A new fluorescence-based approach for direct visualization of coat formation during sporulation in Bacillus cereus.
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
13 09 2023
13 09 2023
Historique:
received:
12
05
2023
accepted:
06
09
2023
medline:
15
9
2023
pubmed:
14
9
2023
entrez:
13
9
2023
Statut:
epublish
Résumé
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
Identifiants
pubmed: 37704668
doi: 10.1038/s41598-023-42143-9
pii: 10.1038/s41598-023-42143-9
pmc: PMC10499802
doi:
Substances chimiques
Coloring Agents
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
15136Informations de copyright
© 2023. Springer Nature Limited.
Références
Henriques, A. O. & Moran, C. P. Jr. Structure, assembly, and function of the spore surface layers. Annu. Rev. Microbiol. 61, 555–588 (2007).
pubmed: 18035610
Stewart, G. C. The exosporium layer of bacterial spores: A connection to the environment and the infected host. Microbiol. Mol. Biol. Rev. 79, 437–457 (2015).
pubmed: 26512126
pmcid: 4651027
Khanna, K., Lopez-Garrido, J. & Pogliano, K. Shaping an endospore: Architectural transformations during Bacillus subtilis sporulation. Annu. Rev. Microbiol. 74, 361–386 (2020).
pubmed: 32660383
pmcid: 7610358
Walker, P. D. (Symposium on bacterial spores: Paper I). Cytology of spore formation and germination. J. Appl. Bacteriol. 33, 1–12 (1970).
pubmed: 4986702
Driks, A. & Eichenberger, P. The Spore Coat. The Bacterial Spore 179–200 (Wiley, 2016).
Delerue, T. et al. Bacterial developmental checkpoint that directly monitors cell surface morphogenesis. Dev. Cell 57, 344-360.e6 (2022).
pubmed: 35065768
pmcid: 8991396
Landajuela, A. et al. FisB relies on homo-oligomerization and lipid binding to catalyze membrane fission in bacteria. PLoS Biol. 19, e3001314 (2021).
pubmed: 34185788
pmcid: 8274934
Andrade Cavalcante, D. et al. Ultrastructural analysis of spores from diverse Bacillales species isolated from Brazilian soil. Environ. Microbiol. Rep. 11, 155–164 (2019).
pubmed: 30421850
Browne, H. P. et al. Culturing of ‘unculturable’ human microbiota reveals novel taxa and extensive sporulation. Nature 533, 543–546 (2016).
pubmed: 27144353
pmcid: 4890681
Lehmann, D. et al. Role of novel polysaccharide layers in assembly of the exosporium, the outermost protein layer of the Bacillus anthracis spore. Mol. Microbiol. 118, 258–277 (2022).
pubmed: 35900297
pmcid: 9549345
McKenney, P. T., Driks, A. & Eichenberger, P. The Bacillus subtilis endospore: Assembly and functions of the multilayered coat. Nat. Rev. Microbiol. 11, 33–44 (2013).
pubmed: 23202530
Riley, E. P., Schwarz, C., Derman, A. I. & Lopez-Garrido, J. Milestones in Bacillus subtilis sporulation research. Microb. Cell 8, 1–16 (2020).
pubmed: 33490228
pmcid: 7780723
Eijlander, R. T. & Kuipers, O. P. Live-cell imaging tool optimization to study gene expression levels and dynamics in single cells of Bacillus cereus. Appl. Environ. Microbiol. 79, 5643–5651 (2013).
pubmed: 23851094
pmcid: 3754164
Arnaud, M., Chastanet, A. & Débarbouillé, M. New vector for efficient allelic replacement in naturally nontransformable, Low-GC-content, Gram-positive bacteria. Appl. Environ. Microbiol. 70, 6887–6891 (2004).
pubmed: 15528558
pmcid: 525206
Ghosh, A. et al. Proteins encoded by the gerP operon are localized to the inner coat in Bacillus cereus spores and are dependent on GerPA and SafA for assembly. Appl. Environ. Microbiol. 84, 14 (2018).
Motomura, K. et al. The C-Terminal zwitterionic sequence of CotB1 is essential for biosilicification of the Bacillus cereus spore coat. J. Bacteriol. 198, 276–282 (2016).
pubmed: 26503850
Lablaine, A. et al. The morphogenetic protein CotE positions exosporium proteins CotY and ExsY during sporulation of Bacillus cereus. mSphere 6, 2 (2021).
