Mutations of DnaA-boxes in the oriR region increase replication frequency of the MiniR1–1 plasmid

Yuan Yao, Sukhbold Enkhtsetseg, Ingvild Odsbu, Lifei Fan, Morigen Morigen

The MiniR1-1 plasmid is a derivative of the R1 plasmid, a low copy cloning vector. Nucleotide sequencing analysis shows that the MiniR1-1 plasmid is a 6316 bp circular double-stranded DNA molecule with an oriR1 (origin for replication). The plasmid carries the repA, tap, copA and bla genes, and genes for ORF1 and ORF2. MiniR1-1 contains eight DnaA-binding sites (DnaA-boxes). DnaA-box1 is in the oriR1 region and fully matched to the DnaA-box consensus sequence, and DnaA-box8, with one mismatch, is close to the copA gene. The presence of the MiniR1-1 plasmid leads to an accumulation of the D-period cells and an increase in cell size of slowly growing Escherichia coli cells, suggesting that the presence of MiniR1-1 delays cell division. Mutations in the MiniR1-1 DnaA-box1 and DnaA-box8 significantly increase the copy number of the plasmid and the mutations in DnaA-box1 also affect cell size. It is likely that titration of DnaA to DnaA-boxes negatively controls replication of the MiniR1-1 plasmid and delays cell division. Interestingly, DnaA weakly interacts with the initiator protein RepA in vivo. DnaA regulates the copy number of MiniR1-1 as a negative factor through interacting with the RepA protein.