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Biomedical subjects

A J Driessen

Publications and source records attributed to A J Driessen.

At least 91 records · Page 5Linked to original sources
PubMed1996

Mechanism of lantibiotic-induced pore-formation.

Nisin and other lantibiotics have a bacteriocidal effect against Gram-positive bacteria, and also inhibit the outgrowth of bacterial spores. The bacteriocidal effect appears to be due to the formation of pores in the bacterial membrane. In the absence of anionic membrane phospholipids, the lantibiotic nisin acts as an anion selective carrier. In the presence of anionic phospholipids, nisin forms nonselective, transient, multi-state pores in cells, proteoliposomes, liposomes and black lipid membranes. Pore formation involves distinct steps. First, nisin associates tightly with the anionic membrane surface leading to a high local concentration. This results in a disturbance of the lipid dynamics near the phospholipid polar head group-water interface, and an immobilization of lipids. In the presence of a transmembrane electrical potential above the threshold level, the molecules reorient, presumably as an aggregate, from a surface-bound into a membrane-inserted configuration. Co-insertion of bound, anionic phospholipids results in bending of the lipid surface giving rise to a wedge-like, nonspecific, water-filled pore.

Amino Acid Sequence↗
PubMed1996

Sec-dependent preprotein translocation in bacteria.

Translocation of precursor proteins across the cytoplasmic membrane in bacteria is mediated by a multisubunit protein complex termed translocase, which consists of the integral membrane heterotrimer Sec YEG and the peripheral homodimeric ATPase SecA. Preproteins are bound by the cytosolic molecular chaperone SecB and targeted in a complex with SecA to the translocation site at the cytoplasmic membrane. This interaction with Sec YEG allows the SecA/preprotein complex to insert into the membrane by binding of ATP to the high affinity nucleotide binding site of SecA. At that stage, presumably recognition and proofreading of the signal sequence occurs. Hydrolysis of ATP causes the release of the preprotein in the translocation channel and drives the withdrawal of SecA from the membrane-integrated state. Hydrolysis of ATP at the low-affinity nucleotide binding site of SecA converts the protein into a compact conformational state and releases it from the membrane. In the absence of the proton motive force, SecA is able to complete the translocation stepwise by multiple nucleotide modulated cycles.

Adenosine Triphosphatases↗
PubMed1996

SecA is an intrinsic subunit of the Escherichia coli preprotein translocase and exposes its carboxyl terminus to the periplasm.

SecA is the dissociable ATPase subunit of the Escherichia coli preprotein translocase, and cycles in a nucleotide-modulated manner between the cytosol and the membrane. Overproduction of the integral subunits of the translocase, the SecY, SecE and SecG polypeptides, results in an increased level of membrane-bound SecA. This fraction of SecA is firmly associated with the membrane as it is resistant to extraction with the chaotropic agent urea, and appears to be anchored by SecYEG rather than by lipids. Topology analysis of this membrane-associated form of SecA indicates that it exposes a carboxy-terminal domain to the periplasmic face of the membrane.

Adenosine Triphosphatases↗
PubMed1996

Sulfate transport in Penicillium chrysogenum plasma membranes.

Transport studies with Penicillium chrysogenum plasma membranes fused with cytochrome c oxidase liposomes demonstrate that sulfate uptake is driven by the transmembrane pH gradient and not by the transmembrane electrical potential. Ca2+ and other divalent cations are not required. It is concluded that the sulfate transport system catalyzes the symport of two protons with one sulfate anion.

Biological Transport↗
PubMed1996

Basic amino acid transport in plasma membrane vesicles of Penicillium chrysogenum.

The characteristics of the basic amino acid permease (system VI) of the filamentous fungus Penicillium chrysogenum were studied in plasma membranes fused with liposomes containing the beef heart mitochondrial cytochrome c oxidase. In the presence of reduced cytochrome c, the hybrid membranes accumulated the basic amino acids arginine and lysine. Inhibition studies with analogs revealed a narrow substrate specificity. Within the external pH range of 5.5 to 7.5, the transmembrane electrical potential (delta psi) functions as the main driving force for uphill transport of arginine, although a low level of uptake was observed when only a transmembrane pH gradient was present. It is concluded that the basic amino acid permease is a H+ symporter. Quantitative analysis of the steady-state levels of arginine uptake in relation to the proton motive force suggests a H+-arginine symport stoichiometry of one to one. Efflux studies demonstrated that the basic amino acid permease functions in a reversible manner.

