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L Letellier

Publications and source records attributed to L Letellier.

At least 37 records · Page 2Linked to original sources
PubMed1996

FhuA, a transporter of the Escherichia coli outer membrane, is converted into a channel upon binding of bacteriophage T5.

The Escherichia coli outer membrane protein FhuA catalyzes the transport of Fe3+(-)ferrichrome and is the receptor of phage T5 and phi 80. The purified protein inserted into planar lipid bilayers showed no channel activity. Binding of phage T5 and FhuA resulted in the appearance of high conductance ion channels. The electrophysiological characteristics of the channels (conductance, kinetic behavior, substates, ion selectivity including the effect of ferrichrome) showed similarities with those of the channel formed by a FhuA derivative from which the 'gating loop' (delta 322-355) had been removed. binding of phage T5 to FhuA in E.coli cells conferred SDS sensitivity to the bacteria, suggesting that such channels also exist in vivo. These data suggest that binding of T5 to loop 322-355 of FhuA, which constitutes the T5 binding site, unmasks an inner channel in FhuA. Both T5 and ferrichrome bind to the closed state of the channel but only T5 can trigger its opening.

Bacterial Outer Membrane Proteins↗
PubMed1996

Structure-activity analysis of thanatin, a 21-residue inducible insect defense peptide with sequence homology to frog skin antimicrobial peptides.

Immune challenge to the insect Podisus maculiventris induces synthesis of a 21-residue peptide with sequence homology to frog skin antimicrobial peptides of the brevinin family. The insect and frog peptides have in common a C-terminally located disulfide bridge delineating a cationic loop. The peptide is bactericidal and fungicidal, exhibiting the largest antimicrobial spectrum observed so far for an insect defense peptide. An all-D-enantiomer is nearly inactive against Gram-negative bacteria and some Gram-positive strains but is fully active against fungi and other Gram-positive bacteria, suggesting that more than one mechanism accounts for the antimicrobial activity of this peptide. Studies with truncated synthetic isoforms underline the role of the C-terminal loop and flanking residues for the activity of this molecule for which we propose the name thanatin.

Amino Acid Sequence↗
PubMed1995

Calcium controls phage T5 infection at the level of the Escherichia coli cytoplasmic membrane.

Phage T5 requires 0.1 mM calcium to produce phage progeny in Escherichia coli cells. Decreasing calcium below 0.1 mM at time phage DNA was transferred depleted the bacteria of K+, caused membrane depolarization, perturbation of phage DNA transfer and resulted in a low internal ATP level. Our data suggest that calcium controls the conformation of the channel involved in the transfer of phage DNA through the host envelope and that below 0.1 mM calcium the channel remains open. This creates an energetic state of the host unfavorable to the synthesis of phage components and leads to abortion of the infectious process.

Adenosine Triphosphate↗
PubMed1995

In vivo and in vitro studies of the inhibition of the channel activity of colicins by gadolinium.

The primary effects of the ionophoric colicins A, E1 and B on Escherichia coli cells include triggering an efflux of cytoplasmic potassium, and a decrease of internal ATP as consequences of the opening of ionic channels in the cytoplasmic membrane. We report that micromolar concentrations of gadolinium and other members of the lanthanide family inhibited the efflux of potassium and the ATP decrease and that the cells recovered both ATP and potassium within a few minutes. Gadolinium, in the same concentration range also efficiently inhibited the channel activity of colicins A, E1, B and of the isolated channel-forming domain of colicin A in planar lipid bilayers. Colicin N was much less sensitive to the trivalent ion in planar lipid bilayers, consistent with the lack of effect of gadolinium on this colicin in vivo. Our data suggest that lanthanide ions act by direct binding to the colicin molecule and that this binding affects both its single-channel conductance and gating behaviour.

Colicins↗
PubMed1995

Quantification of group A colicin import sites.

