The effect of beta-galactosides on the protonmotive force and growth of Escherichia coli

The effect of three beta-galactosides on the components membrane potential (delta psi) and pH gradient (delta pH) of protonmotive force and growth of Escherichia coli has been examined. A good correlation between the reduction of the protonmotive force and growth inhibition was observed. Thus some galactosides had little effect on either the protonmotive force or growth while lactose diminished the protonmotive force and caused growth inhibition. This effect of lactose was dependent on the ionic composition of the growth media. In Medium A (77 mM-Na+, 85 mM-K+) lactose diminished delta psi but had no effect on delta pH. Growth inhibition was transient at an external pH 6.0 but complete at pH 7.5. In medium KA (approximately 1 mM-Na+, 162 mM-K+) delta pH was diminished and delta psi was not affected and consequently growth inhibition was complete at pH 6.0. In medium NA (163 mM-Na+, 20 mM-K+) lactose had little effect on delta psi, delta pH or growth. These data support Skulachev's hypothesis of buffering of the protonmotive force by K+ and Na+ gradients.     

The requirement for energy during export of beta-lactamase in Escherichia coli is fulfilled by the total protonmotive force.

The energy requirement for the maturation and export of the plasmid-encoded TEM beta-lactamase in Escherichia coli K12 was shown to be fulfilled by the total protonmotive force. This was demonstrated by assessing the inhibition of proteolytic processing of the precursor form of beta-lactamase caused by perturbation of the energized state of the membrane in cells treated with valinomycin. The magnitude of the membrane potential was manipulated by varying the concentration of KCl in the medium and the pH gradient was manipulated by varying the external pH. Both components were simultaneously affected by addition of the protonophore carbonylcyanide-p- trifluoromethoxy phenylhydrazone (FCCP). Inhibition of processing was demonstrated in a mutant strain having a defective ATP synthase where protonmotive force could be dissipated without altering the intracellular level of ATP, indicating that the observed inhibition was not the result of decreased ATP concentration. Half-maximal accumulation of precursor of beta-lactamase was observed in all cases when the level of protonmotive force was decreased to approximately 150 mV. Under those conditions the membrane potential varied from 65 to 140 mV (internally negative) and the pH gradient from 95 to 25 mV (internally alkaline). Thus, the energy requirement is satisfied by the total protonmotive force, with no specificity for either the membrane potential or the pH gradient.     

Amplification of the riboflavin operon genes of Bacillus subtilis in Escherichia coli cells]

Amplification of Bacillus subtilis DNA fragments was performed in Escherichia coli using plasmid RSF2124. The main principle of isolation and cloning hybrid plasmids was described using genes of riboflavin operon as a model. Bac. subtilis DNA was treated with restriction endonuclease EcoR; followed by the agarose gel electrophoretic separation of the resulting fragments. Gels were sliced, DNA was eluted from the corresponding slices and used to transform Bac. subtilis auxotrophs rib A72, rib S110 and rib D107. DNA fraction with the molecular weight 7 . 10(6) daltons restored prototrophy of these mutants. DNA of this fraction was ligated with EcoRI treated plasmid RSF2124 DNA and used for transformation of E. coli rk-mk+. Ampicillin resistant transformants which had lost the colicin production ability, were selected. The presence of riboflavin genes within the hybrid plasmids was detected by transformation of B. subtilis auxotrophs. Three hybrid plasmids (pPR1, pPR2 and pPR3), containing a fragment of Bac. subtilis DNA with the molecular weight 6.8 . 10(-6) daltons including riboflavin operon, were selected. The analysis of the transformation activity of Bac. subtilis DNA and plasmid pPR1 DNA revealed, that there was no restriction activity of Bac. subtilis cells against plasmid DNA amplified in E. coli. Heteroduplex analysis has shown that plasmids pPR1 and pPR2 differ in the orientation of Bac. subtilis DNA fragment. DNA of these plasmids restored prototrophy of the several studied E. coli riboflavin auxotrophs.     

The effect of sorbic acid and esters of p-hydroxybenzoic acid on the protonmotive force in Escherichia coli membrane vesicles

The effect of three food preservatives, sorbic acid and methyl and butyl esters of p-hydroxybenzoic acid, on the protonmotive force in Escherichia coli membrane vesicles was investigated. Radioactive chemical probes were used to determine the two components of the protonmotive force: delta pH (pH difference) and delta psi (membrane potential). Both types of compound selectively eliminated delta pH across the membrane, while leaving delta psi much less disturbed indicating that transport inhibition by neutralization of the protonmotive force cannot be the only mechanism of action for the food preservatives tested.     

