Influence of growth conditions on production and activity of mesenterocin 5 by a strain of Leuconostoc mesenteroides

The effects of various parameters on production and activity of mesenterocin 5, a bacteriocin produced by Leuconostoc mesenteroides subsp. mesenteroides UL5, were investigated. Titres of bacteriocin and minimum inhibitory concentration values were determined by a critical dilution micromethod, using a sensitive strain of Listeria ivanovii as an indicator. Production of the antimicrobial compound was optimal at 37 and 40°C after 9 h of incubation, and was maximized in an aerobic fermentor maintained at pH 5.0. Tween 80 was a major factor in increasing mesenteroxin 5 production and specific production. Large quantities of bacteriocin could be obtained in whey and in whey permeate supplemented with yeast extract in the presence of the surfactant (0.1%). Most of the Listeria strains tested including L. monocytogenes were highly sensitive to the bacteriocin in the pH range 5.5 to 6.0 and at a temperature of 20 to 25°C.     

A model of photosystem II for the analysis of fast fluorescence rise in plant leaves

The polyphasic patterns of fluorescence induction rise in pea leaves in vivo and after the treatment with ionophores have been studied using a Plant Efficiency Analyzer. To analyze in detail photosystem II (PS II) electron transfer processes, an extended PS II model was applied, which included the sums of exponential functions to specify explicitly the light-driven formation of the transmembrane electric potential (ΔΨ(t)) as well as pH in the lumen (pH_L(t)) and stroma (pH_S(t)). PS II model parameters and numerical coefficients in ΔΨ(t), pH_L(t), and pH_S(t) were evaluated to fit fluorescence induction data for different experimental conditions: leaf in vivo or after ionophore treatment at low or high light intensity. The model imitated changes in the pattern of fluorescence induction rise due to the elimination of transmembrane potential in the presence of ionophores, when ΔΨ = 0 and pH_L(t), pH_S(t) changed to small extent relative to control values in vivo, with maximum ΔΨ(t) ∼ 90 mV and ΔΨ(t) ∼ 40 mV for the stationary state at ΔpH ≅ 1.8. As the light intensity was increased from 300 to 1200 μmol m⁻² s⁻¹, the heat dissipation rate constants increased threefold for nonradiative recombination of P680⁺Phe⁻ and by ∼30% for P680⁺Q_A⁻. The parameters ΔΨ, pH_S and pH_L were analyzed as factors of PS II redox state populations and fluorescence yield. The kinetic mechanism of fluorescence quenching is discussed, which is related with light-induced lumen acidification, when ⁺Q_A⁻ and P680⁺ recombination probability increases to regulate the Q_A reduction.     

Membrane oxidative damage induced by ionizing radiation detected by fluorescence polarization

Effects of ionizing radiation on biological membranes include alterations in membrane proteins, peroxidation of unsaturated lipids accompanied by perturbations of the lipid bilayer polarity. We have measured radiation-induced membrane modifications using two fluorescent lipophilic membrane probes (TMA-DPH and DPH) by the technique of fluorescence polarization on two different cell lines (Chinese hamster ovary CHO-K1 and lymphoblastic RPMI 1788 cell lines). γ-Irradiation was performed using a ⁶⁰Co source with dose rates of 0.1 and 1 Gy/min for final doses of 4 and 8 Gy. Irradiation induced a decrease of fluorescence intensity and anisotropy of DPH and TMA-DPH in both cell lines, which was dose-dependent but varied inversely with the dose rate. Moreover, the fluorescence anisotropy measured in lymphoblastic cells using TMA-DPH was found to decrease as early as 1 h after irradiation, and remained significantly lower 24 h after irradiation. This study indicates that some alterations of membrane fluidity are observed after low irradiation doses and for some time thereafter. The changes in membrane fluidity might reflect oxidative damage, thus confirming a radiation-induced fluidization of biological membranes. The use of membrane fluidity changes as a potential biological indicator of radiation injury is discussed. Received: 14 May 1996 / Accepted in revised form: 30 September 1996     

Characterization of the mesB Gene and Expression of Bacteriocins by Leuconostoc mesenteroides Y105

