A mutational analysis of the ColE1-encoded cell cycle regulator Rcd confirms its role in plasmid stability

Multimers of multicopy plasmids cause instability. They arise by homologous recombination and accumulate by over-replication in a process known as the dimer catastrophe. Dimers are resolved to monomers by site-specific recombination systems such as Xer-cer of plasmid ColE1. In addition, the Rcd checkpoint hypothesis proposes that a short transcript (Rcd) coded within ColE1 cer delays the division of multimer-containing cells. The crucial observation underpinning the checkpoint hypothesis is that when the Rcd promoter (Pcer) is inactivated by mutation of its invariant T, the plasmid becomes unstable. Recently, we discovered that this mutation also alters a potential Fis binding site in cer. ColE1-like plasmids are less stable in fis mutant hosts and it is conceivable that instability caused by the mutation is due to altered Fis binding, rather than the loss of Rcd expression per se. We have therefore undertaken an independent test of the role of Pcer-Rcd in multicopy plasmid stability. We have generated a series of loss-of-function mutants of Rcd and detailed analysis of two of these shows that they cause a level of instability indistinguishable from Pcer inactivation. This result is consistent with the predictions of the checkpoint hypothesis and confirms the role of Rcd in plasmid stability.