DNA phosphorothioation in Streptomyces lividans: mutational analysis of the dnd locus
DNA phosphorothioation in Streptomyces lividans: mutational analysis of the dnd locus
复制标题
浅青紫链霉菌中的 DNA 硫代磷酸化：dnd 位点的突变分析
DOI：
10.1186/1471-2180-9-41
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发表时间：
2009-02-20
期刊：
影响因子：
4.2
通讯作者：
Deng, Zixin
中科院分区：
文献类型：
--
作者：
Xu, Tiegang;Liang, Jingdan;Deng, Zixin
背景资料：一种新的DNA硫代磷酸酯修饰（DNA硫修饰），其中连接DNA核苷酸的磷酸二酯键中的一个非桥接氧原子被硫交换，被发现是由DND或DND-对应基因座在广泛的细菌从不同的栖息地遗传决定。在变铅青链霉菌中负责DNA硫代磷酸化的dnd基因座内的单个基因的详细突变分析进行，并在这里描述。结果：一个6，665 bp的DNA区域（仅含5个开放阅读框（dndA-E））被确定为S.形成硫代磷酸酯。这提供了诊断上可靠且易于测定的Dnd（DNA降解）表型。RT-PCR分析表明，dndA是独立转录的，而dndB-E是一个操纵子。一个有效的突变整合互补系统的开发，使这些dnd基因的详细功能分析。由dndA、C、D和E基因的特异性框内缺失引起的Dnd表型或由dndB的框内缺失引起的增强的Dnd表型可以通过携带相应dnd基因的表达载体恢复。有趣的是，过量的DndC或DndD，而不是其他Dnd蛋白，在体内被发现是有害的细胞violence.Conclusion：DNA硫代磷酸化是一个多酶和高度协调的过程中控制的五个dnd基因。在体内过表达的一些蛋白质阻止宿主菌株的生长，表明基因簇的表达在天然宿主中受到严格的调控。
Background: A novel DNA phosphorothioate modification (DNA sulfur modification), in which one of the non-bridging oxygen atoms in the phosphodiester bond linking DNA nucleotides is exchanged by sulphur, was found to be genetically determined by dnd or dnd-counterpart loci in a wide spectrum of bacteria from diverse habitats. A detailed mutational analysis of the individual genes within the dnd locus in Streptomyces lividans responsible for DNA phosphorothioation was performed and is described here. It should be of great help for the mechanistic study of this intriguing system.Results: A 6,665-bp DNA region carrying just five ORFs (dndA-E) was defined as the sole determinant for modification of the DNA backbone in S. lividans to form phosphorothioate. This provides a diagnostically reliable and easily assayable Dnd (DNA degradation) phenotype. While dndA is clearly transcribed independently, dndB-E constitute an operon, as revealed by RT-PCR analysis. An efficient mutation-integration-complementation system was developed to allow for detailed functional analysis of these dnd genes. The Dnd-phenotype caused by specific in-frame deletion of the dndA, C, D, and E genes or the enhanced Dnd phenotype resulting from in- frame deletion of dndB could be restored by expression vectors carrying the corresponding dnd genes. Interestingly, overdosage of DndC or DndD, but not other Dnd proteins, in vivo was found to be detrimental to cell viability.Conclusion: DNA phosphorothioation is a multi-enzymatic and highly coordinated process controlled by five dnd genes. Overexpression of some proteins in vivo prevented growth of host strain, suggesting that expression of the gene cluster is strictly regulated in the native host.