TEMPERATURE-SENSITIVE MUTANTS FOR THE REPLICATION OF PLASMIDS IN ESCHERICHIA COLI I. ISOLATION AND SPECIFICITY OF HOST AND PLASMID MUTATIONS

David T. Kingsbury ¹ and Donald R. Helinski ¹

¹ Department of Biology, University of California, San Diego, La Jolla, California 92037

Temperature-sensitive mutants of Escherichia coli defective in the replication of the plasmid colicinogenic factor E1 (ColE₁) were isolated following mutagenesis of E. coli K12 strain carrying the ColE₁ factor. Following the mutagenic treatment an enrichment procedure utilizing the replacement of thymine with bromouracil in the ColE₁ DNA duplicated at the restrictive temperature was used. The mutants isolated following this enrichment step were the result of a mutation event either in the host chromosome or in the ColE₁ plasmid. The host mutants fell into three phenotypic classes based on the effect each mutation had on the maintenance of a variety of other extrachromosomal DNA elements. Phenotypic class I mutations affected all E. coli plasmids, both the I and F sex factor types as well as the ColE₁ factor. Phenotypic class II mutations affected the maintenance of the ColE₁ and the F sex factor type plasmids and not the I type, while phenotypic class III mutations affected only ColE₁ replication. None of these mutations was found to have a significant effect on the replication of the E. coli chromosome. The plasmid-linked mutations fell into two phenotypic classes on the basis of the ability of the Flac episome to complement the mutation in the ColE₁ plasmid.