Effect of EcoRI restrictase on the transforming DNA of different bacilli]

The transforming activity of DNA from Bacillus subtilis 168, Bac. subtilis W23, Bac. subtilis NRS and Bac. aterrimus after the EcoRI restrictase treatment was studied. The auxotrophic strains of Bac. subtilis 168 were used as recipients in bacterial transformation. The transforming activity for different markers decreased up to 0.001--6 per cent from the initial level for different Bacilli DNAs. In some cases the sensitivity of the same marker from different Bacilli differed in more than 50 times. Bac. subtilis and Bac. aterrimus have demonstrated the maximal differences. Such differences can be used for the identification of close related Bacilli.     

Construction of plasmids integrating into the Bacillus subtilis chromosome through homologous recombination and their use as integration vectors

We constructed a number of plasmids which integrate into the chromosome of Bacillus subtilis through homology recombination. Plasmids consist of pBR322 replicon, different fragments of Bac. subtilis chromosomal DNA, Cm resistance marker from pBD64 plasmid. Frequency of transformation was 10(-4) per bacterial cell. Foreign DNA (genes for tryptophan metabolism of Bac. mesentericus) was introduced into the chromosome of Bac. subtilis with the help of these plasmids.     

Protonmotive force as the source of energy for adenosine 5''-triphosphate synthesis in Escherichia coli.

Net synthesis of adenosine 5'-triphosphate (ATP) in energy-depleted cells of Escherichia coli was observed when an inwardly directed protonmotive force was artificially imposed. In wild-type cells, ATP synthesis occurred whether the protonmotive force was dominated by the membrane potential (negative inside) or the pH gradient (alkaline inside). Formation of ATP did not occur unless the protonmotive force exceeded a value of 200 mV. Under these conditions, no ATP synthesis was found when cells were exposed to an inhibitor of the membrane-bound Ca2+- and Mg2+- stimulated adenosine triphosphatase (EC 3.6.1.3), dicyclohexylcarbodiimide, or to a proton conductor, carbonylcyanide-p-trifluoromethoxyphenyl-hydrazone. Adenosine triphosphatase-negative mutants failed to show ATP synthesis in response to either a membrane potential or a pH gradient. ATP synthesis driven by a protonmotive force was observed in a cytochrome-deficient mutant. These observations are consistent with the chemiosmotic hypothesis of Mitchell (1961, 1966, 1974).     

Bacillus licheniformis as an object for the propagaton of heterologous genetic material from bacilli

Bacillus licheniformis was transformed with plasmids pUB110 and pJJ10 (pUB110 - pBR322) isolated from Bac. subtilis and Escherichia coli, respectively. It was revealed that the structure and genetic properties of the plasmids did not change during the transformation process. pJJ101 (pJJ10-rib) DNA isolated from E. coli and containing helper pJJ10 plasmid was used, as a recipient. It was shown that pJJ101 rib markers were "rescued" by the resident plasmid during transformation of Bac. licheniformis (pJJ10). Plasmid pLP1 containing ribB, ribD, Kmr genes and the pUB110 replicator, was isolated from the transformants. pLP1 plasmid might be considered as a detected derivative of the parental pJJ101 plasmid. The deletion is presented by 3,9 MD segment that contains the pBR322 replicator. pLP1 DNA is capable of transforming plasmidless strains of Bac. licheniformis and Bac. subtilis.     

Evidence for an electrogenic 3-deoxy-2-oxo-D-gluconate--proton co-transport driven by the protonmotive force in Escherichia coli K12.

Evidence is presented indicating that the carrier-mediated uptake of 3-deoxy-2-oxo-D-gluconate and D-glucuronate in Escherichia coli K12 is driven by the deltapH and deltapsi components of the protonmotive force. 1. Approximately two protons enter the cells with each sugar molecule, independent of the sugar and the strain used. 2. In respiring cells, the magnitude of the pH gradient alone, as measured by distribution of [3H]acetate, appears to be insufficient to account for the chemical gradient of 3-deoxy-2-oxo-D-gluconate that is developed between pH 6.0 and 8.0. 3. If the external pH is varied between 5.5 and 8.0, 3-deoxy-2-oxo-D-gluconate uptake is gradually inhibited by valinomycin plus K+ ions, whereas the inhibition caused by nigericin is concomitantly relieved, thus reflecting the relative contribution of deltapH and deltapsi to the total protonmotive force at each external pH. 4. 3-Deoxy-2-oxo-D-gluconate can be transiently accumulated into isolated membrane vesicles in response to an artificially induced pH gradient. The process is stimulated when the membrane potential is collapsed by valinomycin in the presence of K+ ions.     

Ca2+-induced permeabilization of the Escherichia coli outer membrane: comparison of transformation and reconstitution of binding-protein-dependent transport.

