Regulation of plasmid DNA synthesis: Isolation and characterization of copy number mutants of miniR6-5 and miniF plasmids

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Abstract
Copy number (Cop) mutants of miniR6-5 and miniF plasmid derivatives containing a β-lactamase gene were isolated by selection for increased ampicillin-resistance. Mutants which exhibited an increased copy number and a reduced incompatibility response as compared to the respective parent mini-plasmid were obtained from both miniR6-5 and miniF. Characterization of these mutant plasmids has provided the first description of the replicative properties of miniF Cop mutants, and has also facilitated a comparison of plasmids representing the IncFI and IncFII incompatibility groups. Cop mutants from these groups differed in several respects: MiniF Cop mutants were considerably more difficult to obtain and showed a markedly lower transforming efficiency than the corresponding miniR6-5 mutants. MiniR6-5 Cop mutants were stably maintained in a polAI strain without selective pressure, whereas miniF Cop mutants severely reduced the viability of this host. MiniR6-5 replication stopped within a few minutes after inhibition of protein synthesis, whereas miniF replication continued at a declining rate for about one hour in the presence of chloramphenicol. In contrast to miniR6-5 replication, miniF DNA synthesis was blocked faster by rifampicin than by chloramphenicol. The copy number of miniR6-5 plasmids (but not of miniF) was reduced by about 50% in an rnc strain deficient in RNAase III.