Leuconostoc mesenteroides Y105, previously described for production of mesentericin Y105, an anti-Listeria bacteriocin, was shown to secrete a second bacteriocin. The latter was purified, and its molecular mass of 3446 Da, obtained by mass spectrometric analysis, indicates that this bacteriocin should be identical to mesenterocin 52B [Revol-Junelles et al., Lett Appl Microbiol 23:120, 1996]. This second bacteriocin produced by L. mesenteroides Y105 was named mesentericin B105. Its structural gene, mesB, was then localized by a reverse genetic approach, cloned, and sequenced. MesB was found on the pHY30 plasmid, next to mesY gene clusters. Curing experiments led to isolation of two L. mesenteroides Y105 derivatives, named L. mesenteroides Y29 and Y30. The latter had lost pHY30 plasmid, encoding bacteriocin determinants, therefore explaining its phenotype (MesY-, MesB-). On the contrary, Y29 derivative still harbors the pHY30 but did not produce any bacteriocin. Thus, its phenotype could likely result from a point mutation within a gene, probably encoding a protein involved in production of both mesentericin Y105 and mesentericin B105. Received: 9 May 1999 / Accepted: 8 June 1999     

Synergistic Mode of Action of Mesenterocins 52A and 52B Produced by <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> subsp. <Emphasis Type="Italic">mesenteroides</Emphasis> FR 52

Few studies have been published on the effects of two bacteriocins combinations and particularly on combinations of two bacteriocins with different structures produced by the same strain. In this work, the actions of mesenterocin 52A (class IIa) and mesenterocin 52B (class II), produced by Leuconostoc mesenteroides subsp. mesenteroides FR 52, were studied on strains susceptible to only one bacteriocin or to both. In broth, combination of mesenterocins enhanced the adaptation time of the strain susceptible to the both mesenterocins (48 h vs 17 h with only one bacteriocin). In agar medium, mesenterocins displayed, as expected, a synergistic effect on this strain (FIC_index < 1), but also on the two strains susceptible to only one mesenterocin. This original result was probably due to membrane composition modifications induced by the mesenterocin that enhanced bacteriocin action. Thus, this hurdle technique seems to be interesting in food preservation in terms of minimizing bacteriocin concentrations.     

Properties of two bacteriocins synthesized byLeuconostoc strains

Mesenterocin 52 (Mes-52), was produced byLeuconostoc mesenteroides ssp.mesenteroides strain FR52 and dextranicin 24 (Dex-24) byLeuconostoc mesenteroides ssp.dextranicum strain J24. Dex-24 had a very narrow spectrum of antibacterial activity: it inhibited only some other strains ofLeuconostoc, contrary to Mes-52, which antagonized alsoListeria andEnterococcus sp. Mes-52 was partially purified by ammonium sulfate precipitation, gel filtration, cation exchange, and hydrophobic interaction chromatography. Mes-52 was biosynthesized and excreted into the medium during growth phase. At different temperatures media, there was an inverse relationship between the final bacteriocin-specific productivity and the mean growth rate. After novobiocin treatment, different mutants of strain FR 52 were obtained: nonproducing-sensitive (Bac^–Imm^–) and nonproducingimmune (Bac^–Imm⁺) strains. Many changes in the plasmid profiles of several mutants were detected by electrophoresis.     

Mechanism of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> death induced by <Emphasis Type="Italic">Cratylia mollis</Emphasis> seed lectin

Incubation of T. cruzi epimastigotes with the lectin Cramoll 1,4 in Ca²⁺ containing medium led to agglutination and inhibition of cell proliferation. The lectin (50 μg/ml) induced plasma membrane permeabilization followed by Ca²⁺ influx and mitochondrial Ca²⁺ accumulation, a result that resembles the classical effect of digitonin. Cramoll 1,4 stimulated (five-fold) mitochondrial reactive oxygen species (ROS) production, significantly decreased the electrical mitochondrial membrane potential (ΔΨ_m) and impaired ADP phosphorylation. The rate of uncoupled respiration in epimastigotes was not affected by Cramoll 1,4 plus Ca²⁺ treatment, but oligomycin-induced resting respiration was 65% higher in treated cells than in controls. Experiments using T. cruzi mitochondrial fractions showed that, in contrast to digitonin, the lectin significantly decreased ΔΨ_m by a mechanism sensitive to EGTA. In agreement with the results showing plasma membrane permeabilization and impairment of oxidative phosphorylation by the lectin, fluorescence microscopy experiments using propidium iodide revealed that Cramoll 1,4 induced epimastigotes death by necrosis.     