Ca2+ treatment renders the outer membrane of Escherichia coli reversibly permeable for macromolecules. We investigated whether Ca2+-induced uptake of exogenous protein into the periplasm occurs by mechanisms similar to Ca2+-induced uptake of DNA into the cytoplasm during transformation. Protein import through the outer membrane was monitored by measuring reconstitution of maltose transport after the addition of shock fluid containing maltose-binding protein. DNA import through the outer and inner membrane was measured by determining the efficiency of transformation with plasmid DNA. Both processes were stimulated by increasing Ca2+ concentrations up to 400 mM. Plasmolysis was essential for a high efficiency; reconstitution and transformation could be stimulated 5- and 40-fold, respectively, by a high concentration of sucrose (400 mM) in cells incubated with a suboptimal Ca2+ concentration (50 mM). The same divalent cations that promote import of DNA (Ca2+, Ba2+, Sr2+, Mg2+, and Ni2+) also induced import of protein. Ca2+ alone was found to be inefficient in promoting reconstitution; successive treatment with phosphate and Ca2+ ions was essential. Transformation also was observed in the absence of phosphate, but could be stimulated by pretreatment with phosphate. The optimal phosphate concentrations were 100 mM and 1 to 10 mM for reconstitution and transformation, respectively. Heat shock, in which the cells are rapidly transferred from 0 to 42 degrees C, affected the two processes differently. Incubation of cells at 0 degrees C in Ca2+ alone allows rapid entry of protein, but not of DNA. Transformation was observed only when exogenous DNA was still present during the heat shock. Shock fluid containing maltose-binding protein inhibited transformation (with 6 microgram of DNA per ml, half-maximal inhibition occurred at around 300 microgram of shock fluid per ml). DNA inhibited reconstitution (with 5 microgram of shock fluid per ml, half-maximal inhibition occurred at around 3 mg of DNA per ml).     

Stimulation of menaquinone-dependent electron transfer in the respiratory chain of Bacillus subtilis by membrane energization

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The NRAMP family of metal-ion transporters

The family of NRAMP metal ion transporters functions in diverse organisms from bacteria to human. NRAMP1 functions in metal transport across the phagosomal membrane of macrophages, and defective NRAMP1 causes sensitivity to several intracellular pathogens. DCT1 (NRAMP2) transport metal ions at the plasma membrane of cells of both the duodenum and in peripheral tissues, and defective DCT1 cause anemia. The driving force for the metal-ion transport is proton gradient (protonmotive force). In DCT1 the stoichiometry between metal ion and proton varied at different conditions due to a mechanistic proton slip. Though the metal ion transport by Smf1p, the yeast homolog of DCT1, is also a protonmotive force, a slippage of sodium ions was observed. The mechanism of the above phenomena could be explained by a combination between transporter and channel mechanisms.     

A novel type of coupling between proline and galactoside transport in Escherichia coli.

Exit of thiomethylgalactoside (TMG) from preloaded cells induced the accumulation of proline. Likewise, proline exit stimulated TMG accumulation. Since a proton ionophore (carbonylcyanide-m-chlorophenylhydrazone) abolished these effects, a protonmotive force was implicated as the "intermediate" in the coupling reaction. The evidence suggests that the exit of TMG resulted in proton exit, which produced either a membrane potential (inside negative or a pH gradient (outside acid) or both. This inwardly directed protonmotive force provided the energy for proline entry and accumulation. Thus the energy coupling was not via a common transport protein but by proton movements which coupled the two separate H+-dependent transport processes.     

Effect of osmotic pressure on membrane energy-linked functions in Escherichia coli

Osmotic upshock of E. coli cells in NaCl or sucrose medium resulted in a large decrease in the cytoplasmic volume and the inhibition of growth, of the electron transfer chain and of four different types of sugar transport system: the lactose proton symport, the glucose phosphotransferase system, the binding-protein dependent maltose transport system and the glycerol facilitator. In contrast to NaCl and sucrose, the permeant solute glycerol had no marked effect. These inhibitions could be partially relieved by glycine betaine. Despite these inhibitions, the internal pH, the protonmotive force and the ATP pool were maintained. It is concluded that inhibition of electron transfer and of sugar transport is the consequence of conformational changes caused by the deformation of the membrane. It is also concluded that the arrest of growth observed upon osmotic upshock is not due to energy limitations and that it cannot be explained by the inhibition of carbohydrate transport.     

Speculations on the evolution of ion transport mechanisms.

