Requirement of heat and metabolic energy for the expression of inhibitory action of colicin K

Escherichia coli B, induced for β-galactoside permease, can accumulate thiomethyl-β-galactoside in the cell even at 0 °C. At this temperature, cells adsorb colicin K but the adsorbed colicin does not inhibit thiomethyl-β-galactoside uptake. Inhibition by colicin K is, however, seen at 0 °C after exposure of the colicin K-cell complex to a high temperature: a greater degree of inhibition occurs with increasing temperature or duration of exposure. There is a transition point at around 21 °C in Arrhenius plots of this colicin K activation reaction.If inhibitors of energy yielding reactions are present during the heat treatment, the inhibitory action of colicin K (as measured by thiomethyl-β-galactoside uptake after returning the colicin K-cell complex to 0 °C and removal of the inhibitors) is prevented.These results indicate that adsorbed colicin K is converted into the active state only in the presence of metabolic energy and that cell surface fluidity appears to be concerned in this process.     

Genetical and physiological studies on a thermosensitive mutant of Escherichia coli defective in cell division.

A new temperature-sensitive mutant of E. coli, defective in cell division, was isolated after selection for tolerance to colicin E2. The mutant strain, ASHI24, growing in either minimal or complex medium, commences filament formation immediately upon shift to high temperature. High densities of bacteria or the presence of 0-44 M-sucrose prevents filament formation at 42 degrees C and division continues. Filament formation in the mutant is reversible and upon return to 29 degrees C the multinucleate filaments divide up into normal-sized bacteria by a series of rapid but sequential divisions. In the presence of chloramphenicol at 29 degrees C, 25% of these division sites are still expressed. A genetic locus designated ftsH, apparently controlling both temperature sensitivity and filament formation, was provisionally mapped at minute 80 on the E. coli K12 map.     

Temperature-sensitive, colicin M-tolerant mutant of Escherichia coli.

A mutant sensitive to colicin M at 30 degrees C and tolerant at 42 degrees C to high concentrations of colicin M was isolated from Escherichia coli K-12. A temperature shift from 30 to 42 degrees C rescued all cells up to the time they started to lyse at 30 degrees C (25 min after addition of colicin M). The growth rate at 42 degrees C remained unaffected by colicin M. AT 42 degrees C the cell-bound colicin M was inactivated by trypsin, sodium dodecyl sulfate, and antiserum against colicin M. Ferrichrome competed with colicin M at 42 degrees C only during the initial adsorption to the common receptor protein in the outer membrane. Since cells lysed earlier at 30 degrees C when they had been preincubated with colicin M at 42 degrees C, we conclude that the process leading finally to cell lysis is initiated at 42 degrees C and stops at a later stage of colicin M trypsin, dodecyl sulfate, and antiserum when cells were transferred from 30 to 42 degrees C, we assume that colicin M is translocated from its target site towards the cell surface. The mutation conferring tolerance was mapped close to the rpsL gene.     

Effects of temperature and energy inhibitors on complex formation between Escherichia coli male cells and filamentous phage fd

The effect of temperature and various energy inhibitors on the formation of a complex between Escherichia coli male cells and filamentous phage fd was studied by a novel filtration method. Centrifuged male cells were observed by electron microscopy to have lost the majority of pili and to produce complexes with fd only above 25 degrees C. After preincubation of the cells at 37 degrees C without addition of the phage, nearly half the level of complex formation observed at 37 degrees C was detected at 0 degrees C and fd was at a minimum at about 20 degrees C. Several energy inhibitors and uncouplers drastically reduced complex formation at 37 degrees C, and also at 0 degrees C if the cells were briefly exposed to the reagents at the end of preincubation. Alteration of the cellular ATP concentration, either by shift-down of temperature or by the addition of the reagents, accompanied alteration in the ability of cells to form a complex with fd as well as alteration of the number of pili on the cell surface. In contrast to earlier reports, these results indicate that the complex formation between male cells and filamentous phage does not proceed either when pili disappear from the cell surface because of a decrease in the cellular energy level or when pili are removed by mechanical forces. The results also show that phage fd adsorption itself is not energy-dependent.     

