Phasmid P4: manipulation of plasmid copy number and induction from the integrated state.

"Phasmid" P4 is unusual in that it is capable of (i) temperate, (ii) lytic, helper-dependent, and (iii) plasmid modes of propagation. In this report we characterize most of the known P4 genetic functions as to their essential or nonessential roles in the stable maintenance of plasmid P4 vir1 (pP4 vir1 (pP4 vir1). We also identify growth conditions that can be used to stably maintain pP4 vir1 at any one of several different copy number levels (n = 1 to 3, n = 10 to 15, or n = 30 to 40). Analyses of a temperature-sensitive alpha derivative of pP4 vir1 show that shifting the temperature from 37 to 42 degrees C allows this mutant to maintain an integrated copy of the plasmid, whereas replication of free copies is repressed because of the nonpermissive condition for their DNA synthesis. Conversely, a shift from 42 to 37 degrees C can be used to reinstate plasmid propagation. The utility of the inducible states of pP4 vir1 is discussed with respect to its attributes as a vector with the potential for cloning inserts of DNA up to 33,000 base pairs in a wide range of bacterial hosts.