A new fluorescence-based approach for direct visualization of coat formation during sporulation in Bacillus cereus

Funding Information: The Ph.D. thesis of A.L. was funded by INRAE and the PACA Region and was partly supported by a grant of the MICA division and a Perdiguier grant of Avignon University. Part of this work was supported by the microscopy facilities of the Platform 3A, funded by the European Regional Development Fund, the French Ministry of Research, Higher Education and Innovation, the Provence-Alpes-Côte d’Azur region, the Departmental Council of Vaucluse and the Urban Community of Avignon. This work was also funded through grants PEst-OE/EQB/LA0004/2011 to AOH, by project LISBOA-01-0145-FEDER-007660 (“Microbiologia Molecular, Estrutural e Celular”) funded by FEDER funds through COMPETE2020 – “Programa Operacional Competitividade e Internacionalização”, and by project PPBI—Portuguese Platform of BioImaging (PPBI-POCI-01-0145-FEDER-022122) co-funded by national funds from OE—"Orçamento de Estado" and by European funds from FEDER—"Fundo Europeu de Desenvolvimento Regional". Work and Lattice SIM imaging in the R.C-L. lab was supported by funding from the European Research Council (ERC) under the Horizon 2020 research and innovation program (grant agreement No 772178, ERC Consolidator grant to R.C.-L.). Publisher Copyright: © 2023, Springer Nature Limited.The human pathogenic bacteria Bacillus cereus, Bacillus anthracis and the entomopathogenic Bacillus thuringiensis form spores encased in a protein coat surrounded by a balloon-like exosporium. These structures mediate spore interactions with its environment, including the host immune system, control the transit of molecules that trigger germination and thus are essential for the spore life cycle. Formation of the coat and exosporium has been traditionally visualized by transmission electronic microscopy on fixed cells. Recently, we showed that assembly of the exosporium can be directly observed in live B. cereus cells by super resolution-structured illumination microscopy (SR-SIM) using the membrane MitoTrackerGreen (MTG) dye. Here, we demonstrate that the different steps of coat formation can also be visualized by SR-SIM using MTG and SNAP-cell TMR-star dyes during B. cereus sporulation. We used these markers to characterize a subpopulation of engulfment-defective B. cereus cells that develops at a suboptimal sporulation temperature. Importantly, we predicted and confirmed that synthesis and accumulation of coat material, as well as synthesis of the σK-dependent protein BxpB, occur in cells arrested during engulfment. These results suggest that, unlike the well-studied model organism Bacillus subtilis, the activity of σK is not strictly linked to the state of forespore development in B. cereus.publishersversionpublishe

La microscopie électronique à balayage appliquée à la microbiologie: Complémentarité de techniques de microscopie

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Deposition of Bacillus subtilis spores using an airbrush-spray or spots to study surface decontamination by pulsed light

International audienceMicrobial contamination on surfaces of food processing equipment is a major concern in industries. A new method to inoculate a single-cell layer (monolayer) of microorganisms onto polystyrene was developed, using a deposition with an airbrush. A homogeneous dispersion of Bacillus subtilis DSM 402 spores sprayed on the surface was observed using both plate count and scanning electron microscopy. No clusters were found, even with high spore concentrations (10 7 spores/inoculated surface). A monolayer of microorganisms was also obtained after deposition of 10 μL droplets containing 3 × 10 4 spores/spot on polystyrene disks, but not with a higher spore concentration. Pulsed light (PL) applied to monolayers of B. subtilis spores allowed log reductions higher than 6. As a consequence of clusters formation in spots of 10 μL containing more than 3 × 10 5 spores, log reductions obtained by PL were significantly lower. The comparative advantages of spot and spray depositions were discussed

<em>Coniothyrium minitans</em> parasite les sclérotes de <em>Sclerotinia minor</em>

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Chemical changes in virgin olive oils as a function of crushing systems: Stone mill and hammer crusher

International audienceOlive oils were tested for their chemical composition in polyphenols, free fatty acids and volatile compounds as a function of the crushing systems, i.e. the stone mill and hammer crusher. The qualitative and quantitative HPLC/DAD analyses of the olive oils showed that luteolin and tyrosol were the most abundant identified phenolic compounds. The olive oil obtained by the hammer crusher had the highest concentration of phenolic compounds and ultimately the strongest antioxidant activity. Olives treated by the two crushing systems were observed by scanning electronic microscopy. Micrographs provide more evidence of the better cell cuts of olive fruits treated by hammer crushing, in contrast to stone mill where olive cell layers have been broken and damaged.Des huiles d'olive issues de deux procédés de broyage (meule de granit ou broyeur à marteaux) ont été comparées en terme de composés polyphénoliques, composés volatiles et acidité libre. Les analyses qualitatives et quantitatives par CLHP avec une détection à barrette de photodiodes des huiles montrent que la lutéoline et le tyrosol sont les composés biophénoliques identifiés majoritaires. L'huile d'olive issue du broyeur à marteaux a des concentrations plus fortes en composés biophénoliques et montre une plus forte activité antioxydante. Les olives broyées par les deux systèmes de broyage ont également été observées par microscopie électronique à balayage. Les photographies mettent en évidence une meilleure découpe des cellules de fragments d'olives issues du broyeur à marteaux, par opposition à la meule de granit pour laquelle les cellules ont été simplement cassées et endommagée

The spore coat is essential for Bacillus subtilis spore resistance to pulsed light, and pulsed light treatment eliminates some spore coat proteins

International audienceMicrobial surface contamination of equipment or of food contact material is a recurring problem in the food industry. Spore-forming bacteria are far more resistant to a wide variety of treatments than their vegetative forms. Understanding the mechanisms underlying decontamination processes is needed to improve surface decontamination strategies against endospores potentially at the source of foodborne diseases or food-spoilage. Pulsed light (PL) with xenon lamps delivers high-energy short-time pulses of light with wavelengths in the range 200 nm–1100 nm and a high UV-C fraction. Bacillus subtilis spores were exposed to either PL or to continuous UV-C. Gel electrophoresis and western blotting revealed elimination of various proteins of the spore coat, an essential outer structure that protects spores from a wide variety of environmental conditions and inactivation treatments. Proteomic analysis confirmed the elimination of some spore coat proteins after PL treatment. Transmission electron microscopy of PL treated spores revealed a gap between the lamellar inner spore coat and the outer spore coat. Overall, spores of mutant strains with defects in genes coding for spore coat proteins were more sensitive to PL than to continuous UV-C. This study demonstrates that radiations delivered by PL contribute to specific damage to the spore coat, and overall to spore inactivation

Improved microwave steam distillation apparatus for isolation of essential oils: Comparison with conventional steam distillation

International audienceSteam distillation (SD) is routinely used by analysts for the isolation of essential oils from herbs, flowers and spices prior to gas chromatographic analysis. In this work, a new process design and operation for an improved microwave steam distillation (MSD) of essential oils from aromatic natural products was developed. To demonstrate its feasibility, MSD was compared with the conventional technique, SD, for the analysis of volatile compounds from dry lavender flowers (Lavandula angustifolia Mill., Lamiaceae). Essential oils isolated by MSD were quantitatively (yield) and qualitatively (aromatic profile) similar to those obtained by SD, but MSD was better than SD in terms of rapidity (6 min versus 30 min for lavender flowers), thereby allowing substantial savings of costs in terms of time and energy. Lavender flowers treated by MSD and SD were observed by scanning electron microscopy. Micrographs provide evidence of more rapid opening of essential oil glands treated by MSD, in contrast to conventional S