Salmonella enteric硫修饰蛋白DptC半胱氨酸定点突变对Dnd表型的影响

Mutagenesis of cysteine residues in dptC from Salmonella enteric serovar Cerro 87 and its effects on DNA phosphorothioate modification

【目的】Salmonella enterica serovar Cerro 87是具有硫修饰现象即Dnd表型的细菌之一,其硫修饰受dptBCDE基因簇控制,将dptBCDE克隆、转化Escherichia coli DH10B后,可赋予后者Dnd表型,而当其中dptC缺失后,Dnd表型随之丧失。本文探索S.enterica serovar Cerro 87硫修饰基因dptC在硫修饰发生过程中的作用。【方法】将DptC中6个半胱氨酸(Cys)在DNA水平上逐个定点突变,转化携带dptBCDE及其衍生系列的质粒至E.coli DH10B,检测相应受体菌Dnd表型。【结果】在DptC的6个Cys中,除C39外,其余5个突变均可导致Dnd表型丧失,说明DptC中C146、C262、C273、C280和C283均与硫修饰有关。生物信息学分析表明,这5个Cys在硫修饰细菌科属间高度保守,佐证了S.enteric DptC中这5个Cys与硫修饰有关的结论。【结论】S.enteric DptC中C146、C262、C273、C280和C283任何一个的突变,都会导致受体菌Dnd表型丧失。该结果为进一步探索DNA硫修饰的发生机制提供了线索。

[Objective]DNA phosphorothioate modification（DNA sulfur modification,a non-bridging oxygen swapped with a sulfur） exists in diverse bacteria.Salmonella enterica serovar Cerro 87 is one of the bacteria that harbor the DNA sulfur modification.The modification is carried out by the products of a four-membered gene cluster,dptBCDE.Transformation of Escherichia coli DH10B with the dptBCDE gene cluster endows the strain with DNA sulfur modification capability.Deletion of dptC abolished the modification.Here,we studied the function of dptC in DNA sulfur modification.[Methods] Six cysteine residues in dptC were mutated individually within the dptBCDE gene cluster.Mutants were then tested for DNA sulfur modification.[Results] Among the 6 cysteine mutations（C39,C146,C262,C273,C280,and C283）,5 abolished DNA modification except for C39,suggesting that C146,C262,C273,C280,and C283 are essential for DNA sulfur modification.Sequence alignment shows that these five cysteine residues are conserved among different strains.[Conclusion] Mutation at anyone of C146,C262,C273,C280 and C283 of dptC abolished DNA modification.Our results shed light on further study of DNA sulfur modification biochemical pathway.