Influence of temperature and pH on production of two bacteriocins by Leuconostoc mesenteroides subsp. mesenteroides FR52 during batch fermentation

The influence of temperature and pH on growth of Leuconostoc mesenteroides subsp. mesenteroides FR52 and production of its two bacteriocins, mesenterocin 52A and mesenterocin 52B, was studied during batch fermentation. Temperature and pH had a strong influence on the production of the two bacteriocins which was stimulated by slow growth rates. The optimal temperature was 20 °C for production of mesenterocin 52A and 25 °C for mesenterocin 52B. Optimal pH values were 5.5 and 5.0 for production of mesenterocin 52A and mesenterocin 52B respectively. Thus, by changing the culture conditions, production of one bacteriocin can be favoured in relation to the other. The relationship between growth and specific production rates of the two bacteriocins, as a function of the culture conditions, showed different kinetics of production and the presence of several peaks in the specific production rates during growth. Received: 13 February 1998 / Received revision: 27 May 1998 / Accepted: 1 June 1998     

Asymmetry of membrane fluidity in the lipid bilayer of blood platelets: Fluorescence study with diphenylhexatriene and analogs

Summary Membrane fluidity of bovine platelets was examined with diphenylhexatriene (DPH), its cationic trimethylammonium derivative (TMA-DPH) and anionic propionic acid derivative (DPH-PA). After addition of these probes to platelet suspensions at 37°C, the fluorescence intensity of DPH-PA reached equilibrium within 2 min, whereas those of DPH and TMA-DPH increased gradually. With increase in the fluorescence intensity of TMA-DPH, its fluorescence anisotropy decreased significantly, but the fluorescence anisotropies of DPH-PA and DPH did not change during incubation. The gradual increase of fluorescence intensity of TMA-DPH was due to its penetration into the cytoplasmic side of the platelet membrane, as shown quantitatively by monitoring decrease in its extractability with albumin. Transbilayer movement of TMA-DPH was markedly temperature-dependent, and was scarcely observed at 15°C. The fluorescence intensity of TMA-DPH was much higher in platelet membranes and vesicles of extracted membrane lipids than the initial intensity in intact platelets. Moreover, the fluorescence anisotropy of TMA-DPH was much lower in the former preparations than the initial value in intact platelets. These results suggest that binding sites for TMA-DPH in the cytoplasmic side of the platelet membrane are more fluid than those in the outer leaflet of the plasma membrane. Platelet activation by ionomycin induced specific change in the fluorescence properties of TMA-DPH without causing transbilayer incorporation of the probe.     

Interstrains comparison of the antimicrobial effect and mode of action of a Vietnamese Cinnamomum cassia essential oil from leaves and its principal component against Listeria monocytogenes

The antibacterial activity of a Cinnamomum cassia essential oil (EO) and of its main component trans-cinnamaldehyde (90% w/w) was examined against five Listeria monocytogenes strains. The minimal inhibitory concentrations (MICs) of C. cassia EO against the five L. monocytogenes strains were identical (250 µg ml⁻¹), while the minimal bactericidal concentrations (MBCs) ranged between 800 and 1200 µg ml⁻¹. In order to study if this EO and trans-cinnamaldehyde altered the five strains at the membrane level, fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) was measured in presence of different concentrations (1/2MIC, MIC, 2MIC) of these antibacterial agents. A concentration-dependent increase of fluorescence anisotropy of DPH in their presence reflecting a rigidification of the membrane was observed for the five strains. This modification of the membrane fluidity was associated with a perturbation of the selective membrane permeability, as a perturbation of the gradient between intracellular and extracellular pH was also observed.     