Amino Acid Transport Systems↗
PubMed1996

Sodium-coupled energy transduction in the newly isolated thermoalkaliphilic strain LBS3.

Strain LBS3 is a novel anaerobic thermoalkaliphilic bacterium that grows optimally at pH 9.5 and 50 degrees C. Since a high concentration of Na+ ions is required for growth, we have analyzed the primary bioenergetic mechanism of energy transduction in this organism. For this purpose, a method was devised for the isolation of right-side-out membrane vesicles that are functional for the energy-dependent uptake of solutes. A strict requirement for Na+ was observed for the uptake of several amino acids, and in the case of L-leucine, it was concluded that amino acid uptake occurs in symport with Na+ ions. Further characterization of the leucine transport system revealed that its pH and temperature optima closely match the conditions that support the growth of strain LBS3. The ATPase activity associated with inside-out membrane vesicles was found to be stimulated by both Na+ and Li+ ions. These data suggest that the primary mechanism of energy transduction in the anaerobic thermoalkaliphilic strain LBS3 is dependent on sodium cycling. The implications of this finding for the mechanism of intracellular pH regulation are discussed.

Adenosine Triphosphatases↗
PubMed1996

Lactococcin G is a potassium ion-conducting, two-component bacteriocin.

Lactococcin G is a novel lactococcal bacteriocin whose activity depends on the complementary action of two peptides, termed alpha and beta. Peptide synthesis of the alpha and beta peptides yielded biologically active lactococcin G, which was used in mode-of-action studies on sensitive cells of Lactococcus lactis. Approximately equivalent amounts of both peptides were required for optimal bactericidal effect. No effect was observed with either the alpha or beta peptide in the absence of the complementary peptide. The combination of alpha and beta peptides (lactococcin G) dissipates the membrane potential (delta omega), and as a consequence cells release alpha-aminoisobutyrate, a non-metabolizable alanine analog that is accumulated through a proton motive-force dependent mechanism. In addition, the cellular ATP level is dramatically reduced, which results in a drastic decrease of the ATP-driven glutamate uptake. Lactococcin G does not form a proton-conducting pore, as it has no effect on the transmembrane pH gradient. Dissipation of the membrane potential by uncouplers causes a slow release of potassium (rubidium) ions. However, rapid release of potassium was observed in the presence of lactococcin G. These data suggest that the bactericidal effect of lactococcin G is due to the formation of potassium-selective channels by the alpha and beta peptides in the target bacterial membrane.

Adenosine Triphosphate↗
PubMed1995

The Lactococcal lmrP gene encodes a proton motive force-dependent drug transporter.

To genetically dissect the drug extrusion systems of Lactococcus lactis, a chromosomal DNA library was made in Escherichia coli and recombinant strains were selected for resistance to high concentrations of ethidium bromide. Recombinant strains were found to be resistant not only to ethidium bromide but also to daunomycin and tetraphenylphosphonium. The drug resistance is conferred by the lmrP gene, which encodes a hydrophobic polypeptide of 408 amino acid residues with 12 putative membrane-spanning segments. Some sequence elements in this novel membrane protein share similarity to regions in the transposon Tn10-encoded tetracycline resistance determinant TetA, the multidrug transporter Bmr from Bacillus subtilis, and the bicyclomycin resistance determinant Bcr from E. coli. Drug resistance associated with lmrP expression correlated with energy-dependent extrusion of the molecules. Drug extrusion was inhibited by ionophores that dissipate the proton motive force but not by the ATPase inhibitor ortho-vanadate. These observations are indicative for a drug-proton antiport system. A lmrP deletion mutant was constructed via homologous recombinant using DNA fragments of the flanking region of the gene. The L. lactis (delta lmrP) strain exhibited residual ethidium extrusion activity, which in contrast to the parent strain was inhibited by ortho-vanadate. The results indicate that in the absence of the functional drug-proton anti-porter LmrP, L. lactis is able to overexpress another, ATP-dependent, drug extrusion system. These findings substantiate earlier studies on the isolation and characterization of drug-resistant mutants of L. lactis (Bolhuis, H., Molenaar, D., Poelarends, G., van Veen, H. W., Poolman, B., Driessen, A. J. M., and Konings, W. N. (1994) J. Bacteriol. 176, 6957-6964).

