Joint research on regulation of chromosomal replication in bacteria

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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.