Thompson, B. M., Hsieh, H.-Y., Spreng, K. A. & Stewart, G. C. The co-dependence of BxpB/ExsFA and BclA for proper incorporation into the exosporium of Bacillus anthracis. Mol. Microbiol. 79, 799–813 (2011).
pubmed: 21255119
pmcid: 3044595
Thompson, B. M., Hoelscher, B. C., Driks, A. & Stewart, G. C. Assembly of the BclB glycoprotein into the exosporium and evidence for its role in the formation of the exosporium ‘cap’ structure in Bacillus anthracis. Mol. Microbiol. 86, 1073–1084 (2012).
pubmed: 22989026
pmcid: 3508365
Boone, T. J. et al. Coordinated assembly of the Bacillus anthracis Coat and exosporium during bacterial spore outer layer formation. MBio 9, 6 (2018).
Durand-Heredia, J., Hsieh, H.-Y., Spreng, K. A. & Stewart, G. C. Roles and organization of BxpB (ExsFA) and ExsFB in the exosporium outer basal layer of Bacillus anthracis. J. Bacteriol. 204, e00290-e322 (2022).
pubmed: 36394311
pmcid: 9765029
Durand-Heredia, J., Hsieh, H.-Y., Thompson, B. M. & Stewart, G. C. ExsY, CotY, and CotE effects on Bacillus anthracis outer spore layer architecture. J. Bacteriol. 204, e00290-e322 (2022).
pubmed: 36394311
pmcid: 9765029
Durand-Heredia, J. & Stewart, G. C. Localization of the CotY and ExsY proteins to the exosporium basal layer of Bacillus anthracis. Microbiol. Open 11, e1327 (2022).
Giorno, R. et al. Localization and assembly of proteins comprising the outer structures of the Bacillus anthracis spore. Microbiol. (Read.) 155, 1133–1145 (2009).
Ohye, D. F. & Murrell, W. G. Exosporium and spore coat formation in Bacillus cereus T. J. Bacteriol. 115, 1179–1190 (1973).
pubmed: 4199508
pmcid: 246368
Giorno, R. et al. Morphogenesis of the Bacillus anthracis spore. J. Bacteriol. 189, 691–705 (2007).
pubmed: 17114257
Boydston, J. A., Yue, L., Kearney, J. F. & Turnbough, C. L. The ExsY protein is required for complete formation of the exosporium of Bacillus anthracis. J. Bacteriol. 188, 7440–7448 (2006).
pubmed: 16936017
pmcid: 1636282
Johnson, M. J. et al. ExsY and CotY are required for the correct assembly of the exosporium and spore coat of Bacillus cereus. J. Bacteriol. 188, 7905–7913 (2006).
pubmed: 16980471
pmcid: 1636315
Ryter, A. Morphologic study of the sporulation of Bacillus subtilis. Ann. Inst. Pasteur (Paris) 108, 40–60 (1965).
pubmed: 14289982
Al-Hinai, M. A., Jones, S. W. & Papoutsakis, E. T. The Clostridium sporulation programs: Diversity and preservation of endospore differentiation. Microbiol. Mol. Biol. R 79, 19–37 (2015).
Fimlaid, K. A. & Shen, A. Diverse mechanisms regulate sporulation sigma factor activity in the Firmicutes. Curr. Opin. Microbiol. 24, 88–95 (2015).
pubmed: 25646759
pmcid: 4380625
Kroos, L., Zhang, B., Ichikawa, H. & Yu, Y.-T.N. Control of σ factor activity during Bacillus subtilis sporulation. Mol. Microbiol. 31, 1285–1294 (1999).
pubmed: 10200951
Piggot, P. J. & Hilbert, D. W. Sporulation of Bacillus subtilis. Curr. Opin. Microbiol. 7, 579–586 (2004).
pubmed: 15556029
Serrano, M. et al. Dual-specificity anti-sigma factor reinforces control of cell-type specific gene expression in Bacillus subtilis. PLoS Genet. 11, e1005104 (2015).
pubmed: 25835496
pmcid: 4383634
Campo, N. & Rudner, D. Z. SpoIVB and CtpB are both forespore signals in the activation of the sporulation transcription factor σ
pubmed: 17557826
pmcid: 1952037
Serrano, M. et al. The SpoIIQ-SpoIIIAH complex of Clostridium difficile controls forespore engulfment and late stages of gene expression and spore morphogenesis. Mol. Microbiol. 100, 204–228 (2016).
pubmed: 26690930
pmcid: 4982068
Fimlaid, K. A., Jensen, O., Donnelly, M. L., Siegrist, M. S. & Shen, A. Regulation of Clostridium difficile spore formation by the SpoIIQ and SpoIIIA proteins. PLoS Genet. 11, e1005562 (2015).