The effect of nonreceptor adsorption on the lethal action of colicin E1

The survivability of Escherichia coli K12s cells has been studied after treatment with 125I-labeled colicin E1. It has been shown that for low amounts of adsorbed colicin the survivability follows single-hit kinetics. When the number of colicin molecules adsorbed exceeds approx. 50 per cell, deviation from single-hit kinetics occurs towards higher survivability. Colicin E1 adsorbed nonreceptorwise by the cell's surface has been shown to inhibit the lethal action of colicin E1 molecules adsorbed at specific receptors. This fact has been used in accounting for the elevated survivability of cells at high colicin doses. The functional significance of the phenomenon is discussed.     

Reversal by trypsin of the inhibition of active transport by colicin E1.

The time course for inhibition of proline transport and irreversible loss of cell viability after treatment with colicin E1 was measured as a function of temperature between 13 and 33 degrees C, using a thermostatted flow dialysis system. Complete inhibition of proline transport at 33 and 13 degrees C occurred in 0.5 min and 3 to 5 min, respectively, after addition of colicin E1 at an effective multiplicity of about 4. At these times, the fractional cell survival, assayed by dilution directly from the flow dialysis vessel into trypsin, ranged from 35 to 80%, with viability always greater than 50% at the lower incubation temperatures. Further studies were carried out at 15 degrees C. Complete inhibition of proline transport, which required 2 to 3 min, occurred much more rapidly at 15 degrees C than did the decay of trypsin rescue, which required 10 to 15 min to reach a survival level of 10 to 20%. The direct addition of trypsin to the flow dialysis vessel, after an addition of colicin E1 that caused complete inhibition of proline or glutamine transport, resulted in restoration of net transport. The restored level was typically about 40% of the control rate, and was very similar to the fractional cell viability measured after incubation in trypsin in the same vessel. It is concluded that trypsin can restore active transport to a significant fraction of a cell population in which transport has been initially inhibited by colicin E1.     

Temperature-Sensitive Mutants for the Replication of Plasmids in ESCHERICHIA COLI II. Properties of Host and Plasmid Mutations

Host mutations in Escherichia coli K12 selected for the temperature-sensitive replication of the bacterial plasmid colicinogenic factor E(1) (ColE(1)) exhibit a pleiotropic effect with respect to the effect of the mutation on other extra-chromosomal elements. The mutations also vary with respect to the time of incubation of the cells at 43 degrees C required for complete cessation of ColE(1) DNA synthesis. While the synthesis of the bacterial chromosome appears unaffected, supercoiled ColE(1) DNA replication stops immediately in some mutants and gradually decreases during several generations of cell growth before stopping in others. Mutations isolated in the ColE(1) plasmid resulted in only a gradual cessation of ColE(1) DNA synthesis over several generations of cell growth at 43 degrees C. Conjugal transfer of the ColE(1) and ColV factors occurs normally in the host mutants when the transfer is carried out at the permissive temperature; however, the presence of a group I mutation in the donor cell prohibited conjugal transfer of either plasmid DNA at 43 degrees C to a normal recipient cell. Similarly, the presence of this mutation in the recipient prevented the establishment of ColE(1) or ColV in the mutant recipient cell upon conjugation with a normal donor at 43 degrees C. Various host ColE(1) replication mutants carrying either ColE(1) or ColE(2) were also defective in the mitomycin C-induced production of colicin E(1) or colicin E(2) at 43 degrees C. The majority of the host mutations examined exhibited a temperature sensitivity to growth in deoxycholate in addition to the inhibition of plasmid DNA replication, suggesting a membrane alteration in these mutants when grown at the restrictive temperature.     

Mechanism of colicin action: early events

The kinetics and the temperature dependence of potassium loss from Escherichia coli cells treated with colicin K have been examined. At 37 C, after a single lethal hit, essentially all of the intracellular potassium is lost within the first few minutes of treatment. The initial rate of loss is linearly related to colicin concentration up to a multiplicity of 30. As the temperature is decreased over the range from 37 to 1 C, an increasing delay is seen in the initiation of potassium loss after colicin adsorption. This delay can be overcome by increasing colicin multiplicity and probably reflects an alteration of the cell membrane at these temperatures. A comparison of this effect with an apparently related effect of temperature on the action of irehdiamine A indicates that the delay may represent the inhibition of a transmission process occurring in the membrane.     

Effect of temperature on alanine uptake by membrane vesicles isolated from a psychrophilic marine bacterium.