Studies elucidating the mechanisms of calcium induced mitochondrial depolarization in individual isolated brain mitochondria

Among other mitochondrial functions, energy production and Ca²⁺ uptake are crucial for maintaining neuronal viability. Both of these functions are critically dependent on mitochondrial membrane potential (ΔΨ_m). Mitochondrial Ca²⁺ overload causing a dissipation of ΔΨ_m is a key component of several neuronal pathologies. However, the mechanism of Ca²⁺-induced depolarization in neuronal mitochondria remains unclear. Typically, ΔΨ_m has been evaluated as a single overall estimate from all mitochondria present in a given cell or tissue. However, recent data showed that the population of mitochondria isolated from tissues is not homogeneous, and averaged parameters from the whole population do not necessarily reflect the processes taking place in a single organelle. This review summarizes our recent studies of Ca²⁺-induced depolarization in individual mitochondria isolated from rat forebrain and immobilized to coverslips. Fluorescence imaging techniques and potentiometric fluorescent dyes were effectively used to study ΔΨ_m changes. The data have shown that Ca²⁺ triggers ΔΨ_m oscillations in brain mitochondria followed by a complete depolarization. Further investigation of this phenomenon led us to suggest that Ca²⁺-induced ΔΨ_m oscillations can represent an intermediate unstable state that may lead to irreversible mitochondrial dysfunction. Therefore, further study of this phenomenon would help to understand what causes the irreversible damage of mitochondria during cytosolic/mitochondrial Ca²⁺ overload. Here we discuss the effects of different modulators of the mitochondrial permeability transition pore on Ca²⁺-induced depolarization in brain mitochondria and in liver mitochondria, where the mechanism of Ca²⁺-depolarization is better understood. A comparison of these effects in brain and liver mitochondria led us to conclude that Ca²⁺ can induce reversible “low conductance” permeability transition in brain mitochondria, the phenomenon which requires a transient conformational change of the adenine nucleotide translocator from a specific transporter to a non-specific pore. The article is published in the original.     

Induced Transmembrane Voltage and Its Correlation with Electroporation-Mediated Molecular Transport

Exposure of a cell to an electric field results in inducement of a voltage across its membrane (induced transmembrane voltage, ΔΨ _m) and, for sufficiently strong fields, in a transient increase of membrane permeability (electroporation). We review the analytical, numerical and experimental methods for determination of ΔΨ _m and a method for monitoring of transmembrane transport. We then combine these methods to investigate the correlation between ΔΨ _m and molecular transport through an electroporated membrane for isolated cells of regular and irregular shapes, for cells in dense suspensions as well as for cells in monolayer clusters. Our experiments on isolated cells of both regular and irregular shapes confirm the theoretical prediction that the highest absolute values of ΔΨ _m are found in the membrane regions facing the electrodes and that electroporation-mediated transport is confined to these same regions. For cells in clusters, the location of transport regions implies that, at the field strengths sufficient for electroporation, the cells behave as electrically insulated (i.e., as individual) cells. In contrast, with substantially weaker, nonelectroporating fields, potentiometric measurements show that the cells in these same clusters behave as electrically interconnected cells (i.e., as one large cell). These results suggest that sufficiently high electric fields affect the intercellular pathways and thus alter the electric behavior of the cells with respect to their normal physiological state.     

Inhibition ofListeria monocytogenes by plantaricin NA, an antibacterial substance fromLactobacillus plantarum

ALactobacillus plantarum of vegetable origin produced a bacteriocin inhibitory toListeria monocytogenes. The antimicrobial agent was inactivated by proteolytic enzymes, was resistant to heat (100°C for 30 min) and stable over a wide pH range (pH 2–10), and displayed a bactericidal mode of action. Growth inhibition ofL. monocytogenes depend on bacteriocin concentration. The antilisterial efficiency depended on the strain ofL. monocytogenes used but was not influenced by the growth phase of this strain. A decrease in absorbance overtime, indicative of cell lysis, was also observed. The significance of the results is discussed in relation to the potential of the bacteriocin in controllingListeria-associated food-borne hazards in foods.     

