FIG. 1.

Construction of strains with dnaA or dnaA46 in the chromosomal attB site. (A) The dnaA/dnaA46 gene was ligated to a nonreplicating attP-carrying cassette (bottom). Lambda Int protein-dependent site-specific recombination with the chromosomal attB site resulted in cells carrying an additional dnaA gene inserted in attB. Subsequently the dnaA gene was removed from its normal location. (B) In wild-type cells dnaA is replicated shortly after oriC. Consequently, both oriC and dnaA are hemimethylated and sequestered by the SeqA protein (•) in the same part of the cell cycle (left). In cells carrying a dnaA gene in attB only, this is replicated with significant delay relative to oriC. Consequently, the dnaA gene is not sequestered for the same part of the cell cycle as the origin (right).