The effect of temperature on the membranes of Ant-300, a psychrophilic marine bacterium, was studied by measuring alanine uptake by isolated membrane vesicles. Uptake was observed from 0 to 35 degrees C. The maximum initial rate of uptake occurred at 25 degrees C although more alanine was ultimately taken up at temperatures from 10 to 20 degrees C. An ARRHENIUS plot of these data shows a single infection point at 7.8 degrees C. Within 10 min, over 50% of the alpha-aminoisobutyric acid taken up by whole cells at 5 degrees C was lost after a temperature shift to 25 degrees C. Vesicles preloaded with alanine at 5 degrees C did not become leaky when shifted to 25 degrees C. In addition, exposure of the vesicles to 25 degrees C for 30 min did not affect subsequent alanine uptake at 5 degrees C. The data obtained suggest that the loss of the uptake and permeability control functions of membranes from psychrophilic bacteria at elevated temperatures is not due to degeneration of the membrane itself, but rather to a control or regulatory mechanism associated with whole cells.     

Characteristics of ion transport in kidney cortex of mammalian hibernators

Slices of kidney cortex of two species of hibernating mammals (hamsters and ground squirrels) have been leached of K, and their subsequent ability to reaccumulate K in vitro has been determined at temperatures between 38° and 0°C. At 5°C (body temperature of a hibernating mammal) uptake is appreciable in kidney cortex of both species. In the kidney cortex of hamsters, for example, the tissue K of slices incubated at 5°C reaches the same steady-state concentration after 2 hours that is observed in slices at 38°C after 20 minutes. At 0°C there is also a measurable uptake. This K transport is blocked by metabolic inhibitors and, in ground squirrel kidneys, by ouabain. In kidney cortex slices from guinea pigs net K accumulation is slight at 5°C and absent at 0°C. The initial rapid uptake of K at 38°C occurs at the same rate in kidney cortex slices of hamsters as in those of rabbits. Lowering the temperature of incubation decreases this initial rate of uptake in hamster kidney slices with a Q ₁₀ of 1.8 between 38° and 15° and of 5.7 between 15° and 0°C. In hamsters this uptake of K has been shown to require the outward extrusion of Na. Conversely, about half of the outward extrusion of Na requires K in the medium, while the remainder appears to be independent of K. The conclusions warranted are that kidney cells of hibernators possess an unusual ability to transport ions at low temperature, that this ability does not depend upon a more rapid rate at higher temperatures, and that the characteristics of transport at low temperature are qualitatively similar to those at 38°C in cells of nonhibernators.     

Involvement of OmpF during reception and translocation steps of colicin N entry

[125I]-colicin N binds to OmpF receptor sites (70,000 per cell) with an average Kassoc of 3.2 x 10(6) M-1 at 23 degrees C. Monoclonal antibody directed against a cell-surface-exposed epitope of OmpF is able to complete with the binding of the colicin in vitro and also to protect against colicin N in vivo. OmpF is an absolute requirement for colicin N uptake. OmpC cannot serve as a substitute for OmpF during translocation across the outer membrane under receptor bypass conditions, which is in contrast to colicin A. Colicin N does not cross-react with various monoclonal antibodies directed against colicin A.     

Cultured Chinese hamster cells undergo apoptosis after exposure to cold but nonfreezing temperatures

Cultured Chinese hamster V79 fibroblast cells at the transition from logarithmic to stationary growth have been shown to undergo apoptosis (programmed cell death) after cold shock [B. L. Soloff, W. A. Nagle, A. J. Moss, Jr., K. J. Henle, and J. T. Crawford, Biochem. Biophys. Res. Commun. 145, 876-883 (1987)]. In this report, we show that about 95% of the cell population was susceptible to cold-induced apoptosis, and the amount of cell killing was dependent on the duration of hypothermia. Cells treated for 0-90 min at 0 degrees C exhibited an exponential survival curve with a D0 of 32 min; thus, even short exposures to the cold (e.g., 5 min) produced measurable cell killing. The cold-induced injury was not produced by freezing, because similar results were observed at 6 degrees C, and cell killing was not influenced by the cryoprotective agent dimethyl sulfoxide. Cold-induced apoptosis was inhibited by rewarming at 23 degrees C, compared to 37 degrees C, by inhibitors of macromolecular synthesis, such as cycloheximide, and by 0.8 mM zinc sulfate. The results suggest that apoptosis represents a new manifestation of cell injury after brief exposure to 0-6 degrees C hypothermia.     

