Characterization and complementation of pMB1 copy number mutant: effect of RNA I gene dosage on plasmid copy number and incompatibility

Abstract

A 16-base-pair insertion has been identified as the mutation responsible for the high-copy-number phenotype of the plasmid copy number mutant pFH118. The mutation is located near the plasmid origin of replication in a region of the genome that encodes two overlapping RNA transcripts. One of these transcripts, RNA I, acts as a negative regulator of plasmid replication. The second transcript is the precursor to the primer for the initiation of DNA synthesis. We demonstrate through complementation that the pFH118 DNA overproduction phenotype is a consequence of the reduced effectiveness of the mutant RNA I at inhibiting plasmid replication and not a consequence of an altered target site on the primer precursor. In addition, a series of plasmids containing multiple RNA I-coding genes was constructed for investigating the effects of RNA I gene dosage on plasmid copy number and incompatibility. The results of this study strongly support the inhibitor dilution model of plasmid copy control with RNA I as the plasmid-specified inhibitor responsible for both copy number control and incompatibility.