Pore-forming colicins are soluble bacteriocins which form voltage-gated ion channels in the inner membrane of Escherichia coli. To reach their target, these colicins first bind to a receptor located on the outer membrane and then are translocated through the envelope. Colicins are subdivided into two groups according to the envelope proteins involved in their translocation: group A colicins use the Tol proteins; group B colicins use the proteins TonB, ExbB, and ExbD. We have previously shown that a double-cysteine colicin A mutant which possesses a disulfide bond in its pore-forming domain is translocated through the envelope but is unable to form a channel in the inner membrane (D. Duché, D. Baty, M. Chartier, and L. Letellier, J. Biol. Chem. 269:24820-24825, 1994). Measurements of colicin-induced K+ efflux reveal that preincubation of the cells with the double-cysteine mutant prevents binding of colicins of group A but not of group B. Moreover, we show that the mutant is still in contact with its receptor and import machinery when it interacts with the inner membrane. From these competition experiments, we conclude that each Escherichia coli cell contains approximately 400 and 1,000 colicin A receptors and translocation sites, respectively.

Biological Transport↗
PubMed1994

Unfolding of colicin A during its translocation through the Escherichia coli envelope as demonstrated by disulfide bond engineering.

Three double cysteine mutants, each possessing a disulfide bond in its pore-forming domain, were used to study the translocation of colicin A through the Escherichia coli envelope. These mutated colicins were able to exert their in vivo channel activity only after their disulfide bonds had been reduced by dithiothreitol. In solution, the reduction of the disulfide bonds by dithiothreitol was a slow process whose kinetics depended on the position of the disulfide bond (t1/2 varying between 35 and 100 s). This t1/2 was strongly decreased (t1/2 = 8-9 s) upon predenaturation of the mutated colicins with urea. The t1/2 values of reduction of the mutants bound to E. coli-sensitive cells were similar to those of predenatured colicins. This suggested that the interaction of the oxidized double cysteine mutants with the E. coli envelope triggered their unfolding. The disulfide bonds did not prevent but delayed the translocation of the colicins. The amplitude of the delay and the time at which it occurred during translocation depended on the position of the disulfide bond. We could discriminate between the delays accumulated during binding to the receptor and those during the translocation via OmpF and the Tol proteins.

Bacterial Outer Membrane Proteins↗
PubMed1994

Colicin A and the Tol proteins involved in its translocation are preferentially located in the contact sites between the inner and outer membranes of Escherichia coli cells.

Colicin A is a bacterial toxin which forms channels in the cytoplasmic membrane of Escherichia coli. Its translocation through the envelope requires the participation of bacterial proteins encoded by the tolQ, -R, -A, and -B genes. Overproduction of the Tol proteins decreased the time needed for colicin A translocation and increased the number of channels formed in vivo. Cells overproducing radioactively labeled Tol proteins and containing or not colicin A were fractionated. The Tol proteins were mainly recovered in the inner membrane and in the contact sites between the two membranes. The presence of colicin A increased the specific radioactivity of the Tol proteins in the contact sites. Our data suggest that the Tol proteins form a complex of definite stoichiometry in the membranes and that colicin A is associated to this complex upon channel formation. We discuss the possibility that the channel activity determined in vivo is due to the colicin A-Tol proteins complex.

Bacterial Proteins↗
PubMed1994

Kinetic studies of the action of lactacin F, a bacteriocin produced by Lactobacillus johnsonii that forms poration complexes in the cytoplasmic membrane.