The mechanism of [3H]dexamethasone uptake into prolactin producing rat pituitary cells (GH3 cells) in culture

Glucocorticosteroids stimulate growth hormone (GH) synthesis and inhibit prolactin (PRL) synthesis and cell growth in cultured GH3 cells, a clonal cell strain derived from a rat pituitary tumour. This model system was used to study the mechanism by which glucocorticosteroids enter target cells. The cellular uptake of [3H]dexamethasone was temperature dependent and was further inhibited by addition of an excess amount of cold dexamethasone. Half maximal uptake was obtained after about 5 min at 37 degrees C. The initial rates of [3H]dexamethasone uptake were a linear function of the extracellular hormone concentration. The uptake of [3H]dexamethasone in intact cells studied at different temperatures resulted in linear Arrhenius plots, with a calculated energy of activation of 91.0 kJ x mole-1 x degree-1. Scatchard analysis of specifically cell bound [3H]dexamethasone at equilibrium (0 degrees C) showed a straight line with a calculated dissociation constant (Kd) of 1.6 x 10(-9) M and a maximal uptake of 180 x 10(-15) mole/mg cell protein. Specific binding of [3H]dexamethasone to cytosol proteins could only be demonstrated at 0 degrees C. These results indicate that [3H]dexamethasone diffuses passively into the cell, and binds to specific receptors in an energy dependent way.     

Temperature Dependence of Drug Interaction with the Platelet 5-Hydroxytryptamine Transporter: A Clue to the Imipramine Selectivity Paradox

Although [3H]imipramine is a selective radioligand for the 5-hydroxytryptamine (5-HT) transporter in human platelets, its affinity for binding to the 5-HT transporter complex at 0 degrees C (0.6 nM) is significantly higher than its potency for inhibition of [3H]5-HT uptake at the physiological temperature of 37 degrees C (Ki = 29 nM). As this apparent discrepancy could be related to the assay temperature, we studied the thermodynamics of drug interaction with the 5-HT transporter at assay temperatures between 0 degrees C and 37 degrees C, using as radioligands [3H]imipramine (0 degrees C and 20 degrees C) and [3H]paroxetine (20 degrees C and 37 degrees C), a newly available probe for the 5-HT transporter. At 20 degrees C, Ki values of 14 tricyclic and nontricyclic drugs for inhibition of [3H]imipramine and [3H]paroxetine binding to human platelet membranes were highly significantly correlated (r = 0.98, p less than 0.001), validating the use of these two radioligands to study the 5-HT transporter over a temperature range larger than was previously possible with [3H]imipramine alone. The affinity of imipramine for the 5-HT transporter is progressively enhanced with decreasing incubation temperature, thus favoring the selectivity of [3H]imipramine for the 5-HT transporter at 0 degrees C. At 37 degrees C, the Ki of imipramine for inhibition of [3H]paroxetine binding is 32 nM, and equals its Ki value for inhibition of 5-HT uptake into human platelets. With the exception of chlorimipramine, other tricyclic 5-HT uptake inhibitors showed a temperature sensitivity in their interaction with the 5-HT transporter similar to that of imipramine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulatory Properties of Acetokinase from Veillonella alcalescens

Ghosts of T4 bacteriophage inhibit the uptake of thiomethyl-beta-galactoside (TMG), alpha-methylglucoside, glucose-6-phosphate, and glycerol in Escherichia coli B. The transport of orthonitrophenyl-beta-galactoside (ONPG) is also inhibited to a lesser degree and without alteration of the apparent K(m) of transport. These effects of ghosts parallel those of energy poisons on these systems. However, no one energy poison can produce such pronounced inhibitory effects in all these systems. The effect of the intact phage in these systems was either absent or very slight relative to the ghost. The effect of ghosts on the uptake of TMG was not immediate; at 10 C, no effect of the ghosts was apparent for at least 2 min. This suggests that a step, more temperature dependent than the attachment of the ghost, is necessary for the inhibitory action. The intracellular level of adenosine triphosphate (ATP) in the ghost-infected cells fell to less than 25% of the control value, and the ATP lost from the cell appeared in extracellular medium. Phage, on the other hand, caused no decrease in the intracellular ATP level. This loss of ATP from the cells after ghost infection suggests an alteration of the barrier properties of the membrane so that ATP can leave the cell; however, the accessibility of extracellular ONPG to intracellular beta-galactosidase does not increase. The dissimilarity of the actions of phage and ghosts on all properties examined does not support the model that the initial events in their infections are identical but that the intact phage, unlike the ghost, can provide information for the repair of its effects.     

