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Instability of a high-copy-number mutant of a miniplasmid derived from broad host range IncP plasmid RK2
Abstract:
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.
Insights
Mini-RK2 plasmids exhibit instability in Escherichia coli, particularly pCT461, due to a deletion in oriVRK2. This instability, dependent on recA+, involves plasmid DNA loss and the formation of deletion derivatives that stabilize replication.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- Mini-RK2 plasmids are derivatives of the broad-host-range plasmid RK2, engineered for specific applications.
- Plasmid stability is crucial for maintaining genetic elements in bacterial populations, especially during storage or selective pressure.
Purpose of the Study:
- To investigate the instability of mini-RK2 plasmids, specifically pCT460 and pCT461, in Escherichia coli.
- To elucidate the mechanisms underlying plasmid instability and the role of specific plasmid regions and host factors.
Main Methods:
- Construction and characterization of mini-RK2 plasmids (pCT460, pCT461) with defined RK2 regions.
- Assessment of plasmid copy number and stability in Escherichia coli under different conditions (batch culture, stock plates).
- Analysis of plasmid DNA integrity and derivative formation in unstable strains using recA+ and recA- backgrounds.
Main Results:
- pCT461, with a deletion in oriVRK2, showed higher instability than pCT460 in Escherichia coli.
- Instability in recA+ strains involved recA+-dependent loss of plasmid DNA and formation of deletion derivatives.
- A plasmid region including the kilD gene contributed to instability in a gene dosage-dependent manner.
Conclusions:
- Plasmid instability in mini-RK2 systems is linked to specific DNA sequences and host factors like recA.
- The formation of deletion derivatives, dependent on replication genes like trfA, can mitigate instability by reducing plasmid copy number.
- Understanding these mechanisms is vital for the effective use and maintenance of RK2-based plasmids in molecular biology.