The bacteriocin lactacin F is bactericidal against Lactobacillus delbrueckii, Lactobacillus helveticus, and Enterococcus faecalis. Activity against L. delbrueckii was recently shown to be dependent on two peptides, LafA and LafX, which are encoded within the lactacin F operon (T. R. Klaenhammer, FEMS Microbiol. Rev. 12:39-87, 1993). It has been proposed that two peptides form an active lactacin F complex. In this study, the action of lactacin F against E. faecalis ATCC 19443 and the effects of various environmental parameters were investigated in detail. Addition of lactacin F induced the loss of K+ from cells of L. delbrueckii, Lactobacillus johnsonii 88-4, and E. faecalis, while the lactacin F producer L. johnsonii VPI 11088 was not affected by the bacteriocin. Lactacin F caused an immediate loss of cellular K+, depolarization of the cytoplasmic membrane, and hydrolysis of internal ATP in E. faecalis. Lactacin F induced loss of K+ in 3,3',4',5-tetrachlorosalicylanilide-treated cells, indicating that pores are formed in the absence of a proton motive force. ATP hydrolysis was not due to dissipation of the proton motive force but was most likely caused by efflux of inorganic phosphate, resulting in a shift of the ATP hydrolysis equilibrium. Action of lactacin F was optimal at acidic pH values and was reduced in the presence of di- and trivalent cations. The lanthanide gadolinium (Gd3+) prevented action of lactacin F completely at a concentration of 0.2 mM. Lactacin F-induced loss of cell K+ was severely reduced at low temperatures, presumably as a result of increased ordering of the lipid hydrocarbon chains in the cytoplasmic membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1994

Mode of Action of Nisin Z against Listeria monocytogenes Scott A Grown at High and Low Temperatures.

Nisin Z, a natural nisin variant, was recently isolated from Lactococcus lactis subspecies lactis NIZO 22186. The gene for this lantibiotic, designated nisZ, has been cloned, and its nucleotide sequence was found to be identical to that of the precursor nisin gene with the exception of a single mutation resulting in the substitution of Asn-27 for His-27 in the mature polypeptide (J. W. M. Mulders, I. J. Boerrigter, H. S. Rollema, R. J. Siezen, and W. M. de Vos, Eur. J. Biochem. 201:581-584, 1991). A K electrode was used to investigate the effect of various environmental parameters on the action of nisin Z against Listeria monocytogenes. Addition of nisin Z resulted in immediate loss of cell K, depolarization of the cytoplasmic membrane, inhibition of respiratory activity, and hydrolysis and partial efflux of cellular ATP. The action of nisin Z was optimal at pH 6.0 and was significantly reduced by di- and trivalent cations. The lanthanide gadolinium (Gd) was an efficient inhibitor and prevented nisin Z activity completely at a concentration of 0.2 mM. Nisin Z-induced loss of cell K was reduced at low temperatures, presumably as a result of the increased ordering of the lipid hydrocarbon chains in the cytoplasmic membrane. In cells grown at 30 degrees C, the action of nisin Z was prevented below 7 degrees C, whereas in cells grown at 4 degrees C nisin Z was able to induce K leakage at this low temperature.

Journal Article↗
PubMed1993

Insect defensin, an inducible antibacterial peptide, forms voltage-dependent channels in Micrococcus luteus.

Insect defensins are cationic, cysteine-rich peptides (approximately 4 kDa) that appear after bacterial challenge or injury in the hemolymph of insects belonging to a large variety of orders. These peptides possess anti-Gram-positive activity and participate in the potent antibacterial defense reactions of insects. Using recombinant insect defensin and the strain Micrococcus luteus as a test organism, we have investigated the mode of action of this peptide. We show that defensin disrupts the permeability barrier of the cytoplasmic membrane of M. luteus, resulting in a loss of cytoplasmic potassium, a partial depolarization of the inner membrane, a decrease in cytoplasmic ATP, and an inhibition of respiration. Potassium loss is inhibited below the order-disorder transition of the lipid hydrocarbon chains. It is also inhibited by divalent cations and by a decrease in the membrane potential below a threshold of 110 mV. We propose that these permeability changes reflect the formation of channels in the cytoplasmic membrane by defensin oligomers. This proposal is supported by patch-clamp experiments that show that insect defensins form channels in giant liposomes.

