Viability of Escherichia coli treated with colicin K.

Abstract

(3) have shown the arrest of synthesis of macromolecules to be a consequence of the lowering of cellular ATP: in uncA mutants, which lack the Mg2+,Ca2+-activated ATPase (ATP phosphohydrolase, EC 3.6.1.3), the colicin failed to lower ATP levels and synthesis of macromolecules continued. Both transport of proline and of glutamine, apparently coupled to different forms of energy (4), were uncoupled by colicin K in the uncA mutants.Since synthesis of macromolecules continued in colicintreated uncA cells, it was paradoxical that they were not viable. At low multiplicities of colicin, two or three killing units per cell, growth and cell division should have generated daughter cells free of colicin, which then would produce colonies. Death might have resulted from an effect of the colicin not reversible in the daughter cells, or from direct inhibition of cell division. Alternatively, the inability of colicin-treated cells to concentrate metabolites or ions such as K+ and Mg2+ (5,6) Preparation of Colicins. Strain K235 (Col K) was induced with mitomycin C and the colicin purified according to Kunugita and Matsuhashi (8). Both fractions eluted from the CM-Sephadex column were used with similar results; each showed several protein bands upon polyacrylamide gel electrophoresis. Colicins El, E2, E3, and Ib were also induced with 0.2 mg of mitomycin C per liter. The colicinogenic bacteria were washed with 0.05 M sodium phosphate at pH 7.0, resuspended in the same buffer, and broken by sonication. The crude extracts were dialyzed against the same buffer and stored frozen.Assay of ATP. Samples (0.1 ml) of a bacterial suspension were added to 1.9 ml of boiling water and boiled for 15 min. One milliliter of the extract was added to 2 ml of 0.05 M potassium arsenate, pH 7.4, and 0.02 M MgSO4. Firefly lantern extract (Sigma), 0.05 ml, was added and the emitted light counted in a Packard model 314-EX2 liquid scintillation counter using the "analyser" mode. RESULTSConditional viability of colicin-treated uncA cells High ATP levels and synthesis of macromolecules are maintained in colicin-treated uncA cells in spite of the uncoupling of active transport (3). In the K+ phosphate-buffered medium employed, however, high levels of intracellular K+ would have remained. Inability to retain K+ after transfer to NaCl-based solid medium could have prevented the colicintreated cells from forming colonies. Fig. 1 shows that a large fraction of colicin-treated uncA cells can survive to form colonies on LBK agar, in which KC1 has replaced the usual NaCl. The optimal pH of LBK for survival of colicin-treated cells was 6.6; half as many colonies appeared at pH 6.0 or 7.1 (data not shown).

Role of Ionic Strength in Colicin K Adsorption

“…A specific role for K+ and Mg2+ ions in protecting bacteria that have adsorbed colicin against subsequent death has been described recently (Kopecky, Copeland & Lusk, 1975). In contrast, the results presented here demonstrate that in general all ions have a similar protective effect on colicin-treated bacteria.…”

Mode of Action of Morganocin 174

“…For colicins El and K, which result in a rapid effliux of K+ and Mg2+ (4,10,16), viability could be restored by growth on K+-, Mg2+-supplemented medium thus artificially maintaining physiological levels intracellularly (13). K+ leakage from morganocin-treated cells was gradual, and growth on supplemented medium did not restore viability.…”

“…K+ leakage from morganocin-treated cells was gradual, and growth on supplemented medium did not restore viability. In this respect it appeared that the mode of action of morganocin 174 more closely resembled that of colicin Ib (13).…”

“…The response of a Ca2+, Mg2+-activated adenosine triphosphatase I Present address: Department of Microbiology, University of Iowa, Iowa City, IA 52242. mutant, uncA, to the action of colicin K suggests that the primary effect is deenergization of the membrane (21). Viability may be retained if cells are grown in medium containing physiological concentrations of K+ and Mg2+ (13).…”

Effects of potassium and sodium ions on the killing action of a Pichia kluyveri toxin in cells of Saccharomyces cerevisiae

“…These authors observed an enhancement of toxin action when adenosine 5'-diphosphate was incorporated in the plate medium, and the enhancement was reversed by the addition of calcium ions. Furthermore, the killing effect of several bacteriocins is strongly affected by the ionic composition of the plate medium; in particular, monovalent cations (4,11) and magnesium ions (4) Preparation ofthe killer toxin and toxin assay. Cells of P. kluyveri 1002 were grown, and 100-fold concentrated solutions of the toxin were prepared as described previously (6a).…”