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            <title xml:lang="en">A new fluorescence-based approach for direct visualization of coat formation during sporulation in Bacillus cereus</title>
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            <funder>grants PEst-OE/EQB/LA0004/2011</funder>
            <funder>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.</funder>
            <funder>project LISBOA-01-0145-FEDER-007660 (“Microbiologia Molecular, Estrutural e Celular”) funded by FEDER funds through COMPETE2020 – “Programa Operacional Competitividade e Internacionalização”</funder>
            <funder>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".</funder>
            <funder>Work  and Lattice SIM imaging 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.)</funder>
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                <term xml:lang="en">microscopy</term>
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              <p>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 σ K -dependent protein BxpB, occur in cells arrested during engulfment. These results suggest that, unlike the well-studied model organism Bacillus subtilis , the activity of σ K is not strictly linked to the state of forespore development in B. cereus .</p>
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