Effects of Colicins E1 and K on Cellular Metabolism

Colicins E1 and K inhibited a whole series of energy-dependent reactions in Escherichia coli cells, including motility, biosynthesis of nucleic acids, proteins and polysaccharides, and the conversion of ornithine to citrulline. Respiration was only partially affected, and substrates such as glucose continued to be catabolized through the normal pathways, albeit with reduced CO(2) production. The soluble products of aerobic glucose catabolism by colicin-treated cells were analyzed. Pyruvate replaced acetate as the major excreted product, and the following intermediates of glycolysis were excreted in significant amounts: glucose-6-phosphate, fructose-1,6-diphosphate, dihydroxyacetone phosphate, and 3-phosphoglycerate. Anaerobically growing cells manifested a somewhat enhanced tolerance to the colicins. This protection by anaerobiosis appeared to depend on the exclusion of oxygen more than on the extent of fermentative catabolism versus catabolism of the respiratory type. These results are interpreted in terms of possible functions of colicin in lowering the adenosine triphosphate (ATP) content of the cells and in terms of the role of lowered ATP levels in inhibiting many of the energy-requiring reactions.     

Mode of Action of Colicins of Types E1, E2, E3, and K

The effect of colicins on deoxyribonucleic acid and protein synthesis, and also their effect on the ability of T4 phage to replicate in Escherichia coli K-12, were studied. Colicins of type K inhibited deoxyribonucleic acid synthesis, protein synthesis, and phage growth. Among colicins of type E, there was an absolute correlation between mode of action and subdivision into types E(1), E(2), and E(3).     

Comparison of the Action of Colicins E1 and K on Escherichia coli with the Effects of Abortive Infection by Virulent Bacteriophages

Abortive infection of certain strains of Escherichia coli or Shigella dysenteriae with phages of the T-even group or with phage T5 resembles the action of colicin E1 or K on sensitive bacteria, especially in the effects on biosynthetic processes. Tests on transport systems and on adenosine triphosphate levels suggest, however, that different mechanisms are involved in the two cases. Abortive infection appears to cause damage to the permeability barrier of the cell, whereas the colicins interfere more directly with the energy metabolism of the bacteria.     

Effects of colicins E1 and K on transport systems

The effect of colicins E1 and K on active transport of beta-d-galactosides and of alpha-methyl-d-glucoside (alphaMG) by Escherichia coli was studied. These colicins strongly inhibited the accumulation of thio-methyl-galactoside (TMG) by bacteria and caused rapid exit of previously accumulated TMG. The inhibition effect was limited to the accumulation phase of galactoside transport; the rate of hydrolysis of o-nitrophenyl galactoside, which is dependent on transport of the substrate by the lactose-permease system, was only slightly affected. The accumulation of alphaMG was highly resistant to inhibition by these colicins under conditions which caused complete suppression of TMG accumulation. These effects of the colicins on transport resemble qualitatively those of sodium azide. The findings were interpreted by assuming that colicins E1 and K inhibit the energy-dependent steps in the accumulation of TMG but do not affect facilitated diffusion of galactosides mediated by the specific transport mechanism. The continued accumulation of alphaMG was attributed to the fact that this compound is stored by E. coli cells as a phosphorylated compound by a phosphoenolpyruvate-dependent transport system rather than by an adenosine triphosphate-linked accumulation mechanism.     

Plasmid Replication and the Induced Synthesis of Colicins E1 and E2 in Escherichia coli

Induction of colicins E1 and E2 in Escherichia coli occurs when plasmid synthesis has been inhibited either by nalidixic acid or by lack of deoxyribonucleic acid polymerase I. Moreover, colicin E1 and E2 synthesis induced by mitomycin C and exposure to chloramphenicol is not associated with a large increase in circular plasmid deoxyribonucleic acid. The mean plasmid content of cells in populations having a low spontaneous frequency of colicin-producing cells because of growth at low temperature or because of the presence of recA(-) or crp(-) alleles, is not significantly different to that in wild-type cells grown at 37 C.     

