Identification of the central regulatory segment of plasmid R6K complexed with the membranes of Escherichia coli.

Understanding the mechanisms which control replication of chromosomes with multiple origins of replication have been the subject of many investigations. The plasmid, R6K, has three origins of replication, alpha, beta, and gamma. In vivo, alpha and beta are utilized with equal frequencies while the gamma origin is rarely used in the parental plasmid, or remains silent in several miniplasmids. Hence, in the present study, the multiple origin plasmid, R6K, was utilized as a model system to investigate DNA replication. A 1.6 kb PCR (polymerase chain reaction) amplified region of R6K containing the central regulatory segment (CRS) was examined to determine if membrane complexing is required for maintenance. Crude membrane extracts from exponentially growing cultures were prepared and sedimented through 30-50% (w/v) linear sucrose gradients. Fractions collected were probed using quantitative PCR which revealed two fractions containing CRS DNA. These complexes were localized in a region between the inner and outer membranes of Escherichia coli.