Fermenting Cucumber, a Potential Source for the Isolation of Pediocin-Like Bacteriocin Producers

Summary A strain of Pediococcus acidilactici CFR K7 isolated from cucumber, produced an antimicrobial peptide which acted against Leuconostoc mesenteroides, selected strains of Lactobacillus spp., Pediococcus spp. and Enterococcus spp. The partially purified bacteriocin had molecular weight of ~4.6 kDa, heat stability in a range of 40–121 °C and was active over a wide range of pH (2.0–9.0). This bacteriocin possessed strong antilisterial activity and was susceptible to proteolytic enzymes. Southern hybridization using the PCR-generated pedA probe established that the gene for the bacteriocin was plasmid-borne as in the case of pediocin PA-1. Nucleotide sequence of the pedAB gene indicated 100% homology to a pediocin AcH/PA-1. Certain bacteriocinogenic strains isolated from naturally fermented cucumber were tested by colony hybridization using the pedA gene probe. Nine out of twenty colonies reacted with the probe indicating their ability to produce the pediocin-like bacteriocin. These nine colonies were further tested for their antimicrobial spectrum, proteolytic inactivation and plasmid profile. It was found that a few of them were active against Bacillus cereus, Micrococcus luteus and Listeria monocytogenes. Their proteolytic inactivation showed that the antimicrobial compound was susceptible to proteinase K. Colony hybridization could thus enable rapid detection of pediocin and pediocin-like bacteriocin producers among a population of bacteriocinogenic strains.     

High vapour pressure deficit exacerbates xylem cavitation and photoinhibition in shade-grown Piper auritum H.B. & K. during prolonged sunflecks

Water relations dynamics during simulated sunflecks at high (36°C) and medium (27°C) temperatures and high and low vapour pressure deficits beween leaf and air (VPD) were studied on shade-grown Piper auritum H.B. & K. plants, a pioneer tree, common in gaps and clearings of tropical rain forests. The leaves of P. auritum wilt rapidly when exposed to high light. Exposure to high VPD and high light caused substantial and rapid dehydration of leaves. Dehydration could be prevented under high humidity irrespective of temperature. Water stored in leaf cells served as initial source for transpiration upon high light exposure. This effect increased with increasing VPD and temperature. The pronounced decrease in leaf water content over time in high light caused a rapid decrease in leaf water potential (Ψ_l) and a concomitant increase in water potential gradient (ΔΨ/Δx) between trunk and leaf, yet the high leaf elasticity (small bulk elastic modulus, ε) allowed turgor maintenance under most conditions. Under high VPD and high temperature, stomata remained open and ΔΨ/Δx frequently exceeded 0.95 MPa · m⁻¹, the cavitation-inducing threshold (ΔΨ/Δx _cav) causing high rates of acoustic emissions from stems and leaf petioles and leading to concomitant losses in hydraulic conductance per leaf area (k _l). At medium temperature (high VPD), stomatal closure contained xylem embolism by keeping ΔΨ/Δx at or below this threshold. We argue that wilting substantially contributes to creating a sufficient driving force for water uptake from the soil, and reducing the VPD (through a decrease in radiation load and thus leaf temperature) to avoid excessive dehydration. Received: 3 March 1996 / Accepted: 10 November 1996     

Combined Effect of Bacteriocins on the Survival of Various Listeria Species in Broth and Meat System

The antilisterial efficiency of three bacteriocins from lactic acid bacteria, lactocin 705 (produced by L. casei CRL705, 17000 AU/ml), enterocin CRL35 (produced by E. faecium CRL35, 17000 AU/ml), and nisin (2000 IU/ml), was tested in broth, individually and in combination against Listeria monocytogenes and Listeria innocua. Both Listeria species showed an initial decrease in viable counts followed by the regrowth of the survivors after 1 h in the presence of each bacteriocin. A greater antilisterial effect was observed when the bacteriocins were combined in pairs, maximal inhibition being reached when nisin was involved. When a mix of the three bacteriocins was used, no survivors were observed after 24 h of incubation. Similar results were obtained when the bacteriocin combinations were tested in a meat system, indicating that the use of more than one LAB bacteriocin in combination may be effective in preventing the spontaneous emergence of a bacteriocin-resistant Listeria population. Received: 17 March 2000 / Accepted: 26 June 2000     

