Thermal events associated with active membrane transport in Escherichia coli

Active transport of non-metabolizable compounds by $\text{Escherichia coli}$ resulted in thermogenesis. With substrates of the lactose permease (thiomethyl galactoside, lactose) and of the glucose transport system (α-methylglucoside) the rate of heat production was largest on initial addition, but then decreased. The kinetics of heat production varied with the transport system. For the lactose transport system, more than turnover of the permease was required since heat was not produced in azide treated cells, where facilitated diffusion is known to take place. The lactose permease thermal effects are suggested to reflect operation of the energy coupling system. The thermal effects are considered to represent a useful approach in studying transport energetics and mechanisms.     

Genetics of the glutamine transport system in Escherichia coli.

The active transport of glutamine by Escherichia coli occurs via a single osmotic shock-sensitive transport system which is known to be dependent upon a periplasmic binding protein specific for glutamine. We obtained a mutant that had elevated levels of glutamine transport and overproduced the glutamine binding protein. From this strain many point mutants and deletion-carrying strains defective in glutamine transport were isolated by a variety of techniques. The genetic locus coding for the glutamine transport system, glnP, and the regulatory mutation which causes overproduction of the transport system were both shown to map at 17.7 min on the E. coli chromosome, and it was demonstrated that the glnP locus contains the structural gene for the glutamine binding protein. Evidence was also obtained that the glutamine transport system, by an unknown mechanism, plays a direct role in the catabolism of glutamate and, hence, of glutamine and proline as well.     

Stimulation of glutamine transport by osmotic stress in Escherichia coli K-12.

Osmotic stress produced by high concentrations of sucrose stimulated the high-affinity transport of glutamine in Escherichia coli cells. Glutamine transport via a low-affinity system was not affected. Osmotic stress produced by NaCl, in contrast, inhibited the transport of glutamine and some other amino acids. Maltose transport was strongly inhibited by osmotic stress.     

Energetic studies of lactose active transport in Escherichia coli membrane vesicles

The energetics of D-lactate-driven active transport of lactose in right-side-out Escherichia coli membrane vesicles has been investigated with a microcalorimetric method. Changes of enthalpy (delta Hox), free energy (delta Gox), and entropy (delta Sox) during the D-lactate oxidation reaction in the presence of membrane vesicles are -39.9 kcal, -46.4 kcal, and 22 cal/deg per mole of D-lactate, respectively. The free energy released by this reaction is utilized to form a proton electrochemical potential (delta-microH+) across the membrane. The higher observed heat in the D-lactate oxidation reaction in the presence of carbonylcyanide m-chlorophenylhydrazone (a proton ionophore) supports the postulate that delta-microH+ is formed across the membrane vesicles. Thermodynamic quantities for the formation of delta-microH+ are delta Hm = 14.1 kcal, delta Gm = 0.6 kcal, and delta Sm = 45 cal/deg per mole of D-lactate. The efficiency in the free energy transfer from the oxidation reaction to the formation of delta-microH+ (defined by delta Gm/delta Gox) was 2%, as compared to that in the heat transfer (defined by delta Hm/delta Hox) of 35%. The energetics of the movement of lactose in symport with proton across the membrane as a consequence of the formation of delta-microH+ are delta H1 = -19 kcal, delta G1 = -0.5 kcal, and delta S1 = -62 cal/deg per mole of lactose. No heat of reaction is contributed by lactose movement across the membrane without symport with H+.     

Studies on energy supply for genetic processes. Requirement for membrane potential in Escherichia coli infection by phage T4

In this study the hypothesis considering the requirement for an electrochemical proton gradient in the injection of phage T4 DNA into Escherichia coli cell has been verified experimentally. The phage caused a reversible depolarization of cell membrane, while phage 'ghosts' induced an irreversible depolarization. The phage infection was strictly dependent on E. coli membrane potential value when phage/cell ratio was 5 and higher. When the ratio was close to 1, the decrease in the membrane potential up to -100 mV caused practically no effect on the phage infection. The infection inhibition was observed when the membrane potential was lowered below this 'threshold' value. On the other hand, the decrease in the membrane potential caused no effect on the phage infection under conditions promoting a concomitant increase in the value of the transmembranous pH gradient. The phage DNA transfer through the membrane of ATPase-deficient cells was reversibly inhibited by switching off the respiratory chain - the sole generator of a protonmotive force in these mutant cells. The membrane should be kept in the energized state during the phage DNA entrance into the cell. Adsorption of the phage on E. coli was followed by the reversible release of the respiratory control. Thus the results presented here indicate the requirement of the electrochemical proton gradient across the plasma membrane for injection of phage T4 DNA into E. coli. They support the concept postulating an expenditure of host cell metabolic energy for phage T4 DNA transfer through the membrane.     

