Instability of a high-copy-number mutant of a miniplasmid derived from broad host range IncP plasmid RK2.

Mini-RK2 plasmids pCT460 and pCT461 which contain the oriVRK2, trfA and trfB regions of RK2 in addition to tetracycline and kanamycin resistance determinants, have copy numbers of 17 and 35 copies per chromosome equivalent, respectively. The difference in copy number is due to a 56-bp deletion in oriVRK2 in pCT461. In Escherichia coli only pCT461 is markedly unstable in batch culture while both are unstable (although pCT461 is more so) in bacteria on stock plates. The instability of pCT461 in bacteria on stock plates is recA+ dependent and appears to involve loss of plasmid DNA from bacteria rather than selective cell death. After storage of recA+ bacteria carrying pCT461 for a few weeks the remaining antibiotic-resistant bacteria carry a mixture of plasmid DNA species including parental pCT461, transposable element insertion derivatives, and, by far the majority, deletion derivatives. It appears that one particular plasmid region, which includes the kilD gene (which inhibits plasmid maintenance in the absence of korD which, however, is present on pCT460 and pCT461), is responsible for this instability in a gene dosage-dependent way. Most of these deletion derivatives are dependent on pCT461-specified trfA gene (essential for replication) so that they do not displace pCT461 entirely. Their presence reduces the copy number of pCT461, thus reducing the instability, and is probably ultimately responsible for pCT461 survival on stock plates. In many bacteria the same process which gives rise to deletion derivatives may result in degradation of plasmid DNA extensive enough to cause loss of pCT461.