假单胞菌Pseudomonas fluorescens Pf0-1中转录调控因子SpfB负调控DNA磷硫酰化修饰

The transcriptional regulator SpfB negatively regulates DNA phosphorothioate modification in Pseudomonas fluorescens Pf0-1

摘要:

[目的]DNA磷硫酰化修饰是DNA骨架上非桥接的氧原子以序列选择性和R-构型被硫取代的一种新型DNA修饰。目前，磷硫酰化修饰在多种细菌、古生菌以及人类致病菌中多有发现，但其分子调控机制尚不清楚。为了全面解析磷硫酰化修饰的调控机制，本文选择荧光假单胞菌Pf0-1为研究对象，开展了其DNA磷硫酰化修饰的调控机制研究。[方法]首先，构建了spfB基因缺失和回补菌株，使用碘能特异性断裂磷硫酰化修饰DNA的方法，研究了该基因缺失对修饰表型的影响。利用cDNA在相邻同方向的基因间隔区进行PCR，确定了磷硫酰化修饰基因簇spfBCDE内的共转录单元。通过荧光定量RT-PCR，分析了spfB基因缺失突变株中磷硫酰化修饰基因的转录量。利用异源表达并纯化得到的重组蛋白SpfB进行了体外功能研究。通过EMSA实验，验证了SpfB蛋白具有与spfB启动子序列结合活性。通过DNase I footprinting实验，精确定位了SpfB蛋白与DNA结合序列。[结果]spfB基因的缺失加剧了磷硫酰化修饰DNA断裂所致电泳条带弥散的表型，spfB基因的回补能够恢复该表型，证明spfB基因负调控磷硫酰化修饰。鉴定了spf基因簇中只含有1个共转录单元，且该共转录单元在△spfB突变株中转录水平明显上升。通过EMSA和DNase I footprint实验，检测了SpfB蛋白与磷硫酰化修饰基因spfBCDE的启动子区域5'-TGTTTGT-3'相结合。[结论]SpfB作为转录调控因子负调控磷硫酰化修饰基因spfBCDE的表达，为解析磷硫酰化修饰的调控机制和全面理解基因组上的部分修饰特征奠定了基础。

Abstract:

[Objective] DNA phosphorothioate (PT) modification, in which the sulfur replaces a nonbridging oxygen, occurs naturally in diverse bacteria, archaea and human pathogens as a new kind of epigenetic modification. However, the regulatory mechanism of PT modification has not been fully characterized. In this study, the regulatory mechanism of spfB on DNA phosphorothioate (PT) modification in Pseudomonas fluorescens Pf0-1 was demonstrated. [Methods] Firstly, the spfB genetic interruption and complementary strains were constructed by homologous recombination and then tested the modification frequency in these strains by iodine cleavage. The operons within spf gene cluster were grouped by RT-PCR and the transcriptional level was analyzed in the ΔspfB mutant by quantitative real-time PCR. Finally, the possible regulatory region of SpfB on the spf operon was characterized by EMSA and DNase I footprinting assay.[Results] The inactivation of spfB led to more dispersed small fragments in genomic DNA of ΔspfB mutant and its complementation obviously restored the phenotype of wild type strain. Genes in spf gene cluster were assigned into one co-transcription unit, and the disruption of spfB directly up-regulated the transcription of the operon. In vitro SpfB directly protected two separate sequences within the spf promoter region from DNase I cleavage, and each protected sequence contained a direct repeat (5'-TGTTTGT-3'). [Conclusion] SpfB in Pseudomonas fluorescens Pf0-1 was a negative regulator in DNA phosphorothioate modification.