Joint research on regulation of chromosomal replication in bacteria

我们已经发现，dnaA基因及其结合序列（DnaA-box）在各种细菌的染色体复制起始区是保守的。这项国际联合研究的目的是通过比较枯草芽孢杆菌和大肠杆菌中DnaA蛋白和DnaA-box的作用，分析其功能的保守性和多样性。在与汉森博士和阿特龙博士（丹麦技术大学，丹麦）的合作中，我们成功地从枯草B染色体的复制起始区分离出了一个自主复制序列。最小必需片段包含由dnaA基因分开的两个DnaA-box区域。ARS的两个区域的要求和oriC质粒的低拷贝数与E. coli oriC.与Firshein博士（Wesleyan University，USA）和Messer博士（Max-Planck Institute，柏林）合作，进一步研究了体外B.subtilis oriC质粒的复制，揭示了两个DnaA盒区域通过与它们结合的DnaA蛋白之间的相互作用而相互作用。与Rasmussen博士（丹麦技术大学，丹麦）合作，我们分析了B.subtilis细胞的细胞周期控制，发现染色体复制起始的时间像大肠杆菌细胞一样受到严格调控。发现枯草B的DnaA和Dna B蛋白负责控制。与Alonso博士（马克斯-普朗克研究所，柏林）合作，建造了一个新的B。枯草杆菌突变体细胞，其中染色体复制是独立的dnaA基因，现在正在进行中，以分析的DnaA蛋白在体内的起始调节的确切作用。

We have found that dnaA gene and its binding sequences(DnaA-box)are conserved in the replication origin region of the chromosome among various bacteria. The aim of this international joint research was to analyze the functional conservation and diversity of DnaA protein and DnaA-boxes through comparative studies of their roles in Bacillus subtilis and Escherichia coli. In collaboration with Dr.Hansen and Dr.Atlung(Technical University of Denmark, Denmark), we have succeeded to isolate an autonomously replicating sequence from the replication origin region of the B.subtilis chromosome. The minimum essential fragment contained two DnaA-box regions separated by the dnaA gene. Requirements of two regions for ARS and the low copy number of the oriC plasmids are in sharp contrast to features exhibited by E. coli oriC. Further studies of replication of the B.subtilis oriC plasmids in vitro, in collaboration with Dr.Firshein (Wesleyan University, USA)and Dr.Messer(Max-Planck Institute, Berlin), revealed the interaction of the two DnaA-box regions through interaction between DnaA proteins bound to them. In collaboration with Dr.Rasmussen(Technical University of Denmark, Denmark), we have analyzed cell cycle control of B.subtilis cells and found that timing of initiation of chromosomal replication is strictly regulated like in E.coli cells. DnaA and DnaB proteins of B.subtilis were found to be responsible for the control. In collaboration with Dr.Alonso(Max-Planck Institute, Berlin), construction of a new B. subtilis mutant cell, in which chromosomal replication is independent of the dnaA gene, is now underway in order to analyze the precise role of DnaA protein in regulation of initiation in vivo.