Use of the fluorescent probe LAURDAN to label and measure inner membrane fluidity of endospores of Clostridium spp.

Hofstetter, Simmon; Denter, Christian; Winter, Roland; McMullen, Lynn M.; Gänzle, Michael G.

doi:10.1016/j.mimet.2012.07.023

Cited by 24 publications

(16 citation statements)

References 46 publications

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“…FT-IR and laurdan measurements confirm a gel state of inner membranes of endospores (16,20). A phase shift toward a liquid-crystalline phase was observed after heating, in keeping with the effects of heat and pressure on model membranes (27).…”

supporting

confidence: 71%

“…Membrane fluidity changes and phase transitions were assessed with the fluorescent dye laurdan (6-dodecanoyl-2-dimethylaminonaphthalene). The laurdan general polarization (GP) values indicate membrane fluidity of bacterial cells and endospores (19,20). Laurdan was previously used to characterize the response of bacterial membranes to high pressure, nisin, and reutericyclin (21,22).…”

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confidence: 99%

“…GP values of laurdan-labeled endospores exposed to nisin or reutericyclin at 90°C or 90°C and 600 MPa were measured ex situ to relate membrane properties to spore inactivation. Spore preparation, treatments, and GP measurements were performed as described previously (11,20). Treatment of spores at 90°C for 8 min reduced viable spore counts by less than 1 log CFU ml Ϫ1 , and the spores released less than 5% DPA (11) but GP values of clostridial endospores were lowered (Fig.…”

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confidence: 99%

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In Situ Determination of Clostridium Endospore Membrane Fluidity during Pressure-Assisted Thermal Processing in Combination with Nisin or Reutericyclin

Hofstetter

Winter

McMullen

et al. 2013

Appl Environ Microbiol

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bThis study determined the membrane fluidity of clostridial endospores during treatment with heat and pressure with nisin or reutericyclin. Heating (90°C) reduced laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) general polarization, corresponding to membrane fluidization. Pressure (200 MPa) stabilized membrane order. Reutericyclin and nisin exhibit divergent effects on heat-and pressure-induced spore inactivation and membrane fluidity. P ressure-assisted thermal sterilization (PATS) of food is an alternative to thermal processing (1). PATS releases dipicolinic acid (DPA) from endospores, rehydrates the core of endospores, and allows for spore inactivation (2,3,4). A combination of 200 to 800 MPa with 120°C eliminates resistant spores of Clostridium botulinum (2, 5). At or above 120°C, however, pressure may fail to accelerate thermal inactivation or even exerts protective effects (2, 5). Antimicrobials acting in concert with PATS may enhance the inactivation of endospores, thus ensuring product safety at reduced treatment intensity. Nisin and reutericyclin exhibit antimicrobial activity against endospores (6, 7). Nisin is a pore-forming lantibiotic (6), reutericyclin is a proton ionophore (8). Remarkably, nisin enhances pressure-induced spore inactivation (9, 10), whereas reutericyclin had no effect or even attenuated pressure-induced inactivation of bacterial endospores (11).Endospores have multiple, distinct layers that contribute to resistance and metabolic dormancy (2, 12, 13). Dehydration of the spore core contributes to endospore resistance (12). Endospores possess an outer membrane, a remnant of sporulation, and an inner membrane separating the dehydrated core from the hydrated exterior (14, 15). Lipids of the inner membrane are compressed, and the surface area expands during germination without lipid synthesis (16). Disruption of the inner membrane of endospores rehydrates the core and allows inactivation of endospores by antimicrobials (17, 18). An improved understanding of the effect of pressure on endospore membranes will improve the control of endospores by pressure and facilitate the selection of antimicrobials to support pressure-assisted sterilization; however, little is known about the Ϫ1 nisin (B), or in the presence of 6.4 mg liter Ϫ1 reutericyclin (C). Measurements were taken every 10 min as samples were heated to 90°C at a rate of 5°C/10 min. The graph highlights the area corresponding to the CH 2 asymmetrical stretching absorbance; i and ii denote the wavenumbers corresponding to gel state and liquid-crystalline membranes, respectively. Comparable results were obtained with C. beijerinckii (data not shown).

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confidence: 99%

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In Situ Determination of Clostridium Endospore Membrane Fluidity during Pressure-Assisted Thermal Processing in Combination with Nisin or Reutericyclin

Hofstetter

Winter

McMullen

et al. 2013

Appl Environ Microbiol

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“…The inner membrane of bacterial spores has been suggested to be in part in a gel state [23,56,57]. Furthermore, according to our previous data, the inner membranes were characterized by an extremely high viscosity of ~1000 cP [23].…”

Section: Discussionmentioning

confidence: 82%

Effect of ethanol perturbation on viscosity and permeability of an inner membrane in Bacillus subtilis spores

Loison

Gervais

Perrier-Cornet

et al. 2016

Biochimica et Biophysica Acta (BBA) - Biomembranes

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In this work, we investigated how a combination of ethanol and high temperature (70 °C), affect the properties of the inner membrane of Bacillus subtilis spores. We observed membrane permeabilization for ethanol concentrations ≥ 50%, as indicated by the staining of the spores' DNA by the cell impermeable dye Propidium Iodide. The loss of membrane integrity was also confirmed by a decrease in the peak corresponding to dipicolinic acid using infrared spectroscopy. Finally, the spore refractivity (as measured by phase contrast microscopy) was decreased after the ethanol-heat treatment, suggesting a partial rehydration of the protoplast. Previously we have used fluorescent lifetime imaging microscopy (FLIM) combined with the fluorescent molecular rotor Bodipy-C12 to study the microscopic viscosity in the inner membrane of Bacillus subtilis spores, and showed that at normal conditions it is characterized by a very high viscosity. Here we demonstrate that the ethanol/high temperature treatment led to a decrease of the viscosity of the inner membrane, from 1000 cP to 860 cP for wild type spores at 50% of ethanol. Altogether, our present work confirms the deleterious effect of ethanol on the structure of Bacillus subtilis spores, as well as demonstrates the ability of FLIM -Bodipy-C12 to measure changes in the microviscosity of the spores upon perturbation.

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“…Based on an adaptation of the method developed previously (Hofstetter, Denter, Winter, McMullen, & G€ anzle, 2012;Hofstetter, 2012), Laurdan fluorescent staining of G. stearothermophilus spores' inner membrane was achieved with the sporulation method described above and as explained elsewhere (Georget et al, 2014). Laurdan (Sigma Aldrich, Hamburg, Germany) was selected as fluorescent dye because of its non-cytotoxicity (Owen, Rentero, Magenau, Abu-Siniyeh, & Gaus, 2012), enabling further evaluation of the physiological state of spores by plate count, for instance.…”

Section: Laurdan Stainingmentioning

confidence: 99%

Geobacillus stearothermophilus ATCC 7953 spore chemical germination mechanisms in model systems

et al. 2015

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Use of the fluorescent probe LAURDAN to label and measure inner membrane fluidity of endospores of Clostridium spp.

Cited by 24 publications

References 46 publications

In Situ Determination of Clostridium Endospore Membrane Fluidity during Pressure-Assisted Thermal Processing in Combination with Nisin or Reutericyclin

In Situ Determination of Clostridium Endospore Membrane Fluidity during Pressure-Assisted Thermal Processing in Combination with Nisin or Reutericyclin

Effect of ethanol perturbation on viscosity and permeability of an inner membrane in Bacillus subtilis spores

Geobacillus stearothermophilus ATCC 7953 spore chemical germination mechanisms in model systems

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