State Key Laboratory of Reproductive Regulation & Breeding of Grassland Livestock,School of Life Sciences, Inner Mongolia University, Hohhot, 010070, China.
Department of Public Health Sciences, Karolinska Institutet, Stockholm, Sweden.
BMC Microbiol. 2018 Apr 3;18(1):27. doi: 10.1186/s12866-018-1162-3.
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.
MiniR1-1 质粒是 R1 质粒的衍生物，是一种低拷贝克隆载体。
核苷酸测序分析表明，MiniR1-1 质粒是一个 6316bp 的圆形双链 DNA 分子，带有 oriR1（复制起点）。该质粒携带 repA、tap、copA 和 bla 基因，以及 ORF1 和 ORF2 基因。MiniR1-1 包含 8 个 DnaA 结合位点（DnaA-box）。DnaA-box1 位于 oriR1 区域，完全符合 DnaA-box 共识序列，而 DnaA-box8 与一个错配接近 copA 基因。MiniR1-1 质粒的存在导致 D 期细胞的积累和缓慢生长的大肠杆菌细胞大小的增加，表明 MiniR1-1 的存在延迟了细胞分裂。MiniR1-1 DnaA-box1 和 DnaA-box8 突变显著增加了质粒的拷贝数，而 DnaA-box1 突变也影响细胞大小。很可能是 DnaA 对 DnaA-box 的滴定负调控了 MiniR1-1 质粒的复制并延迟了细胞分裂。有趣的是，DnaA 在体内与起始蛋白 RepA 弱相互作用。
DnaA 通过与 RepA 蛋白相互作用作为负调控因子调节 MiniR1-1 的拷贝数。
