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L L Grinius

Publications and source records attributed to L L Grinius.

At least 19 recordsLinked to original sources
PubMed1996

The SMR family: a novel family of multidrug efflux proteins involved with the efflux of lipophilic drugs.

The sequenced members of a novel family of small, hydrophobic, bacterial multidrug-resistance efflux proteins, which we have designated the small multidrug resistance (SMR) protein family, are identified and analysed. Two distinct clusters of proteins were identified within this family: (i) small multidrug efflux systems; and (ii) Sug proteins, potentially involved in the suppression of groEL mutations. Hydropathy and residue distribution analyses of this family suggest a structural model in which the polypeptide chain spans the membrane four times as mildly amphipathic alpha-helices. The roles of specific residues, a possible mechanistic model of drug efflux, and the primary physiological role(s) of the SMR proteins are discussed.

Amino Acid Sequence↗
PubMed1994

Bacterial multidrug resistance is due to a single membrane protein which functions as a drug pump.

Multidrug transport system in proteoliposomes was reconstituted using the highly purified membrane transport protein responsible for bacterial multidrug resistance. This protein (named Smr, for staphylococcal multidrug resistance) consists of 107 amino acid residues and displays four putative transmembrane domains. The Smr protein was tagged with a FLAG epitope, and the modified protein was expressed, purified, characterized, and reconstituted into proteoliposomes. With this in vitro experimental system, it has been demonstrated that a highly purified multidrug resistance protein functions as a drug pump, which transports methyltriphenylphosphonium actively against a 10(3)-fold concentration gradient. Delta mu H+ was shown to be a driving force, and an electrogenic drug/proton antiport was suggested as the molecular mechanism of the drug transport. Of the 2 Glu residues in putative extramembrane loops of the Smr polypeptide chain, Glu-24 was shown to be involved in determining the specificity of drug resistance. Replacement of both of these Glu residues with Asp produced active Smr. In contrast, Smr was unable to protect cells from multiple drugs when a Glu-13-->Asp-13 replacement was made. We suggest that Glu-13, a unique acidic residue located in the hydrophobic domain of Smr, is directly involved in the drug/proton antiport.

Amino Acid Sequence↗
PubMed1989

[Magnitude of the proton moving force of Staphylococcus aureus cells and features of the interaction of staphylococci with phages].

The magnitude of the transmembrane electrical potential difference and the proton gradient across the energy-transducing membrane of Staphylococcus aureus were determined. The delta psi value was shown to rise from 100 to 160 mV upon alkalinization of the medium within the pH range of 5.0-8.0; at the same time, the pH value dropped from 90 to 40 mV. The proton motive force magnitude remained within the range of 191-198 mV at the pH values under study. Membrane potential generation took place, when the respiratory chain and H+-ATPase were operative. An addition of phages to cell suspensions resulted in a decrease of the membrane potential magnitude. Phage infection was effectively suppressed by inhibitors which affect the proton motive force generation in cell membranes of staphylococci.

Adenosine Triphosphatases↗
PubMed1989

[Membrane potential of E. coli recipient cells determines the rate of linear transport of DNA during conjugation].

The rate of conjugal DNA transport from donor to recipient cells has been shown to depend on the membrane potential (delta psi) value in the DNA recipient cell. On the other hand, delta psi in the DNA donor cells is required for the formation of stable aggregates of conjugating cells, but not for the RNA transport. Both components of the electrochemical proton gradient on the cytoplasmic membrane of the recipient cells, the delta psi and the pH gradient are equivalent in the conjugal process.

Biological Transport↗
PubMed1987

[Formation of ion channels in the Escherichia coli cytoplasmic membrane after exposure to bacteriophages T4 and lambda].

