DnaA boxes are important elements in setting the initiation mass of Escherichia coli.

The binding of DnaA protein to its DNA binding sites-DnaA boxes-in the chromosomal oriC region is essential for initiation of chromosome replication. In this report, we show that additional DnaA boxes affect chromosome initiation control, i.e., increase the initiation mass. The cellular DnaA box concentration was increased by introducing pBR322-derived plasmids carrying DnaA boxes from the oriC region into Escherichia coli and by growing the strains at different generation times to obtain different plasmid copy numbers. In fast-growing cells, where the DnaA box plasmid copy number per oriC locus was low, the presence of extra DnaA boxes caused only a moderate increase in the initiation mass. In slowly growing cells, where the DnaA box plasmid copy number per oriC locus was higher, we observed more pronounced increases in the initiation mass. Our data clearly show that the presence of extra DnaA boxes increases the initiation mass, supporting the idea that the initiation mass is determined by the normal complement of DnaA protein binding sites in E. coli cells.