Effect of ethanol perturbation on viscosity and permeability of an inner membrane in Bacillus subtilis spores.
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 B. 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 1000cP to 860cP for wild type spores at 50% of ethanol. Altogether, our present work confirms the deleterious effect of ethanol on the structure of B. subtilis spores, as well as demonstrates the ability of FLIM - Bodipy-C12 to measure changes in the microviscosity of the spores upon perturbation.
Univ. Bourgogne Franche-Comté, AgroSup Dijon, PAM UMR A 02.102, PMB, F-21000 Dijon, France.
Pauline
P
Loison
Univ. Bourgogne Franche-Comté, AgroSup Dijon, PAM UMR A 02.102, PMB, F-21000 Dijon, France.
Patrick
P
Gervais
Univ. Bourgogne Franche-Comté, AgroSup Dijon, PAM UMR A 02.102, PMB, F-21000 Dijon, France; Dimacell Imaging Resource Center, Univ. Bourgogne Franche-Comté, AgroSup Dijon, F-21000 Dijon, France. Electronic address: jm.perrier@agrosupdijon.fr.
Jean-Marie
JM
Perrier-Cornet
Department of Chemistry, Imperial College London, Exhibition Road, SW7 2AZ, UK. Electronic address: m.kuimova@imperial.ac.uk.
Marina K
MK
Kuimova
D001412:Bacillus subtilis / Q000737:chemistry* / Q000166:cytology; D001896:Boron Compounds / Q000737:chemistry; D002462:Cell Membrane / Q000737:chemistry*; D000431:Ethanol / Q000737:chemistry*; D008856:Microscopy, Fluorescence; D010539:Permeability; D013171:Spores, Bacterial / Q000737:chemistry* / Q000166:cytology; D014783:Viscosity
C530974:4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid; D001896:Boron Compounds; D000431:Ethanol
Bacillus subtilis spores; Ethanol perturbation; Fluorescence lifetime imaging (FLIM); Inner membrane; Microviscosity decrease; Permeabilization