Effects of Magnesium Ions on Thermal Inactivation of Alkaline Phosphatase

The effect of Mg²⁺ on the thermal inactivation and unfolding of calf intestinal alkaline phosphatase has been studied at different temperatures and Mg²⁺ concentrations. Increasing the Mg²⁺ concentration in the denatured system significantly enhanced the inactivation and unfolding of the enzyme during thermal inactivation. The analysis of the kinetic course of substrate reaction during thermal inactivation showed that at 47°C the increased free Mg²⁺ concentration caused the inactivation rate to increase. Increasing the temperature strengthened the effect of Mg²⁺ on the thermal inactivation. Control experiment showed that this is not due to salt effect. The time course of fluorescence emission spectra showed that the emission maximum for Mg²⁺-containing system was always higher than that of Mg²⁺-free system, and the higher temperature enhanced this difference. In addition, Mg²⁺also enhanced the unfolding rate of the enzyme at 47°C. The potential biological significance of these results are discussed.     

Colicins E7 and E8 degrade DNA in sensitive bacteria

The primary target of colicin E7 in sensitive bacteria are their DNA molecules. In agarose gel electrophoresis of lysates of cells treated with colicin E7, both chromosomal and plasmid DNA bands disappear, in direct relation to E7 concentration and to the duration of treatment. DNA degradation is followed by a cessation of DNA synthesis. In E7-immune bacteria, no damage to DNA due to colicin E7 occurs. The mode of action of colicin E7 thus appears to be equal to that of colicin E2. Also, colicin E8 causes a distinct damage to chromosomal and plasmid DNA in sensitive, but not in immune bacteria. None of the colicins E1, E3, E4, E5, E6 or E9 has any influence on bacterial DNA.     

Role of Ferrous Ions in Synthetic Cobaltous Sulfide Leaching of Thiobacillus ferrooxidans

Microbiological leaching of synthetic cobaltous sulfide (CoS) was investigated with a pure strain of Thiobacillus ferroxidans. The strain could not grow on CoS-salts medium in the absence of ferrous ions (Fe²⁺). However, in CoS-salts medium supplemented with 18 mM Fe²⁺, the strain utilized both Fe²⁺ and the sulfur moiety in CoS for growth, resulting in an enhanced solubilization of Co²⁺. Cell growth on sulfur-salts medium was strongly inhibited by Co²⁺, and this inhibition was completely protected by Fe²⁺. Cobalt-resistant cells, obtained by subculturing the strain in medium supplemented with both Fe²⁺ and Co²⁺, brought a marked decrease in the amount of Fe²⁺ absolutely required for cell growth on CoS-salts medium. As one mechanism of protection by Fe²⁺, it is proposed that the strain utilizes one part of Fe²⁺ externally added to CoS-salts medium to synthesize the cobalt-resistant system. Since a similar protective effect by Fe²⁺ was also observed for cell inhibition by stannous, nickel, zinc, silver, and mercuric ions, a new role of Fe²⁺ in bacterial leaching in T. ferrooxidans is proposed.     

Effects of colicins K and E1 on the glucose phosphotransferase system

1. Glycerol-grown cells of Escherichia coli and its mutant uncA, treated with colicin E1 or K, exhibited a several-fold higher level of α-methylglucoside uptake than untreated cells. This stimulation was independent of the carbon source present during the uptake test. In a mutant strain that has elevated levels of α-methylglucoside accumulation the addition of colicin E1 or carbonylcyanide m-chlorophenylhydrazone (CCCP) did not further enhance the uptake.2. Colicins K and E1 decreased the apparent K_m for α-methylglucoside uptake significantly and increased the V about twofold. The exit of the glucoside was severely inhibited by the colicins.3. In the presence of colicins, α-methylglucoside is still accumulated via the phosphoenolpyruvate-phosphotransferase system since no accumulation or phosphorylation occurs in an enzyme I mutant. The colicins increased the relative intracellular concentration of phosphorylated α-methylglucoside, possibly by inhibiting the dephosphorylation reaction, and caused an excretion of this compound.4. The results are interpreted as indicating that energization of the membrane has an inhibitory effect on the phosphotransferase system. Possible modes of action are discussed.     

