Transcription of Plasmid DNA in Escherichia Coli Minicells

Cellular RNA polymerase in association with plasmid DNA segregates into the minicells of minicell-producing strains. In general, one "plasmid equivalent" of RNA polymerase, reflecting the size of the segregating plasmid DNA and its efficiency of segregation, entered the minicell with the plasmid. The amount of RNA polymerase (measured as the amount of enzyme activity purified from minicells and the rate of RNA synthesis in plasmid-containing minicells), and not the DNA content, appeared to be rate-limiting in plasmid-mediated transcription in minicells. The purified minicell and cellular RNA polymerases showed the same sensitivity to rifampin and streptolydigin; both were associated with sigma factor, although the minicell enzyme appeared to have slightly less than the cellular enzyme. These studies demonstrate that transcription of plasmid DNA in minicells is a function of the efficiency of segregation and the amount of RNA polymerase which enters with the plasmid DNA. Because RNA polymerase is limiting, plasmids with relatively weak promoters for the vector genes should be used when attempting to identify products from inserted foreign DNA.