The effects of phage T4 and lambda on the ion permeability of the E. coli cytoplasmic membrane were studied. It was shown that the phage-induced depolarization of the membrane is coupled with a simultaneous increase in a transmembrane pH gradient. Hence, the total value of the proton-motive force remains unchanged at moderate multiplicity of infection. The rise in the pH gradient occurs due to an increase in the activity of the redox H+-pump of the E. coli membrane. Analysis of the temperature dependence showed that the stimulating effect of the phage is observed at 6-8 degrees C. Apart from the phages, gramicidin is also capable of stimulating the H+-pump under these conditions, while the stimulating effect of valinomycin is diminished. These data suggest that the ion-permeable channels are formed in the membrane during the interaction of E. coli cells with the phages. The experimental results demonstrate that the channels are permeable to ions of monovalent metals. The phage can also increase the permeability of cell membranes to protons; however, the permeability to monovalent ions is higher when these ions are in excess.

Bacteriophage lambda↗
PubMed1983

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.

Biological Transport, Active↗
PubMed1980

[Study of membrane potential of Bacillus subtilis and Escherichia coli cells by the penetration ions methods].

Using the penetrating ions of tetraphenylphosphonium (TPP+) and tetraphenylborone (TPB-), the membrane potential of the Bacillus subtilis and Escherichia coli cells was shown that the TPP+ absorption by the cells is an energy-coupled process. The TPB- anions are released from the cells after addition of an energy substrate. The value of the membrane potential calculated from the distribution pattern of the penetrating ions in the cells and the incubation medium lies within the interval of --100--150 mV (intracellular negative electric potential). The value of the membrane potential strongly depends on pH of the incubation medium; our attempts to measure the membrane potential in the E. coli cells at ph 6.0 were unsuccessful; however, at pH 8.5 it was found to be equal to --100 mV. Treatment of the cells with nigericin partially prevents the decrease of the membrane potential in an acidic medium and increases the potential in neutral and alkaline media. The formation of the membrane potential is suppressed by valinomycin and gramicidine, as well as by the oxidative phosphorylation uncouplers; the inhibiting effect of valinomycin requires the presence of K+ in the incubation medium. The membrane potential of the B. subtilis cells is insensitive to the effect of cyanide in the absence of arsenate. It is concluded that the membrane potential of B. subtilis and E. coli is formed both via respiration and by hydrolysis of intracellular ATP.

Bacillus subtilis↗
PubMed1980

[Energy supply for transport of plasmid R 100-1 during conjugation of Escherichia coli cells].

It was shown that the transfer of plasmid R 100-1 during conjugation of donor and recipient cells of E. coli is suppressed under treatment of the cells by oxidative phosphorylation uncouplers. Studies on recipient cells devoid of their H+-ATPase activity due to mutation showed that the transfer of the plasmid into the cells is repressed after a switch-off of the respiratory chain, the only generator of proton motive force in the mutated cells. In the absence of arsenate the plasmid transfer from the donor into the recipient cells possessing intact H+-ATPase occurs independently of inhibition of the cell respiratory activity by cyanide. However, the presence of arsenate in the conjugation medium induces the sensitivity of the plasmid transfer process to cyanide. In the absence of cyanide the cell conjugation is suppressed by 60 mM arsenate. A kinetic study of different steps of cell conjugation showed that the generation of proton motive force in recipient cells is necessary for the occurrence of plasmid transport. It was assumed that the generation of both proton motive force and phosphorylated high energy compounds is a necessary prerequisite for plasmid transport during conjugation of donor and recipient cells.

Adenosine Triphosphatases↗
PubMed1980

[Nature of membrane ATPase inactivation in an Escherichia coli mutant with genetically impaired ATPase].

Homogeneous preparations of F1 possessing identical subunit composition have been isolated from the mutant strain of E. coli AN 120 with genetically impaired membrane ATPase and from the wild strain of AN 180. Using ion-exchange chromatography, the subunits alpha and beta of F1 were isolated. It was shown that the alpha- and beta-subunits of both active and genetically impaired F1 have similar molecular weights and total electrical charges.

Adenosine Triphosphatases↗
PubMed1979

[Role of proton motive force in the infection of E. coli K-12 cells by bacteriophage T4].

