Determination of the minimum domain II size of Escherichia coli DnaA protein essential for cell viability.

The DnaA protein is the bacterial initiator of replication at a unique chromosomal site, oriC. It is present in all bacterial species and has a conserved structure with four domains. The structures of domains I and III-IV have been solved recently for some bacterial species, and the molecular process leading to the initiation event has been investigated in detail. On the other hand, domain II appears to have no rigid structure and is assumed to be a flexible linker connecting the N-terminal domain I and the C-terminal domains III-IV. It differs significantly in length and amino acid sequence among bacterial species. Whether or not domain II has any function(s) to initiate replication is unknown. The precise borders at both of its ends as well as its essential portions for cell viability are also unknown. In this study, we introduced systematic deletions into the domain II region on the chromosomal dnaA gene of Escherichia coli and examined their effect on cell physiology. Stretches of 30-36 consecutive amino acid residues could be deleted from various portions between the 78th and the 136th residues without affecting cell viability. We propose that domain II of E. coli DnaA is from the 79th to the 135th residues and at least 21-27 residues are required as a spacer to keep domains I and III-IV in the correct positions.