Primate cells evolved a plasma membrane to restrict the loss of important molecules. The osmotic problems that then arose were solved in one of several ways. Of major importance was the evolution of specific ion pumps, to actively extrude those salts whose inward diffusion would have led to swelling and lysis. In addition, these pumps allowed the cell to store energy in the form of ion gradients across the membrane. Thus, even in the earliest stages, the evolution of ion transport systems coincided with the development of mechanisms which catalyzes the energy transformations. It is postulated that an "ATP"-driven proton pump was one of the first ion transport systems. Such a proton pump would extrude hydrogen ions from the cell, establishing both a transmembrane pH gradient (alkaline inside) and a membrane potential (negative inside). This difference in electrochemical potential for protons (the proton-motive force) could then drive a variety of essential membrane functions, such as the active transport of ions and nutrients. A second major advance was the evolution of an ion transport system that converted light energy into a form which could be used by the cell. The modern model for this is the "purple membrane" of Halobacterium halobium, which catalyzes the extrusion of protons after the capture of light. The protonmotive force generated by such a light-driven proton pump could then power net synthesis of ATP by a reversal of the ATP-driven proton pump. A third important evolutionary step associated with ion transport was the development of a system to harness energy released by biological oxidations. Again, the solution of this problem was to conserve energy as a protonmotive force by coupling the activity of a respiratory chain to the extrusion of protons. Finally, with the development of animal cells a more careful regulation of internal and external pH was required. Thus, an ATP-driven Na+-K+ pump replaced the proton-translocating ATPase as the major ion pump found in plasma membranes.     

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.     

The use of valinomycin, nigericin and trichlorocarbanilide in control of the protonmotive force in Escherichia coli cells.

Valinomycin, nigericin and trichlorocarbanilide were assessed for their ability to control the protonmotive force in Escherichia coli cells. Valinomycin, at high K+ concentrations, was found to decrease the membrane potential delta phi and indirectly to decrease the pH gradient delta pH. Nigericin was found to have two modes of action. At low concentrations (0.05-2 microM) it carried out K+/H+ exchange and decreased delta pH. At higher concentrations (50 microM) it carried out a K+-dependent transfer of H+, decreasing both delta phi and delta pH. In EDTA-treated cells only the latter mode of action was evident, whereas in a mutant sensitive to deoxycholate both types of effect were observed. Trichlorocarbanilide is proposed as an alternative to nigericin for the specific control of delta pH, and it can be used in cells not treated with EDTA.     

Restriction and modification in B. subtilis: the role of homology between donor and recipient DNA in transformation and transfection

Non-modified DNAs from phages SPO2 and phi 105, and prophage DNAs extracted from lysogens carrying these phages, were used to transfect isogenic r+m+ B. subtilis recipients which were either non-lysogenic, or had been lysogenized with a homologous or a non-homologous phage. Restriction of transfecting phage and prophage DNA occurred in non-lysogenic recipients and in recipients lysogenic for a non-homologous phage. No effect of restriction was observed when phage or prophage DNA was used to transfect recipients carrying a homologous prophage. This is analogous to the absence of restriction in transformation and indicates that in B. subtilis the distinction between transforming and transforming and transfecting DNA is not made at the initial stages of DNA uptake and processing, but rather at later stages, where recognition of homologous regions in donor and recipient DNA plays an important role.     

Effects of Monovalent Ions on the Transport of Noradrenaline Across the Plasma Membrane of Neuronal Cells (PC-12 Cells)

The dependence on Na+, K+, and Cl- of uptake and accumulation of [3H]noradrenaline was studied in plasma membrane vesicles isolated from PC-12 pheochromocytoma cells. Plasma membrane vesicles accumulated [3H]noradrenaline when an inward-directed gradient for Na+ and an outward-directed gradient for K+ were imposed across the vesicle membrane. Under these conditions, initial rates of uptake of [3H]noradrenaline were saturable (Km = 0.14 microM) and inhibited by a series of substrates and inhibitors of "uptake". The IC50 values were positively correlated with those for inhibition of uptake into intact PC-12 cells. Uptake and accumulation of [3H]noradrenaline in plasma membrane vesicles were absolutely dependent on external Na+ and Cl-; they were dependent on an inwardly directed gradient for Na+ but less dependent on an inwardly directed gradient for Cl-. Internal K+ strongly enhanced uptake and accumulation of [3H]noradrenaline. Rb+, but not Li+, had the capacity to replace internal K+. Two explanations are proposed for this effect of internal K+: (a) creation of a K+ diffusion potential (inside negative) provides a driving force for inward transport, and/or (b) K+ increases the turnover rate by formation of a highly mobile potassium-carrier complex. A hypothetical scheme for the transport of noradrenaline is presented.     

Restriction-modification systems in Bacillus strains related to Bacillus subtilis

127 strains of bacilli sensitive to different phages of Bacillus subtilis were isolated from the soil of Moscow and its country-side. In 6 strains, restriction and modification systems were discovered which differed from these previously described for Bac. subtilis BsuR system. Two strains has identical restriction-modification systems, and one strain possessed two different systems. Using DNA from all 6 strains, it was possible to transform competent cells of Bac. subtilis RUB834. Two of these 6 strains could serve as recipients in transformation and transfection experiments.