Expression of the kil gene of the ColE1 plasmid in Escherichia coli Kilr mutants causes release of periplasmic enzymes and of colicin without cell death.

Expression of the kil gene of the ColE1 plasmid in certain classes of Escherichia coli mutants (Kilr) resistant to kil-caused cell death brought about release of periplasmic enzymes and of colicin. Phospholipase A was present but was not activated by kil expression in any of the mutants. This indicates that in these mutants the various effects of kil gene expression have become dissociated.

Expression of the cloned ColE1 kil gene in normal and Kilr Escherichia coli.

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.

Genetic study of the functional organization of the colicin E1 molecule.

Colicin E1 fragments obtained by genetic manipulations of the ColE1 plasmid were tested for bactericidal activity, binding to bacterial cells, and reactions with a series of anticolicin monoclonal antibodies. Two of the fragments were also tested for ability to form channels in liposomal vesicles. The results are in agreement with studies from chemically and enzymatically derived colicin fragments, assigning the receptor binding activity to the central part of the molecule and the killing activity to a region near the carboxyl terminus.

cea-kil operon of the ColE1 plasmid.

We isolated a series of Tn5 transposon insertion mutants and chemically induced mutants with mutations in the region of the ColE1 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 ColE1 plasmid of an operon containing the cea and kil genes whose product is needed for mitomycin C-induced lethality. Bacteria carrying ColE1 plasmids with Tn5 inserted within the cea gene produced serologically cross-reacting fragments of the colicin E1 molecule, the lengths of which were proportional to the distance between the insertion and the promoter end of the cea gene.

Alternative forms of lethality in mitomycin C-induced bacteria carrying ColE1 plasmids.

We have studied the physiological effects of mitomycin C induction on cells carrying ColE1 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(+) plasmids are killed and macromolecular synthesis stops, capacity for transport of proline, thiomethyl beta-D-galactoside, and alpha-methyl glucoside is lost, and the membrane becomes abnormally permeable as indicated by an increased accessibility of intracellular beta-galactosidase to the substrate o-nitrophenyl beta-D-galactoside. All of these events occur when a cea(-)kil(+)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 of colicin. 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 alpha-methyl glucoside uptake and accumulation and efflux of alpha-methyl glucoside 6-phosphate and by an absence of the alteration in membrane permeability for o-nitrophenyl beta-D-galactoside. These features are typical of colicin E1 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.

The mistaken identity of colicin A.

In a series of published articles, colicin A has been mistakenly labeled as colicin K.

Macromolecular syntheses in an Escherichia coli adk mutant.

Preferential inhibition by high temperatures of synthesis of newly induced enzymes in Escherichia coli K-12 CR341T28 adk is only apparent; syntheses of all macromolecules cease simultaneously.

Reduction of membrane potential, an immediate effect of colicin K.

Colicin K causes a rapid and drastic reduction of the membrane potential of Escherichia coli cells, as measured by the uptake of the lipophilic cation triphenylmethylphosphonium. The colicin causes no major changes in the pH gradient, as measured by the uptake of butyric acid. The decrease in membrane potential following addition of colicin K to the cells is a prompt response that parallels or precedes known physiological effects such as efflux of accumulated substrates. Hence the loss of membrane potential qualifies as the primary action by which the colicin uncouples membrane-associated function from respiration. Certain peculiarities of bacterial cells pretreated with EDTA in their response to uncoupling agents and lipophilic ions are described.

Stages in colicin K action, as revealed by the action of trypsin.

The effects of trypsin on Escherichia coli cells that have been treated with colicins have been examined. By the use of trypsin, it has been possible to demonstrate that the action of several colicins (E1, E2, and K) proceeds through at least two stages. Stage I is a period after colicin adsorption when trypsin can restore colony-forming ability to a colicin-treated cell. Stage I is followed by a period when trypsin is unable to restore colony-forming ability (stage II). The transition between stage I and stage II follows first-order kinetics, with a rate proportional to the number of killing units of colicin adsorbed.A quantitative comparison of the effects of colicin K on colony-forming ability and on several cellular processes indicates that colicin damage to these processes occurs in the stage II period of colicin action and is not subject to reversal by the trypsin treatment that restores viability to cell in stage I. The implications of these findings for an understanding of the mode of action of colicins are discussed.