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

Abstract

Abstract Background The MiniR1–1 plasmid is a derivative of the R1 plasmid, a low copy cloning vector. Results 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. Conclusion DnaA regulates the copy number of MiniR1–1 as a negative factor through interacting with the RepA protein.

Keywords

• MiniR1–1 replication
• DnaA-boxes
• Complete genome sequence
• E. coli Cell cycle