Trans-complementable copy-number mutants of plasmid ColE1

Abstract

Plasmid ColE1, like many other small non-conjugative plasmids, is present in multiple copies (about 15 per chromsome equivalent) in Escherichia coli cells^1,2. Because of their high copy number, the replication of such plasmids has been described as ‘relaxed’, even though there is good evidence that it is strictly controlled: ColE1 derivatives have characteristic but different copy numbers² and ColE1 copy-number mutants have been characterised^3–5. No plasmid-specified protein is essential for the replication of ColE1 and related plasmids, as extensive replication can occur in chloramphenicol-treated cells⁶, in plasmid-free chloramphenicol-treated cells transfected with a hybrid ColE1/phage replicon and in vitro in extracts derived from plasmid-free cells⁸. Nevertheless, it is possible that a plasmid-specified protein is involved in ColE1 replication control in viable cells. Here we show that deletion of a given non-essential region from ColE1-like plasmids results in a raised copy number. Such plasmids are stably maintained and have their copy number returned to normal when a complementing plasmid is present in the same cell, indicating that a plasmid-specified diffusible gene product regulates the plasmid content of ColE1-containing cells. Deletion of the equivalent region from the cloning vector pBR322 gives a derivative which has a raised copy number and which has also lost its origin for conjugal transfer; unlike pBR322, it cannot be mobilised.