Permeability of Squid Axon Membrane to Various Ions

The permeability of the squid axon membrane was determined by the use of radioisotopes of Na, K, Ca, Cs, and Br. Effluxes of these isotopes were measured mainly by the method of intracellular injection. Measurements of influxes were carried out under continuous intracellular perfusion with an isotonic solution of potassium sulfate. The Na permeability of the resting (excitable) axonal membrane was found to be roughly equal to the K permeability. The permeability to anion was far smaller than that to cations. It is emphasized that the axonal membrane has properties of a cation exchanger. The physicochemical nature of the "two stable states" of the excitable membrane is discussed on the basis of ion exchange isotherms.     

Enhancing Effect of Nickel Ions on the Response to Magnesium Ions of Single Fibers of the Frog Glossopharyngeal Nerve: Competitive Inhibition by Calcium Ions of the Nickel-enhanced Response to Magnesium Ions

Single fibers of the frog glossopharyngeal nerve respond toMgCl₂ at concentrations exceeding 10 mM. NiCl₂ at 1 mM enhancedthe Mg²⁺ response. CaCl₂ at 0.5–2 mM induced an inhibitionof the Ni²⁺-enhanced response to Mg²⁺ ions. A quantitative explanationfor these results is provided by the hypothesis that Ni²⁺ ionssecondarily affect a magnesium receptor (designated X^*_Mg) thatis responsible for the Mg²⁺ response and that Ca²⁺ ions inhibitthe Ni²⁺-enhanced response to Mg²⁺ ions by competing with Mg²⁺ions for X^*_Mg. Double-reciprocal plots of the experimental dataindicate that Ni²⁺ ions do not affect the affinities of X^*_Mgfor both Mg²⁺ ions (agonist) and Ca²⁺ ions (competitive antagonist)appreciably, and that Ni²⁺ ions at 1 mM enhanced the maximalresponse to Mg²⁺ ions by 270%. It appears that a magnesium receptorinteracts with an Ni²⁺-binding element that is affected by Ni²⁺ions and, thus, Ni²⁺ ions can induce an enhancement of the Mg²⁺response. Chem. Senses 22: 613–622,1997.     

Effects of colicins K and E1 on the glucose phosphotransferase system.

1. Glycerol-grown cells of Escherichia coli and its mutant uncA, treated with colicin E1 or K, exhibited a several-fold higher level of alpha-methylglucoside uptake than untreated cells. This stimulation was independent of the carbon source present during the uptake test. In a mutant strain that has elevated levels of alpha-methylglucoside accumulation the addition of colicin E1 or carbonylcyanide m-chlorophenylhydrazone (CCCP) did not further enhance the uptake. 2. Colicins K and E1 decreased the apparent Km for alpha-methylglucoside uptake significantly and increased the V about twofold. The exit of the glucoside was severely inhibited by the colicins. 3. In the presence of colicins, alpha-methylglucoside is still accumulated via the phosphoenolpyruvate-phosphotransferase system since no accumulation or phosphorylation occurs in an enzyme I mutant. The colicins increased the relative intracellular concentration of phosphorylated alpha-methylglucoside, possibly by inhibiting the dephosphorylation reaction, and caused an excretion of this compound. 4. The results are interpreted as indicating that energization of the membrane has an inhibitory effect on the phosphotransferase system. Possible modes of action are discussed.     

Reduced Permeability to K+ and Na+ Ions of K+ Channels in the Plasma Membrane of Tobacco Cells in Suspension after Adaptation to 50 mM NaCl

The whole-cell patch-clamp technique was used to study and comparethe characteristics of K⁺-and Na⁺-transport processes acrossthe plasma membrane in two types of protoplast isolated fromNaCl-adapted and -unadapted cells of tobacco (Nicotiana tabacumL. cv. Bright Yellow-2) in suspension culture. In both typesof protoplast, with 100 mM KCl in the bathing solution and inthe pipette solution, depolarization of the plasma membranefrom the holding potential of 0 mV to a positive potential resultedin a relatively large outward current which increased with increasingpositive potential, whereas hyperpolarization to negative potentialsup to –100 mV resulted in only a small inward current.The outward current activated by depolarization was predominantlycarried by K⁺ ions through K⁺ channels. Na⁺ ions also had afinite ability to pass through these K⁺ channels. The outwardK⁺ and Na⁺ currents of the NaCl-adapted cells were considerablysmaller than those of the NaCl-unadapted cells. These resultssuggest that adaptation to salinity results in reduced permeabilityof the plasma membrane to both K⁺ and Na⁺ ions. ¹Present address: Research Laboratory of Applied Biochemistry,Tanabe Seiyaku Co., Ltd., 16-89, Kashima 3-chome, Yodogawa-ku,Osaka, 532 Japan     

