We have studied the physiological effects of mitomycin C induction on cells carrying ColEl plasmids with differing configurations of three genes: the structural gene coding for colicin (cea), a gene responsible for mitomycin C lethality (kil) that we located as part of an operon with cea, and the immunity (imm) gene, which lies near cea but is not in the same operon. kil is close to or overlaps imm. When cea' plasmids are present mitomycin C induction results in 100-fold or greater increases in the level of colicin. Within an hour after induction more than 90% of cells carrying cea' kil' imm' plasmids are killed and macromolecular synthesis stops, capacity for transport ofproline, thiomethyl -fiD-galactoside, and a-methyl glucoside is lost, and the membrane becomes abnormally permeable as indicated by an increased accessibility of intracellular 13-galactosidase to the substrate o-nitrophenyl .8-D-galactoside. All of these events occur when a ceakil+ imm+ plasmid is present and none does when the plasmid is cea+ kil-imm+, so the damage can be attributed solely to the Kil function and not to the presence ofcolicin. However, cells carrying a cea+ kil-imm-plasmid are killed upon induction, apparently by action of endogenous colicin on the nonimmune cytoplasmic membrane. The pattern of accompanying physiological damage is distinguished from the kil+-associated damage by an enhancement of a-methyl glucoside uptake and accumulation and efflux of a-methyl glucoside 6-phosphate and by an absence of the alteration in membrane permeability for o-nitrophenyl P-D-galactoside. These features are typical of colicin El action on the membrane. The induced damage is not prevented by trypsin and occurs in cells of a strain specifically tolerant to exogenous colicin E1, indicating that the attack is from inside the cell. transposon that confers resistance to ampicillin. The relevant genome configurations of this plasmid and its derivatives that were used in this study are diagrammed in Fig. 1. The isolation and mapping of the Tn5 insertion mutants has been described (2). pRLB6 was constructed by cleaving pDMS630 DNA with the restriction endonucleases Sna I and EcoRI (from New England BioLabs), each of which cleaves the plasmid once in the cea gene at sites 1,290 base pairs apart (Fig. 1, ref. 5). The two enzymatic digestions were carried out separately, using the conditions specified by the supplier. After the EcoRI digestion was complete the enzyme was inactivated by heating at 70'C for 5 min and the ends of the DNA fragments were filled in by incubation for 1 hr at 370C with the four deoxyribonucleoside triphosphates dTTP, dATP, dGTP, and dCTP (each 0.25 mM; from Sigma) and DNA polymerase I (25 units/ml; from New England BioLabs). The DNA was then precipitated with ethyl alcohol, redissolved in buffer (final concentrations: 0.1 M Tris at pH 7.5, 6 mM MgCl2, 6 mM NaCl, 0.4 mM ATP, 10 mM dithiothreitol, and bovine serum albumin at 50 Ag/ml), and ligated by action of phage T4 DNA ligase (400 units/ml; from New England BioLa...

The kil gene of the ColE1 plasmid was cloned under control of the lac promoter. Its expression under this promoter gave rise to the same pattern of bacterial cell damage and lethality as that which accompanies induction of the kil gene in the colicin operon by mitomycin C. This confirms that cell damage after induction is solely due to expression of kil and is independent of the cea or imm gene products. Escherichia coli derivatives resistant to the lethal effects of kil gene expression under either the normal or the lac promoter were isolated and found to fall into several classes, some of which were altered in sensitivity to agents that affect the bacterial envelope.

We isolated a series of Tn5 transposon insertion mutants and chemically induced mutants with mutations in the region of the ColEl plasmid that includes the cea (colicin) and imm (immunity) genes. Bacterial cells harboring each of the mutant plasmids were tested for their response to the colicin-inducing agent mitomycin C. All insertion mutations within the cea gene failed to bring about cell killing after mitomycin C treatment. A cea-amber mutation exerted a polar effect on killing by mitomycin C. Two insertions beyond the cea gene but within or near the imm gene also prevented the lethal response to mitomycin C. These findings suggest the presence in the ColEl plasmid of an operon containing the cea and kil genes whose product is needed for mitomycin C-induced lethality. Bacteria carrying ColEl plasmids with TnS inserted within the cea gene produced serologically cross-reacting fragments of the colicin El molecule, the lengths of which were proportional to the distance between the insertion and the promoter end of the cea gene.

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