Role of the rom protein in copy number control of plasmid pBR322 at different growth rates in Escherichia coli K-12

The copy number per cell mass of plasmid pBR322 and a rom(-) derivative was measured as a function of generation time. In fast growing cells the copy number per cell mass was virtually identical for rom(+) and rom(-) derivatives. However, the copy number of pBR322 only increased 3- to 4-fold from a 20- to 80-min generation time, whereas the copy number of the torn derivative increased 7- to 10-fold. The copy number stayed constant for the rom(+) and rom(-) plasmids at generation times longer than 80-100 min. Thus, the presence of the rom gene decreased the copy number of plasmid pBR322 in slowly growing cells at least 2-fold when compared with the rom(-) plasmid. To study the effect of the rom gene in trans we cloned the gene into the compatible P15A-derived rom(-) plasmid pACYC184. In cells carrying both pACYC184 rein; and pBR322 rom(-) the presence of the rom gene in trans had little effect on the copy number of pBR322 rom(-) at fast growth, but it decreased its copy number at slow growth to the same level as found for pBR322, i.e., complemented the pBR322 rom(-) plasmid. The pACYC184 plasmid and its rom(+) derivatives showed copy numbers similar to those of pBR322 rom(-) and pBR322 itself, respectively, at fast and slow growth. We conclude that the rom gene product-the Rom protein-is an important element in copy number control of Co1E1-type plasmids especially in slowly growing cells. (C) 1999 Academic Press.