Ion selectivity of colicin E1: II. Permeability to organic cations

Channels formed by colicin E1 in planar lipid bilayers have large diameters and conduct both cations and anions. The rates at which ions are transported, however, are relatively slow, and the relative anion-to-cation selectivity is modulated over a wide range by the pH of the bathing solutions. We have examined the permeability of these channels to cationic probes having a variety of sizes, shapes, and charge distributions. All of the monovalent probes were found to be permeant, establishing a minimum diameter at the narrowest part of the pore of approximately 9 A. In contrast to this behavior, all of the polyvalent organic cations were shown to be impermeant. This simple exclusionary rule is interpreted as evidence that, when steric restrictions require partial dehydration of an ion, the structure of the channel is able to provide a substitute electrostatic environment for only one charged group at time.     

Cytotoxic effects of colicins E1–E5 and K on hamster fibroblasts

As a further model of mammalian tissue cells, Chinese hamster fibroblasts of the stable line V79 were used to cheek the cytotoxic effects of colicins. The efficiency of plating of cells treated with colicins E1–E5 and with colicin K was followed. The V79 cells were, in general, poorly sensitive to colicins; only colicins E1, E3 and E5. lowered the number of colony-forming cells to some degree. Again, a different action of colicins E1 and K (which is the same in bacteria) was found in eukaryotic cells.     

Inhibition of Anthocyanin Synthesis by Cobaltous Ions in the First Internode of Sorghum bicolor L. Moench

Cobaltous ions (Co²⁺) inhibited light-mediated anthocyanin synthesisin excised first internodes of Sorghum bicolor (L.) Moench.Studies with precursors/intermediates of the anthocyanin biosyntheticpathway, such as acetate, phenylalanine, t-cinnamic acid, tyrosine,and p-coumaric acid were undertaken to identify the metabolicsite at which Co²⁺ ions inhibit anthocyanin synthesis. p-Coumaricacid partially restored anthocyanin synthesis. No other intermediatewas effective in bringing about recovery. It is suggested thatCo²⁺ might interfere with process/es which leads to the formationof p-coumaric acid, an intermediate of the anthocyanin biosyntheticpathway. Key words: Sorghum bicolor (L.) Moench, anthocyanin, cobaltous ions, phenylalanine ammonia-lyase     

Differential Biological Effects of K252 Kinase Inhibitors Are Related to Membrane Solubility but Not to Permeability

Abstract: K252a and K252b are related protein kinase inhibitors that, dependent on conditions, can either inhibit or potentiate the effects of neurotrophic factors. K252a, an ester, is more potent and more cytotoxic on intact cells than K252b, a carboxylic acid. To understand better why these drugs elicit different degrees of biological responses, we analyzed their hydrophobicity, cell permeability, and subcellular distribution. As judged by partitioning between organic and aqueous phases, both compounds are hydrophobic. The partition coefficients were 15.6:1 (organic/aqueous phases) for K252a and 4.4:1 for K252b. The ratio of fluorescence excitation at 352 nm to that at 340 nm for the K252 compounds in the organic alcohol 1-decanol versus water provides a simple assay of binding of these compounds to phospholipid membranes. This ratio shifted for K252a, but not K252b, in the presence of phospholipid vesicles, indicating that K252a dissolved in the hydrophobic interior of the membrane. Using quantitative video fluorescence microscopy, we found that K252a strongly labeled both Sf9 insect cells and PC12 rat pheochromocytoma cells, probably staining intracellular membranes. The uptake of K252a was rapid and apparently irreversible. K252b also quickly entered Sf9 and PC12 cells, but staining was much weaker. Hence, K252a and K252b are similar in that they both rapidly enter cells but greatly differ in their membrane solubility.