大肠杆菌ArcB/ArcA二组分体对DNA复制起始的调控机制

Bacterial two-component system (TCS) is acting as sensors to help bacteria response to environmental changes via regulating transcription of a number of target genes. The ArcB/ArcA two-component system of Escherichia coli regulates gene expression in response to the redox conditions of growth. We found that deletion of the regulator gene arcA of E. coli ArcB/ArcA disturbed initiation of DNA replication; ArcA-GFP fusion protein was found to colocalize with chromosomes; the DnaK、PurL、PurA、OmpA or RpoB protein was co-immunoprecipitated with ArcA-GFP. Interestingly, all these phenomena depend on nutrition supplements; however, the mechanisms behind these observations are still unclear. Here, we plan to define (i) target genes controlled by ArcB/ArcA in a manner of nutrition-dependent, and the effects of these genes on initiation of DNA replication; (ii) the proteins which can interact with ArcA under different nutrition conditions, and effects of these proteins on initiation of replication; (iii) the binding of ArcA to the oriC region in vivo; (iv) effects of 20 amino acids and 4 deoxynucleotides on initiation of replication in arcA mutant. Finally, the results obtained can be expected to explain the mechanisms of how ArcB/ArcA regulates initiation of DNA replication.

细菌通过二组分体感受外界环境因子的变化，继而通过调节其靶基因的表达来适应环境。大肠杆菌ArcB/ArcA二组分体感知环境氧条件变化，影响一系列基因表达。本课题前期实验发现ArcB/ArcA二组分体调节基因arcA缺失显著影响DNA复制起始，用质粒表达ArcA蛋白能将∆arcA突变表型恢复为野生型表型，ArcA-GFP融合蛋白与染色体共定位，DnaK、PurL、PurA、OmpA、RpoB蛋白与ArcA免疫共沉淀。这些现象都依赖于营养条件, 但其分子机制尚不清楚。本项目拟确定由ArcB/ArcA调控的营养特异性靶基因及其对复制起始的影响方式、与ArcA相互作用的营养特异性蛋白及其对复制起始的影响、ArcA对复制原点oriC的结合；以及20氨基酸和4种核苷酸分别对∆arcA突变体复制起始的影响。这将是首次在大肠杆菌中详细探索ArcB/ArcA赖以影响DNA复制起始的营养信号及调控机制。

细菌通过二组分体感受外界环境因子的变化，继而通过调节其靶基因的表达来适应环境。大肠杆菌ArcB/ArcA二组分体感知环境氧条件变化，影响一系列基因表达。本课题发现ArcB/ArcA二组分体号转导系统以依赖营养模式影响DNA复制起始，即营养丰富时，ArcB/ArcA信号转导系统促进DNA复制起始，而外界营养较差时，ArcB/ArcA信号转导系统会抑制DNA复制起始。半胱氨酸在一定程度上补救ArcB/ArcA信号转导系统缺失所引起的染色体复制起始变化。添加脱氧核糖核苷酸可以补救ArcB/ArcA信号转导系统缺失所致的染色体复制式样的改变，其中嘧啶的效果尤其显著。用RNA-seq技术，分别检测了∆arcB和∆arcA细胞在LB或ABTGcasa培养基上基因组表达变化谱，并与野生型细胞比较分析发现，在缺失ArcB/ArcA信号转导系统时，AceF、Fis、GlnQ、RimO、Ffs和NlpD参与的细胞过程可能有助于维持正常细胞活动。综上所述，我们认为ArcB/ArcA信号转导系统感知营养信号如脱氧核苷酸，并结合调节细胞运动对环境营养做出响应，是一种营养依赖响应。这是首次在大肠杆菌中详细探索ArcB/ArcA赖以影响DNA复制起始的营养信号及调控机制。