Isolation of a Derivative ofEscherichia coli–Enterococcus faecalisShuttle Vector pAM401 Temperature Sensitive for Maintenance inE. faecalisand Its Use in Evaluating the Mechanism of pAD1par-Dependent Plasmid Stabilization

A derivative of theEscherichia coli–Enterococcus faecalisshuttle vector pAM401 was isolated by mutagenesis in anE. colimutator strain. This plasmid, designated pAM401ts, was more than an order of magnitude less stable at 38°C than at 30°C in theE. faecalishost strain JH2-2. TheE. faecalisplasmid pAD1-encodedparstability locus was cloned onto pAM401ts, and its effects on plasmid stability and host cell viability were assessed. It was found thatparstabilized pAM401ts at 38°C but also caused a substantial drop in cell viability three to four generations after a temperature shift from 30 to 38°C. After a maximum viability drop of 94%, culture growth recovered as plasmid-free cells began to accumulate. Provision of excess RNAII, the putativeparantidote,in transattenuated cell killing. These characteristics support a postsegregational killing mechanism forpar-mediated plasmid stabilization.