contains dnd cluster dndACDE from Htg.jeotgali A29 into a dnd-archaea Natrinema sp.CJ7.After transformation,DNA PT modification can be observed in transformants,and the contexts are as the same as that in Htg.jeotgali A29.The results confirmed that horizontal gene transfer of dnd clusters do occur among archaea.Moreover,we-verified that dndCDE are necessary for PT modification in CJ7.This study also tried to explore the influences of DNA PT modification in global gene expression and life activities of archaea.In this study,the heterologous expression of archaeal dnd gene clusters in E.coli,functional analysis of DndR protein and archaea knockout system construction have been carried on to the preliminary exploration.This experiment has provided more abundant resources and a new direction for the studies on mechanism of this unusual PT modification-based restriction-modification(R-M)systems,the role of PT modifications in gene regulation and cell physiology and the phylogeny of PT systems.