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Use of a DNA polymerase III bypass mutant of Escherichia coli, pcbA1, to isolate potentially useful mutations of a complex plasmid replicon.

The essential replicon region of plasmid pCU1 has, within 1.2 kb, two origins of replication that can function in the absence of Escherichia coli DNA polymerase I and one that requires this polymerase. To isolate mutants in the replicon pathway that uses the PolI-dependent origin in the presence of the two other origins, we examined the feasibility of exploiting E. coli strains carrying a polymerase c bypass mutation (pcbA) and which can survive and form colonies with the polymerase activity of polC inactivated at 42 degrees C. The selection scheme that is described was successful and resulted in the isolation of a mutant replicon that is not maintained at 42 degrees C in a PcbA-PolC+tsPolA+ strain. Nucleotide sequencing indentified the mutated region to be within the origin (OriV) that was known to be polA-dependent. Electron microscopy of mutant plasmid molecules replicating in a Pcb+ strain confirmed that OriV is inactivated.