It was shown that infection of E. coli cells by phage T4 is suppressed, when the cells are treated by oxidative phosphorylation uncouplers. The inhibiting effects of the uncouplers manifest themselves at the stage of phage DNA entry into the cells. Study of the E. coli cells devoid of their H+-ATPase activity due to mutation showed that the infection is suppressed by a switch-off of the respiratory chain, the only generator of the proton motive force (PMF) in mutated cells. Infection of the E. coli cells containing intact H+-ATPase occured even in the case when the respiratory chain activity was inhibited. The kinetic studies showed that generation of PMF is necessary during phage DNA transport into the cells and is indispensable for phage DNA entry into bacterial cells.

Adenosine Triphosphatases↗
PubMed1979

[Transhydrogenase as an additional site of energy accumulation in the E. coli respiratory chain].

NAD+ reduction catalyzed by transhydrogenase (EC 1.6.1.1) from E. coli membrane particles at the expense of NADPH oxidation is coupled with phenyldicarbaundecaborate (PCB-) absorption by the particles. This process is inhibited by oxidative phosphorylation protonophorous uncouplers and by equilibration of concentrations of the substrates and products of the transhydrogenase reaction. Elimination of the water-soluble part of membrane ATPase results in the inhibition of PCB- absorption at the expense of the transhydrogenase reaction energy. Treatment of the particles by dicyclohexyl carbodiimide increases the transhydrogenase-coupled absorption of PCB-. The transhydrogenase-induced increase of pPCB in the suspension of particles is directly correlated with the ratio of ([NADPH].[NAD+])/([NADP+].[NADH]). When this value is equal to 1, no energy-dependent increase of pPCB was observed. NADP+ reduction at the expense of NADH oxidation leads to a decrease in the amount of PCB- absorbed by the particles at the expense of ATP hydrolysis energy. The experimental data suggest that NADPH oxidation in the course of the transhydrogenase reaction is coupled with the formation of a membrane potential with a positive charge localized inside the particles.

Cell Membrane↗
PubMed1978

[The role of a protonmotive force in genetic transformation of Bacillus subtilis].

The hypothesis on the role of protonmotive force in the transport of DNA through the membrane of Bac. subtilis cell during initial stages of genetic transformation was tested. A genetic transformation of arsenate-treated cells was observed. Treatment of cells by the protonophorous uncoupler of oxidative phosphorylation-carbonylcyanide dichlorophenyl--hydrazone-led to the inhibition of initial stages of genetic transformation having no significant effect on the level of intracellular ATP concentration and on the viability of cells. The dissipation of protonmotive force by means of K+ and H+ fluxes catalyzed by valinomycin and nigericin also caused the inhibition of initial stages of genetic transformation. The inhibitory effect of cationic penetrant tetraphenyl phosphonium was observed, the effect being potentiated by low concentrations of anionic penetrant phenyldicarbaundecaborate. The value of the membrane potential in the energized valinomycin-treated cells calculated from the distribution of K+ was within the range of 70--100 mV (inside minus). These results support the conception that a protonmotive force drives DNA transport through the membrane of Bac. subtilis cells.

Adenosine Triphosphate↗
PubMed1976

[Chemiosmotic mechanism of transport of biological macromolecules through bacterial membranes].

A general mechanism of the nucleic acids transport through bacterial membranes during genetic transformation, transfection, viral infection and bacterial conjugation, has been developed. The uptake of nucleic acid occurs due to the symport with H+ ions down to an electrochemical potential gradient ("minus" inside) generated by respiration or ATP hydrolysis within recipient cells. The nucleic acid anions of non--lethal viruses are extruded from the negatively charged host cell cytoplasm by electrostatic repulsion. The difference of electrochemical potentials between the conjugating cells cytoplasms is considered as a driving force for the transport of DNA from the donor to the recipient cell.

Adenosine Triphosphatases↗