Stability and replication control of Escherichia coli minichromosomes

Abstract

A stabilized minichromosome--a plasmid replicating from the chromosomal origin oriC--was constructed by cloning the sopA,B,C, genes from plasmid F. This minichromosome had a loss frequency of less than 10(-3), while that of the nonstabilized parental plasmid was 2 X 10(-2) to 4 X 10(-2). Both minichromosomes had the same average copy number per chromosomal origin, and the copy numbers were constant over an eightfold range of growth rates. Different mutations in the mioC gene and promoter, from which transcription enters oriC, were constructed, and their effects on minichromosome copy number and stability were tested. The results indicated that normal replication control at oriC was independent of the MioC protein and most of the sequences between the promoter and oriC, but required both transcription from the mioC promoter and probably also the presence of the DnaA box (DnaA protein-binding site) just upstream of the mioC promoter. Transcription from the mioC promoter was shown to be efficiently repressed in vivo after overproduction of DnaA protein and to be derepressed at the nonpermissive temperature in six different dnaA(Ts) mutants.