pubmed: 26465937
pmcid: 4605598
Saujet, L. et al. Genome-wide analysis of cell type-specific gene transcription during spore formation in Clostridium difficile. PLoS Genet. 9, e1003756 (2013).
pubmed: 24098137
pmcid: 3789822
Pereira, F. C. et al. The spore differentiation pathway in the enteric pathogen Clostridium difficile. PLoS Genet. 9, e1003782 (2013).
pubmed: 24098139
pmcid: 3789829
Fimlaid, K. A. et al. Global analysis of the sporulation pathway of Clostridium difficile. PLoS Genet. 9, e1003660 (2013).
pubmed: 23950727
pmcid: 3738446
Ribis, J. W., Ravichandran, P., Putnam, E. E., Pishdadian, K. & Shen, A. The conserved spore coat protein SpoVM is largely dispensable in Clostridium difficile spore formation. mSphere 2, e00315-17 (2017).
pubmed: 28959733
pmcid: 5607322
Terry, C. et al. Molecular tiling on the surface of a bacterial spore—the exosporium of the Bacillus anthracis/cereus/thuringiensis group. Mol. Microbiol. 104, 539–552 (2017).
pubmed: 28214340
pmcid: 5434927
Stewart, G. C. Assembly of the outermost spore layer: Pieces of the puzzle are coming together. Mol. Microbiol. 104, 535–538 (2017).
pubmed: 28207180
pmcid: 5426953
Cassona, C. P., Pereira, F., Serrano, M. & Henriques, A. O. A fluorescent reporter for single cell analysis of gene expression in Clostridium difficile. In Clostridium difficile: Methods and Protocols (eds. Roberts, A. P. & Mullany, P.) 69–90 (Springer, 2016).
Lanzilli, M. et al. The exosporium of Bacillus megaterium QM B1551 is permeable to the red fluorescence protein of the coral Discosoma sp. Front. Microbiol 7, 1752 (2016).
pubmed: 27867376
pmcid: 5095127
Donadio, G., Lanzilli, M., Sirec, T., Ricca, E. & Isticato, R. Localization of a red fluorescence protein adsorbed on wild type and mutant spores of Bacillus subtilis. Microb. Cell Factor. 15, 153 (2016).
Sirec, T., Benarroch, J. M., Buffard, P., Garcia-Ojalvo, J. & Asally, M. Electrical polarization enables integrative quality control during bacterial differentiation into spores. iScience 16, 378–389 (2019).
pubmed: 31226599
pmcid: 6586994
Magge, A., Setlow, B., Cowan, A. E. & Setlow, P. Analysis of dye binding by and membrane potential in spores of Bacillus species. J. Appl. Microbiol. 106, 814–824 (2009).
pubmed: 19187156
pmcid: 2661013
Kuwana, R., Yamazawa, R., Ito, K. & Takamatsu, H. Comparative analysis of thioflavin T and other fluorescent dyes for fluorescent staining of Bacillus subtilis vegetative cell, sporulating cell, and mature spore. Biosci. Biotechnol. Biochem. 87, 338–348 (2023).
pubmed: 36472554
Bressuire-Isoard, C., Bornard, I., Henriques, A. O., Carlin, F. & Broussolle, V. Sporulation temperature reveals a requirement for CotE in the assembly of both the coat and exosporium layers of Bacillus cereus spores. Appl. Environ. Microbiol. 82, 232–243 (2016).
pubmed: 26497467
Sharp, M. D. & Pogliano, K. An in vivo membrane fusion assay implicates SpoIIIE in the final stages of engulfment during Bacillus subtilis sporulation. PNAS 96, 14553–14558 (1999).
pubmed: 10588743
pmcid: 24474
Doan, T. et al. FisB mediates membrane fission during sporulation in Bacillus subtilis. Genes Dev. 27, 322–334 (2013).
pubmed: 23388828
pmcid: 3576517
Perez, A. R., Mello, A.A.-D. & Pogliano, K. SpoIIB localizes to active sites of septal biogenesis and spatially regulates septal thinning during engulfment in Bacillus subtilis. J. Bacteriol. 182, 1096–1108 (2000).
pubmed: 10648537
pmcid: 94387
Mello, A.A.-D., Sun, Y.-L., Aung, S. & Pogliano, K. A cytoskeleton-like role for the bacterial cell wall during engulfment of the Bacillus subtilis forespore. Genes Dev. 16, 3253–3264 (2002).