Effect of temperature on H+-K+ exchange in Escherichia coli bacteria during their anaerobic growth

The H(+)-K(+)-exchange in E.coli grown under anaerobic conditions at temperatures from 17 to 37 degrees C was studied. The Arrhenius plots for both the N,N'-dicyclohexylcarbodiimide-sensitive release of H+ and K+ uptake by cells transferred into a fresh medium containing a carbon source (glucose) are nonlinear. The activation energy values for the transport of these cations at different temperatures significantly differ. It is shown that as the temperature decreases, the accumulation of K+ by cells is reduced. In this process, the initial rate of K+ absorption through the TrkA system, the time of accumulation of these cations by cells and the osmosensitivity of K+ uptake substantially decrease. At temperatures below 20 degrees C, the absorption becomes insensitive to the secondary osmoshock. However, the stoichiometry of N,N'-dicyclohexylcar-bodiimide-sensitive cation fluxes remains unchanged and is equal to 2H+:K+. It is assumed that the H(+)-K(+)-exchange proceeds by the operation of an ensemble of oligomers, formed from the protomers of F0F1 and TrkA, which rearrange by the action of temperature, whereas F0F1 and TrkA in each protomer do not change.     

Binding and internalization of 125I-alpha 2-macroglobulin by cultured fibroblasts

The binding and internalization of 125I-labeled alpha 2-macroglobulin (125I-alpha 2M) was studied in cultured fibroblasts. Two classes of binding sites were detected on cell surfaces. One class corresponds to the previously described, high affinity and low capacity sites. The other class of binding sites may mediate uptake of high physiological blood levels of 125I-alpha 2M and has not been described previously. At 0 degrees C, this lower affinity class saturates at approximately 1,000 micrograms/ml and has a capacity of approximately 600,000 sites/cell. The lower affinity class accounts for the vast majority of cellular receptors for alpha 2M. An assay employing pepsin at pH 4 was developed to distinguish between surface-bound and internalized 125I-alpha 2M. Cellular uptake of 125I-alpha 2M at 37 degrees C has a component which saturates between 200 and 1,000 micrograms/ml and the rate of internalization of this component was approximately 1,700,000 molecules/cell/h. One mM Ca2+ was required for cell uptake of 125I-alpha 2M at 37 degrees C. Ca2+ was also required for binding at 0 degrees C to both low and high affinity classes of binding sites for 125I-alpha 2M. The transglutaminase inhibitors bacitracin, monodansylcadaverine, and N-benzyloxycarboxyl-5-diazo-4-oxonorvaline paranitrophenyl ester all inhibited cellular internalization of 125I-alpha 2M at 37 degrees C. Each of these three compounds selectively reduced 125I-alpha 2M binding to the high affinity, low capacity component at 0 degrees C. Based on the current binding studies and previous studies using electron microscopy which showed that bacitracin and other transglutaminase inhibitors block clustering of alpha 2M-receptor complexes in coated pits, we suggest that the inhibitors block the accumulation of occupied lower affinity alpha 2M receptors in coated pits where they acquire a higher apparent affinity.     

Calcium-ion transport by intact Ehrlich ascites-tumour cells. Role of respiratory substrates, Pi and temperature.

1. The interaction of intact Ehrlich ascites-tumour cells with Ca2+ at 37 degrees C consists of Ca2+ uptake followed by efflux from the cells. Under optimum conditions, two or three cycles of uptake and efflux are observed in the first 15 min after Ca2+ addition. 2. The respiratory substrates malate, succinate and ascorbate plus p-phenylenediamine support Ca2+ uptake. Ca2+ uptake at 37 degrees C is sensitive to the respiratory inhibitors rotenone and antimycin A when appropriate substrates are present. Ca2+ uptake and retention are inhibited by the uncoupler S-13. 3. Increasing extracellular Pi (12 to 30 mM) stimulates uncoupler-sensitive Ca2+ uptake, which reaches a maximum extent of 15 nmol/mg of protein when supported by succinate respiration. Ca2+ efflux is partially inhibited at 30 mM-Pi. 4. Optimum Ca2+ uptake occurs in the presence of succinate and Pi, suggesting that availability of substrate and Pi are rate-limiting. K. Ca2+ uptake occurs at 4 degrees C and is sensitive to uncouplers and oligomycin. Ca2+ efflux at this temperature is minimal. These data are consistent with a model in which passive diffusion of Ca2+ through the plasma membrane is followed by active uptake by the mitochondria. Ca2+ uptake is supported by substrates entering respiration at all three energy-coupling sites. Ca2+ efflux appears to be an active process with a high temperature coefficient.