W3110 cells carrying the ColE3-CA38 plasmid are immune to externally added colicin E8, a newly described member of the E group colicins. By molecular cloning and transposon mutagenesis we localized the colicin E8 immunity gene between the RI site (4.0 kb on the restriction map) and the II site (3.68 kb) of the ColE3-CA38 plasmid. This placed the colicin E8 immunity gene between the colicin E3 immunity gene and , the region which determined mitomycin C sensitivity. Insertion of a transposon into the colicin E3 structural gene prevented the synthesis of active colicin and completely abolished mitomycin C sensitivity, but had no effect on the two immunity genes. In contrast, insertion of a transposon into the colicin E8 immunity gene had no effect upon colicin E3 production or colicin E3 immunity but did abolish mitomycin C sensitivity. The phenotype conferred by plasmids with a transposon inserted into the region of ColE3-CA38 was dependent upon the site of insertion.


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