Meyer, P., Gutierrez, J., Pogliano, K. & Dworkin, J. Cell wall synthesis is necessary for membrane dynamics during sporulation of Bacillus subtilis. Mol. Microbiol. 76, 956–970 (2010).
pubmed: 20444098
pmcid: 2893020
Ojkic, N., López-Garrido, J., Pogliano, K. & Endres, R. G. Cell-wall remodeling drives engulfment during Bacillus subtilis sporulation. Elife 5, e18657 (2016).
pubmed: 27852437
pmcid: 5158138
Chan, H. et al. Genetic screens identify additional genes implicated in envelope remodeling during the engulfment stage of Bacillus subtilis sporulation. MBio 13, e01732-22 (2022).
pubmed: 36066101
pmcid: 9600426
Bravo, A., Agaisse, H., Salamitou, S. & Lereclus, D. Analysis of cryIAa expression in sigE and sigK mutants of Bacillus thuringiensis. Mol. Gen. Genet. 250, 734–741 (1996).
pubmed: 8628234
Peng, Q. et al. The regulation of exosporium-related genes in Bacillus thuringiensis. Sci. Rep. 6, 19005 (2016).
pubmed: 26805020
pmcid: 4750369
Manetsberger, J., Hall, E. A. H. & Christie, G. Plasmid-encoded genes influence exosporium assembly and morphology in Bacillus megaterium QM B1551 spores. FEMS Microbiol. Lett. 362, fnv147 (2015).
pmcid: 4674009
McPherson, S. A., Li, M., Kearney, J. F. & Turnbough, C. L. ExsB, an unusually highly phosphorylated protein required for the stable attachment of the exosporium of Bacillus anthracis. J. Bacteriol. 76, 1527–1538 (2010).
Todd, S. J., Moir, A. J. G., Johnson, M. J. & Moir, A. Genes of Bacillus cereus and Bacillus anthracis encoding proteins of the exosporium. J. Bacteriol. 185, 3373–3378 (2003).
pubmed: 12754235
pmcid: 155386
Kim, H. et al. The Bacillus subtilis spore coat protein interaction network. Mol. Microbiol. 59, 487–502 (2006).
pubmed: 16390444
Freitas, C. et al. A protein phosphorylation module patterns the Bacillus subtilis spore outer coat. Mol. Microbiol. 114, 934–951 (2020).
pubmed: 32592201
pmcid: 7821199
Saggese, A. et al. Antagonistic role of CotG and CotH on spore germination and coat formation in Bacillus subtilis. PLoS ONE 9, e104900 (2014).
pubmed: 25115591
pmcid: 4130616
Sacco, M., Ricca, E., Losick, R. & Cutting, S. An additional GerE-controlled gene encoding an abundant spore coat protein from Bacillus subtilis. J. Bacteriol. 177, 372–377 (1995).
pubmed: 7814326
pmcid: 176600
Saggese, A. et al. CotG Mediates spore surface permeability in Bacillus subtilis. MBio 13, e02760-22 (2022).
pubmed: 36354330
pmcid: 9765600
Haraldsen, J. D. & Sonenshein, A. L. Efficient sporulation in Clostridium difficile requires disruption of the σ
pubmed: 12694623
Abe, K. et al. Regulated DNA rearrangement during sporulation in Bacillus weihenstephanensis KBAB4. Mol. Microbiol. 90, 415–427 (2013).
pubmed: 24015831
Klee, S. R. et al. The genome of a Bacillus isolate causing anthrax in chimpanzees combines chromosomal properties of B. cereus with B. anthracis virulence plasmids. PLoS ONE 5, e10986 (2010).
pubmed: 20634886
pmcid: 2901330
Gustafsson, M. G. L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. Microsc. 198, 82–87 (2000).
pubmed: 10810003
Heintzmann, R. Saturated patterned excitation microscopy with two-dimensional excitation patterns. Micron 34, 283–291 (2003).
pubmed: 12932771
Betzig, E. Excitation strategies for optical lattice microscopy. Opt. Express 13, 3021–3036 (2005).
pubmed: 19495199
Costes, S. V. et al. Automatic and quantitative measurement of protein-protein colocalization in live cells. Biophys. J. 86, 3993–4003 (2004).
pubmed: 15189895
pmcid: 1304300
Lereclus, D., Arantès, O., Chaufaux, J. & Lecadet, M.-M. Transformation and expression of a cloned δ-endotoxin gene in Bacillus thuringiensis. FEMS Microbiol. Lett. 60, 211–217 (1989).
Sanchis, V., Agaisse, H., Chaufaux, J. & Lereclus, D. Construction of new insecticidal Bacillus thuringiensis recombinant strains by using the sporulation non-dependent expression system of cryIIIA and a site specific recombination vector. J. Biotechnol. 48, 81–96 (1996).
pubmed: 8818275