Regions of broad-host-range plasmid RK2 which are essential for replication and maintenance

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Abstract
The sites of cleavage on the map of the broad-host-range plasmid RK2 (56 kbases) were determined for the BglII, PstI and SmaI restriction enzymes, and the determinants for tetracycline and ampicillin resistance were localized. The cleavage sites were clustered at or near the drug resistance genes. To localize regions required for plasmid replication and maintenance in Escherichia coli, nonessential regions of RK2 were deleted by partial digestion with the restriction endonuclease HaeII to produce small derivatives. The smallest stable replicon obtained contained 5 HaeII fragments of RK2 which total 5.4 kbases. These fragments were derived from 3 regions of RK2 that are separated from each other by antibiotic resistance genes. One of these HaeII fragments (0.75 kbases) has the properties expected of the origin of replication. The other 4 fragments, located in 2 separate regions of RK2, provided, in trans, functions that permit the replication of the HaeII fragment carrying the origin of the replication. Apparently, at least 2 plasmid-encoded genes, capable of acting in trans and a replication origin are required for RK2 replication and maintenance.