Characterization of an active transport system for calcium in inverted membrane vesicles of Escherichia coli.

The energy-dependent uptake of calcium by inverted membrane vesicles of Escherichia coli was investigated. Methods for preparation and storage of the vesicles were devised to allow for the maximal activity and stability of the calcium transport system. The pH and temperature optima for the reaction were observed to occur at pH 8.0 AND 30 DEGREES, RESPECTIVELY. The eft was found that the extent of the reaction depended on the presence of phosphate or oxalate. Phosphate was found to enter the vesicles at a rate slower than that of calcium. A Ca2+:Pi ratio of approximately 1.5 was found, suggesting formation of Ca3(PO4)2. Monovalent cations stimulated calcium uptake, with the order of effectiveness being K+ is greater than Na+ is greater than Li+ is greater than NH4+. Inhibition was found with certain divalent cations, but these also inhibited the electron transport chain. Of the divalent cations examined only Mg2+ and Sr2+ inhibited calcium transport without a corresponding inhibition of respiration. Calcium transport exhibited biphasic Kinetics, with a low affinity system and a high affinity system. The low affinity system showed a Km of 0.34 mM and a Vmax of 85 nmol/min/mg of protein. The kinetic constants of the high affinity system were 4.5 muM and 2 nmol/min/mg of protein. The energy for calcium transport could be derived from the electron transport chain by oxidation of NADH, D-lactate, and succinate, in order of their effectiveness. Respiration-driven calcium transport was inhibited by inhibitors of the electron transport chain and by uncouplers of oxidative phosphorylation. ATP could also be used to supply enerty for calcium transport. The ATP-driven reaction was inhibited by inhibitors of the Mg2+ATPase and by an antiserum prepared against that protein, demonstrating that that enzyme is involved in the utilization of ATP for active transport in inverted vesicles.     

Inactivation of membrane transport in Escherichia coli by near-ultraviolet light.

Evidence is presented that near-ultraviolet (near-UV) light can alter galactoside transport in Escherichia coli in several independent ways. It can inactivate the permease system per se, it can interfere with metabolic energy production or transfer, and it can cause an increase in the generalized permeability of the membrane. Earlier publications suggested that near-UV destroys cofactors needed for electron transport and thus places a limitation on energy reserves. In agreement, we found that the active accumulation of [14C]thiomethyl-beta-D-galactopyranoside is decreased after irradiation by a larger factor than that due to action directly on the permease system. The effect on the latter was measured by the decrease in the rate of o-nitrophenyl-beta-D-galactopyranoside (ONPG) transport. As evidence that energy supplies for this "downhill" process did not become rate limiting after irradiation, we found that carbonylcyanide-m-chlorophenyl-hydrazone did not stimulate ONPG transport of irradiated cells. Cells genetically deficient in functional permease or cells treated with formaldehyde still transport ONPG passively, although at much lower rates. With the use of such cells, it was found that high fluences (doses) made the cells leaky. Further evidence that the permease system and the metabolic energy system can be inactivated independently is also presented. It is shown that a photoproduct from the irradiation of chloramphenicol inactivates the permease system much more efficiently than the energy system. In addition, it is shown that thio-beta-D-digalactopyranoside protects the permease system, but not the energy system, both against direct inactivation by near-UV and against photosensitized inactivation in the presence of chloramphenicol.     

Feedback inhibition of ammonium (methylammonium) ion transport in Escherichia coli by glutamine and glutamine analogs.