Amino Acid Sequence↗
PubMed1995

Identification of the magnesium-binding domain of the high-affinity ATP-binding site of the Bacillus subtilis and Escherichia coli SecA protein.

The homodimeric SecA protein is the peripheral subunit of the translocase, and couples the hydrolysis of ATP to the translocation of precursor proteins across the bacterial cytoplasmic membrane. The high affinity ATP binding activity of SecA resides in the amino-terminal domain of SecA. This domain contains a tandem repeat of the "so-called" Walker B-motif, hXhhD (Walker, J.E., Saraste, M., Runswick, M.J., and Gay, N.J. (1982) EMBO J. 1, 945-951), that in combination with motif A is responsible for the Mg(2+)-phosphate protein interaction. Two aspartate residues at positions 207 and 215 of the Bacillus subtilis SecA, and Asp-217 in the Escherichia coli SecA, that could be Mg2+ ion ligands, were individually mutated to an asparagine. Mutant SecA proteins were unable to growth-complement an E. coli secA amber mutant strain, and the E. coli SecA mutant interfered with the translocation of precursor proteins in vivo. B. subtilis mutant SecA proteins were expressed to a high level and purified to homogeneity. The high affinity ATP and Mg(2+)-ion binding activity was reduced in the Asp-207 mutant, and completely lost in the Asp-215 mutant. Both SecA proteins were defective in lipid-stimulated ATPase activity. Proteolytic studies suggest that the two subunits of the mutated dimeric SecA proteins are present in different conformational states. These data suggest that Asp-207 and Asp-215 are involved in the binding of the Mg(2+)-ion when Mg(2+)-ATP is bound to SecA, while Asp-207 fulfills an additional catalytic role, possibly in accepting a proton during catalysis.

Adenosine Triphosphatases↗
PubMed1995

Diffusion-limited interaction between unfolded polypeptides and the Escherichia coli chaperone SecB.

SecB is a chaperone dedicated to protein translocation in Escherichia coli. SecB binds to a subset of precursor proteins, and targets them in a translocation-competent state to the SecA subunit of the translocase. The nature and kinetics of the interaction of SecB with polypeptides were studied by spectroscopic techniques using the reduced form of bovine pancreatic trypsin inhibitor (BPTI) as a model substrate. Binding of SecB to BPTI resulted in an increase in the fluorescence of the surface-exposed tryptophan residue 36 of SecB. SecB reversibly binds BPTI in stoichiometric amounts. Labeling of BPTI with the fluorophore acrylodan allowed the analysis of the binding reaction at nanomolar concentrations. High-affinity binding (KD of 5.4 nM) of labeled BPTI to SecB resulted in a blue shift of the acrylodan emission maximum and an increase in the fluorescence quantum yield, suggesting that BPTI binds in an apolar environment. Stopped-flow acquisition of rate constants of complex formation between SecB and BPTI yielded a second-order binding rate constant of 5 x 10(9) M-1 s-1, and a dissociation rate constant of 48 s-1. These data demonstrate that in vitro, the association of SecB with polypeptide substrates is limited by the rate of collision. In vivo, SecB binding is selective, and predominantly occurs with nascent polypeptides. Since these chains are not expected to fold into stable structures, SecB association may be governed by "more or less" specific interactions and be limited by the rate of chain elongation rather than the rate of folding.

2-Naphthylamine↗
PubMed1995

Thermostability of respiratory terminal oxidases in the lipid environment.