Characterization of two bacteriocins produced by Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442, isolated from dry fermented sausages

Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442, isolated from dry fermented sausages, produce bacteriocins antagonistic towards closely related species and pathogens, such as Listeria monocytogenes. The bacteriocins were inactivated by proteolytic enzymes and lipase but not by catalase and lysozyme. They were also heat stable, retaining activity after heating at 100 °C for 60 min. The bacteriocins were stable at pH values ranging from 2.0 to 8.0. Bacteriocin production was observed at low temperatures (10 and 4 °C) and in meat juice. The maximum bacteriocin activity was observed at the end of the exponential growth phase. The bacteriocins were produced in media with initial pH values ranging from 5.0 to 7.5, but not in media with a pH lower than 5.0 (weak bacteriocin activity of the antibacterial compound produced by Ln. mesenteroides L124 was observed at pH 4.5). Both bacteriocins exhibited strong bactericidal activity following cell/bacteriocin contact.     

The fluidity of plasma membranes from ethanol-treated rat liver

Summary Male Wistar rats were maintained for 35–40 days on a liquid diet containing 36% of calories as ethanol. Ethanol was replaced by carbohydrates in the isocaloric diet fed to control animals. The effect of ethanol consumption has been studied on the fluorescence polarization of rat liver plasma membranes and artificial lipid vesicles and on the lipid composition of the membranes. Fluorescence polarization in both membranes and vesicles was determined using DPH and TMA-DPH as fluorescence markers; from these data, the polarization term (r_o/r–1)^–1 and flow activation energy (E) were calculated. The ethanol consumption induces a more fluid environment within the membrane core of liver plasma membranes; the ethanol-fed rat membranes are more resistant to the in vitro effect of ethanol disordering the membrane structure. Vesicles obtained with lipids from either control membranes or ethanol-fed rat membranes were treated with ethanol and the changes in polarization paralleled to those exhibited by the membranes. The absence of phase transitions and of E changes was also shown in temperature-dependence studies. The lower cholesterol content found in ethanol-fed rat plasma membranes might be responsible for observed variations in the microviscosity.Abbreviation OG octyl -D-glucopyranoside     

Activation of human lymphocytes by concanavalin A or purified protein derivative results in no alteration of fluorescence polarization of lipid probes although the electrophoretic mobility of the cells is changed

Upon stimulation with either concanavalin A or the tuberculin antigen, purified protein derivative, human peripheral blood lymphocytes, purified on Ficoll-Hypaque, did not exhibit a concomitant lipid fluidity alteration as measured by fluorescence polarization (P) of the lipid probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). This result was independent of the incubation period, ranging from 10 min to 72 h. However, a general reduction in polarization value, from P = 0.287 (maintained for up to 2 h of incubation) to P = 0.225 after 20 h was observed for both experimental and control samples. Moreover, fluorescence polarization studies of the nonpenetrating modified DPH cationic lipid probe, 1-[4′-trimethylaminophenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), also failed to show any change in lipid fluidity subsequent to a 1–3 h incubation of lymphocytes with concanavalin A. Cell electrophoretic mobility, however, was altered (mean cell mobility increased by 10–15%) in a fast response to stimulation and was observed within several hours of in vitro application of concanavalin A and purified protein derivative. This initial response disappeared with further incubation at 37°C (>3 h) and was followed by a decline of cellular mobility of the concanavalin A-exposed cells after 48 and 72 h of incubation. The unstimulated control cells did not change in mobility as a function of incubation time. The slow decline in mean cell mobility of the experimental cells is believed to be associated with blastogenesis. It is concluded that neither blastogenic transformation nor short term membrane alterations associated with human lymphocyte activation lead to lipid fluidity changes as measured in steady state by the fluorescence polarization of both DPH and TMA-DPH.