Adenosine Triphosphate↗
PubMed1993

Phosphate efflux through the channels formed by colicins and phage T5 in Escherichia coli cells is responsible for the fall in cytoplasmic ATP.

Previous studies have shown that channel formation in the cytoplasmic membrane of Escherichia coli by colicin A and phage T5 leads to an efflux of cytoplasmic potassium and to a membrane depolarization. Here we show that upon opening of these channels, the intracellular ATP concentration is decreased to 10% of its original value in < 5 min. ATP is not found in the external medium, but is hydrolyzed in the cytoplasm into ADP and AMP. The rate of ATP hydrolysis depends on the number of channels and on their activity. ATP hydrolysis takes place if the F1F0-ATPase is absent or inhibited. Depolarization of the inner membrane is not the main cause of ATP hydrolysis. Opening of the phage and colicin channels also leads to an efflux of inorganic phosphate. Conditions that prevent the efflux of phosphate (i.e. depolarization of the cells and high external phosphate concentration) prevent ATP hydrolysis. We propose that ATP is hydrolyzed as a consequence of a shift in the ATP equilibrium due to the efflux of phosphate through the channels. The consequences for the cells of this ATP depletion are discussed.

Adenine Nucleotides↗
PubMed1993

Role of acidic lipids in the translocation and channel activity of colicins A and N in Escherichia coli cells.

Colicins A and N are pore-forming bacterial toxins that kill Escherichia coli cells. Their mode of action involves three steps; binding to specific receptors located in the outer membrane, translocation through this membrane and the periplasm, and channel formation in the inner membrane. In-vitro studies have shown that negatively charged phospholipids are an absolute requirement for the channel formation of colicin A. Using HDL11 strain, in which the phosphatidylglycerol (PtdGro) content was altered by varying the synthesis of the PtdGro-phosphate synthase, the effect of envelope PtdGro content on the activity of colicin A was studied in vivo. The formation by colicin A of a voltage-gated channel in the cytoplasmic membrane results in an efflux of cytoplasmic potassium. This efflux is preceded by a lag time which is related to the time needed by the toxin to cross the cell envelope. This lag time is higher when the cells have a reduced PtdGro level, suggesting that the receptor/translocation machinery of colicin A (OmpF, BtuB and Tol QRAB proteins) is altered in the absence of PtdGro. The rate of potassium efflux is also greatly reduced when the PtdGro content is decreased, suggesting that a certain level of PtdGro is indeed required for proper insertion of the colicin-A channel. In contrast, the activity of colicin N does not show any PtdGro dependence. The difference between the behavior of colicin A and that of colicin N is discussed.

Biological Transport↗
PubMed1992

Ion channels are likely to be involved in the two steps of phage T5 DNA penetration into Escherichia coli cells.

Phage T5 injects its DNA into Escherichia coli cells in two steps; 8% of the chromosome is first injected, and then there is a pause during which proteins encoded by this DNA fragment are synthesized allowing the remaining DNA to be injected. Using a potassium-selective electrode, we show that the injection of the two DNA fragments is associated with an efflux in two steps, of cytoplasmic potassium. The rate of efflux is linearly related to the number of added phages suggesting that each phage induces the formation of at least one channel in the inner membrane. The first efflux occurs even in depolarized cells suggesting that the insertion and the opening of the channel can take place in the absence of the electrochemical gradient of protons (delta mu H+). The channel is in a closed configuration during the time required for the synthesis of the phage-encoded proteins; this closing and the second efflux are prevented by the depolarization of the cell. The insertion of the channel in the inner membrane requires a fluid membrane. The results obtained suggest that the function of this channel is to translocate phage T5 DNA.

DNA, Viral↗
PubMed1992

Involvement of phage T5 tail proteins and contact sites between the outer and inner membrane of Escherichia coli in phage T5 DNA injection.