The effect of the lipid environment on the thermostability of three respiratory terminal oxidases was determined. Cytochrome-c oxidase from beef heart and Bacillus stearothermophilus were used as representative proteins from mesophilic and thermophilic origin, respectively. Quinol oxidase from the archaeon Sulfolobus acidocaldarius represented the model for a extreme thermoacidophilic enzyme. All three integral membrane proteins were tested for their thermal inactivation in detergent and after reconstitution in liposomes composed of phospholipids of Escherichia coli or tetraether lipids from S. acidocaldarius. When preincubated at 0 degrees C, all three enzymes exhibited biphasic thermal inactivation curves. Data could be analysed according to a two-state model that defines two conformations of the enzyme, differing in their thermostability. Monophasic inactivation curves were observed when the enzymes were preincubated at higher temperatures prior to thermal inactivation. Lipids rendered the beef-heart cytochrome-c oxidase and S. acidocaldarius quinol oxidase more thermostable as compared to detergent solution. In contrast, the B. stearothermophilus oxidase, an intrinsically thermostable enzyme, was as thermostable in detergent as in the reconstituted state. These data suggest that the lipid environment can be an important factor in the thermostability of membrane proteins.

Animals↗
PubMed1995

Mechanistic studies of lantibiotic-induced permeabilization of phospholipid vesicles.

Nisin is a cationic polycyclic bacteriocin secreted by some lactic acid bacteria. Nisin has previously been shown to permeabilize liposomes. The interaction of nisin was analyzed with liposomes prepared of the zwitterionic phosphatidylcholine (PC) and the anionic phosphatidylglycerol (PG). Nisin induces the release of 6-carboxyfluorescein and other small anionic fluorescent dyes from PC liposomes in a delta psi-stimulated manner, and not that of neutral and cationic fluorescent dyes. This activity is blocked in PG liposomes. Nisin, however, efficiently dissipates the delta psi in cytochrome c oxidase proteoliposomes reconstituted with PG, with a threshold delta psi requirement of about -100 mV. Nisin associates with the anionic surface of PG liposomes and disturbs the lipid dynamics near the phospholipid polar head group-water interface. Further studies with a novel cationic lantibiotic, epilancin K7, indicate that this molecule penetrates into the hydrophobic carbon region of the lipid bilayer upon the imposition of a delta psi. It is concluded that nisin acts as an anion-selective carrier in the absence of anionic phospholipids. In vivo, however, this activity is likely to be prevented by electrostatic interactions with anionic lipids of the target membrane. It is suggested that pore formation by cationic (type A) lantibiotics involves the local perturbation of the bilayer structure and a delta psi-dependent reorientation of these molecules from a surface-bound into a membrane-inserted configuration.

Anti-Bacterial Agents↗
PubMed1995

Ion permeability of the cytoplasmic membrane limits the maximum growth temperature of bacteria and archaea.

Protons and sodium ions are the most commonly used coupling ions in energy transduction in bacteria and archaea. At their growth temperature, the permeability of the cytoplasmic membrane of thermophilic bacteria to protons is high compared with that of sodium ions. In some thermophiles, sodium is the sole energy-coupling ion. To test whether sodium is the preferred coupling ion at high temperatures, the proton- and sodium permeability was determined in liposomes prepared from lipids isolated from various bacterial and archaeal species that differ in their optimal growth temperature. The proton permeability increased with the temperature and was comparable for most species at their respective growth temperatures. Liposomes of thermophilic bacteria are an exception in the sense that the proton permeability is already high at the growth temperature. In all liposomes, the sodium permeability was lower than the proton permeability and increased with the temperature. The results suggest that the proton permeability of the cytoplasmic membrane is an important parameter in determining the maximum growth temperature.

Archaea↗
PubMed1995

Expression of the gltP gene of Escherichia coli in a glutamate transport-deficient mutant of Rhodobacter sphaeroides restores chemotaxis to glutamate.