When cultured with glutamate or glutamine as the nitrogen source, Escherichia coli expresses a specific ammonium (methylammonium) transport system. Over 95% of the methylammonium transport activity in washed cells was blocked by incubation with 100 microM L-glutamine in the presence of chloramphenicol (100 micrograms/ml). The time course for the onset of this glutamine inhibition followed a first-order rate expression with a t1/2 of 2.8 min. The inhibition of transport by L-glutamine was noncompetitive (Ki = 18 microM) with respect to the [14C]methylammonium substrate. D-Glutamine had no significant effect. The glutamine analogs gamma-L-glutamyl hydroxamate (Ki = 360 microM) and gamma-L-glutamyl hydrazide (Ki = 800 microM) were also noncompetitive inhibitors of methylammonium transport, suggesting that glutamine metabolism is not required. The role of the intracellular glutamine pool in the regulation of ammonium transport was investigated by using mutants carrying defects in the operon of glnP, the gene for the glutamine transporter. The glnP mutants had normal rates of methylammonium transport but were refractory to glutamine inhibition. Glycylglycine, a noncompetitive inhibitor of methylammonium uptake in wild-type cells (Ki = 43 microM), was equipotent in blocking transport in glnP mutants. Although ammonium transport is also subject to repression by growth of E. coli in the presence of ammonia, this phenomenon is unrelated to glutamine inhibition. A GlnL RegC mutant which constitutively expressed ammonium transport activity exhibited a sensitivity to glutamine inhibition similar to that of wild-type cells. These findings indicate that ammonium transport in E. coli is regulated by the internal glutamine pool via feedback inhibition.     

Mechanisms of active transport in isolated bacterial membrane vesicles. XII. Active transport by a mutant of Escherichia coli uncoupled for oxidative phosphorylation

Amino acid and β-galactoside transport activity catalyzed by whole cells and membrane vesicles prepared from an Escherichia coli mutant uncoupled for oxidative phosphorylation is comparable to the activity of analogous preparations from the parent strain. Valinomycin-induced rubidium uptake is also similar in membrane vesicles prepared from wild-type and mutant cells. The properties of the transport systems in mutant vesicles are the same as those of wild-type vesicles with respect to electron donors which stimulate transport, and with respect to inhibition by anoxia, cyanide, and 2,4-dinitrophenol.Magnesium ion markedly stimulates the ATPase activity of wild-type membrane vesicles and ethylenediaminetetraacetate markedly inhibits. However, these compounds have relatively slight effects on either the initial rate or extent of transport. Dicyclohexylcarbodiimide does not inhibit respiration-dependent transport despite inhibition of the calcium, magnesium-activated ATPase activity of wild-type vesicles.These results confirm earlier observations indicating that oxidative phosphorylation is not involved in respiration-linked active transport.     

pH-dependent changes in proton:substrate stoichiometries during active transport in Escherichia coli membrane vesicles.

Experiments are presented in which the proton electrochemical gradient (deltamuH+) IN Escherichia coli membrane vesicles (interior negative and alkaline) was measured under a variety of conditions and compared with steady-state levels of accumulation of lactose, proline, D-lactate, and glucose-6-P measured under identical conditions. Accumulation of lactose and proline is proportional to the magnitude of deltamuH+ at pH 5.5, where the pH gradient (deltapH) and the electrical potential (deltapsi) both contribute to deltamuH+, and at pH 7.5, where deltapsi represents the only component of deltamuH+. Moreover, the proportionality constants between deltamuH+ and lactose or proline accumulation indicate that the proton:substrate stoichiometries are 1:1 at pH 5.5 and 2:1 at pH 7.5. Evidence is also presented which indicates that the functional group responsible for the increase in proton:proline stoichiometry has a pK of approximately 6.8. Accumulation of D-lactate and glucose-6-P is directly related to the magnitude of deltapH at pH 5.5, and stoichiometry values of one and approximately 1.7 are obtained for D-lactate and glucose-6-P, respectively, at this pH. At pH 7.5, on the other hand, accumulation of each organic acid bears a linear relationship to deltapsi, and proton:substrate stoichiometries of unity are observed in both instances. The results are consistent with the models discussed by Rottenberg (Rottenberg, H. (1976), FEBS Lett. 66, 159).