New runaway-replication-plasmid cloning vectors and suppression of runaway replication by novobiocin

Two new cloning vectors (pBEU28 and pBEU50) with temperature-controlled runaway-replication properties are described. pBEU28 is similar to aphA+ (KanR) plasmid pBEU2 but lacks a 1.8-kb duplication which is responsible for plasmid instability. pBEU50 is an analog of pBR313 and pBR322 in that it carries bla+(AmpR), which can be used for selection, and tet+(TetR) which can be inactivated by cloning at HindIII and BamHI restriction sites. Sublethal concentrations of novobiocin were exploited to suppress runaway replication and to restore the viability of the plasmid carriers. By this method copB deletion mutants of two temperature-controlled, conditional runaway-replication plasmids were detected and isolated. The unconditional runaway-replication property of these plasmids leads us to hypothesize that there are at least two controls of plasmid R1 copy number and that the copB-dependent control is temperature-sensitive in the conditional runaway replication mutants. The novobiocin suppression of the runaway replication permitted us to clone dnaN+ on pBEU28 and to identify its presence at 42 degrees C with a dnaN59 transformation recipient which was temperature-sensitive due to a defect in the dnaN gene.