Rhodobacter sphaeroides is chemotactic to glutamate and most other amino acids. In Escherichia coli, chemotaxis involves a membrane-bound sensor that either binds the amino acid directly or interacts with the binding protein loaded with the amino acid. In R. sphaeroides, chemotaxis is thought to require both the uptake and the metabolism of the amino acid. Glutamate is accumulated by the cells via a binding protein-dependent system. To determine the role of the binding protein and transport in glutamate taxis, mutants were created by Tn5 insertion mutagenesis and selected for growth in the presence of the toxic glutamine analogue gamma-glutamyl-hydrazide. One of the mutants, R. sphaeroides MJ7, was defective in glutamate uptake but showed wild-type levels of binding protein. The mutant showed no chemotactic response to glutamate. Both glutamate uptake and chemotaxis were recovered when the gltP gene, coding for the H(+)-linked glutamate carrier of E. coli, was expressed in R. sphaeroides MJ7. It is concluded that the chemotactic response to glutamate strictly requires uptake of glutamate, supporting the view that intracellular metabolism is needed for chemotaxis in R. sphaeroides.

Bacterial Proteins↗
PubMed1995

Mutacin II, a bactericidal antibiotic from Streptococcus mutans.

Mutacin II is an antibiotic that is produced by group II Streptococcus mutans. It inhibits the growth of other streptococci as well as many other gram-positive microorganisms by a hitherto unknown mechanism. Mutacin II possess bactericidal activity against susceptible cells. It transiently depolarizes the transmembrane electrical potential (delta psi) and the transmembrane pH gradient (delta pH) and partially inhibits amino acid transport. However, it rapidly depletes the intracellular ATP pool in glucose-energized cells and prevents the generation of ATP. It is concluded that mutacin II does not belong to the group of pore-forming antibiotics (type A) or to the type B antibiotics, which inhibit phospholipases or interfere with peptidoglycan biosynthesis. Mutacin II acts by inhibiting essential enzyme functions at the level of metabolic energy generation, an activity that has not yet been classified for antibiotics.

Adenosine Triphosphate↗
PubMed1995

Characterization of a binding protein-dependent glutamate transport system of Rhodobacter sphaeroides.

The mechanism of L-glutamate uptake was studied in Rhodobacter sphaeroides. Uptake of L-glutamate is mediated by a high-affinity (Kt of 1.2 microM), shock-sensitive transport system that is inhibited by vanadate and dependent on the internal pH. From the shock fluid, an L-glutamate-binding protein was isolated and purified. The protein binds L-glutamate (apparent Kd of 1.3 microM) and L-glutamine (Ki of 15 microM) with high affinity. The expression level of this binding protein is maximal at limiting concentrations of glutamine in the growth medium. The glutamate-binding protein restores the uptake of L-glutamate in spheroplasts. L-Aspartate is a strong competitive inhibitor of L-glutamate uptake (Ki of 3 microM) but competes only poorly with L-glutamate for binding to the binding protein (Ki of > 200 microM). The uptake of L-aspartate in R. sphaeroides also involves a binding protein which is distinct from the L-glutamate-binding protein. These data suggest that in R. sphaeroides, the L-glutamate- and L-aspartate-binding proteins interact with the same membrane transporter.

Adenosine Triphosphate↗
PubMed1994

Generation of proton-motive force by an archaeal terminal quinol oxidase from Sulfolobus acidocaldarius.

The terminal quinol oxidase of the cytochrome aa3 type was isolated from the extreme thermoacidophilic archaeon Sulfolobus acidocaldarius. In micellar solution, the enzyme oxidized various quinols and exerted the highest activity with the physiological substrate caldariella quinol. The enzyme was functionally reconstituted into monolayer liposomes composed of archaeal tetraether lipids also derived from S. acidocaldarius. With the electron donor system ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine, the reconstituted enzyme was more active in the archaeal lipids as compared to lipids derived from Escherichia coli at temperatures above 50 degrees C. Due to the low proton permeability of the tetraether lipids, it was possible to generate a steady-state transmembrane electrical potential (delta psi, interior negative), and transmembrane pH gradient (delta pH, interior alkaline) at temperatures up to 70 degrees C. The successful functional reconstitution of the cytochrome aa3-type quinol oxidase from Sulfolobus identifies it as the key energy converter in the respiratory system of this hyperthermophilic archaeon.

Catalysis↗