The penetration of phage T5 DNA into the Escherichia coli envelope takes place through ion channels (Boulanger, P., and Letellier, L. (1992) J. Biol. Chem. 267, 3168-3172). To identify putative phage protein(s) involved in the formation of these channels, E. coli cells were infected at 37 degrees C with radioactively labeled phage and their envelopes were fractionated. After a flotation gradient, proteins belonging to the phage tail were recovered both in fractions containing the contact sites between the inner and outer membranes and in the outer membrane. The electrophoretic banding pattern of phage proteins indicates that the contact sites were enriched in the protein pb2. Moreover, infected cells were significantly enriched in contact sites as compared to intact cells. There was no enrichment of contact sites and very little radioactivity was found in this fraction and in the outer membrane when the cells were infected at 4 degrees C (i.e. under conditions where the phage does not inject its DNA). These results suggest that both contact sites and pb2 may play a central role in the translocation of phage T5 DNA.

Centrifugation, Density Gradient↗
PubMed1992

Excretion of glutamate from Corynebacterium glutamicum triggered by amine surfactants.

Corynebacterium glutamicum is used for the industrial production of glutamate. Excretion of the amino acid may be induced by various means. We have analyzed the characteristics of glutamate excretion induced by two amine surfactants, dodecylammonium acetate (DA) and dodecyltrimethylammonium bromide (DTA). Addition of these surfactants induced an immediate efflux of internal glutamate. It also induced a perturbation of the energetic parameters of the cell (decrease of delta mu H, decrease of the internal ATP concentration). The efflux was not the result of these perturbations: glutamate is taken up by the cells via an ATP-dependent unidirectional active transport system and no efflux took place as a consequence of an artificial decrease of the energetic parameters. In addition, amine surfactants also induced an excretion of other species, in particular potassium. We have tested the possibility that the effluxes result from a permeabilization of the lipid bilayer by analyzing the interactions between the surfactants and liposomes.

Adenosine Triphosphate↗
PubMed1992

Colicin A unfolds during its translocation in Escherichia coli cells and spans the whole cell envelope when its pore has formed.

The addition of the pore forming colicin A to Escherichia coli cells results in an efflux of cytoplasmic potassium. This efflux is preceded by a lag time which is related to the time needed for the translocation of the toxin through the envelope. Denaturing the colicin A with urea, before adding it to the cells, did not affect the properties of the pore but decreased the lag time. After renaturation, the lag time was similar to that of the native colicin. This suggests that the unfolding of colicin A accelerates its translocation. The addition of trypsin, which has access neither to the periplasmic space nor to the cytoplasmic membrane, resulted in an immediate arrest of the potassium efflux induced by colicins A and B. The possibility that trypsin may act on a bacterial component required for colicin reception and/or translocation was ruled out. It is thus likely that the arrest of the efflux corresponds to a closing of the pores. This long distance effect of trypsin suggests that part of the polypeptide chain of the colicins may still be in contact with the external medium even when the pore has formed in the inner membrane.

Cell Membrane↗
PubMed1990

Mechanism of penetration and of action of local anesthetics in Escherichia coli cells.

Escherichia coli cells were used to study the mechanism of penetration of local anesthetics and the relationship between permeation and functional properties. We show that both the neutral and the protonated form of dibucaine can be accumulated in the cells. Accumulation of the protonated form occurs in response to a transmembrane electrical potential (negative inside) and results in high trapped concentrations (70 mM). Accumulation can lead to an alkalinization of the internal pH. Low concentrations of dibucaine stimulate the respiration, increase the transmembrane electrical potential and raise the accumulation of solutes. Inhibition of these functions occurs at higher concentrations of the drug. Furthermore, the drug concentration required to inhibit these functions is smaller at alkaline external pH than at acidic external pH, suggesting that the inhibition is mainly due to the neutral form of the anesthetics. Other hydrophobic amines also stimulate and inhibit different membrane functions, their efficiency being correlated to their lipophilicity.

Adenosine Triphosphate↗