Characterization of Spores of Bacillus subtilis That Lack Most Coat Layers

Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT 06030-3305, USA.
Journal of bacteriology (Impact Factor: 2.81). 09/2008; 190(20):6741-8. DOI: 10.1128/JB.00896-08
Source: PubMed


Spores of Bacillus subtilis have a thick outer layer of relatively insoluble protein called the coat, which protects spores against a number of treatments
and may also play roles in spore germination. However, elucidation of precise roles of the coat in spore properties has been
hampered by the inability to prepare spores lacking all or most coat material. In this work, we show that spores of a strain
with mutations in both the cotE and gerE genes, which encode proteins involved in coat assembly and expression of genes encoding coat proteins, respectively, lack
most extractable coat protein as seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as well as the great majority
of the coat as seen by atomic force microscopy. However, the cotE gerE spores did retain a thin layer of insoluble coat material that was most easily seen by microscopy following digestion of
these spores with lysozyme. These severely coat-deficient spores germinated relatively normally with nutrients and even better
with dodecylamine but not with a 1:1 chelate of Ca2+ and dipicolinic acid. These spores were also quite resistant to wet heat, to mechanical disruption, and to treatment with
detergents at an elevated temperature and pH but were exquisitely sensitive to killing by sodium hypochlorite. These results
provide new insight into the role of the coat layer in spore properties.

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    • "We also employed the strain PS4150 in which most of the cotE and gerE coding sequences are deleted, hence this strain lacks most of its coat [26] "
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    ABSTRACT: We utilize the fluorescent molecular rotor Bodipy-C12 to investigate the viscoelastic properties of hydrophobic layers of bacterial spores Bacillus subtilis. The molecular rotor shows a marked increase in fluorescence lifetime, from 0.3 to 4ns, upon viscosity increase from 1 to 1500cP and can be incorporated into the hydrophobic layers within the spores from dormant state through to germination. We use fluorescence lifetime imaging microscopy (FLIM) to visualize the viscosity inside different compartments of the bacterial spore in order to investigate the inner membrane and relate its compaction to the extreme resistance observed during exposure of spores to toxic chemicals. We demonstrate that the bacterial spores possess an inner membrane that is characterized by a very high viscosity, exceeding 1000cP, where the lipid bilayer is likely in a gel state. We also show that this membrane evolves during germination to reach a viscosity value close to that of a vegetative cell membrane, ca. 600cP. The present study demonstrates quantitative imaging of the microscopic viscosity in hydrophobic layers of bacterial spores Bacillus subtilis and shows the potential for further investigation of spore membranes under environmental stress.
    Biochimica et Biophysica Acta 07/2013; DOI:10.1016/j.bbamem.2013.06.028 · 4.66 Impact Factor
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    • "The spore coat has also been identified as a critical resistance mechanism against many chemicals, especially oxidizing agents such as hydrogen peroxide (Riesenman and Nicholson 2000; Young and Setlow 2004b), ozone (Young and Setlow 2004a), peroxynitrite (Genest et al. 2002), chlorine dioxide and hypochlorite (Young and Setlow 2003; Ghosh et al. 2008), all of which kill spores more rapidly when the coat layer is absent. This protective role was perhaps most clearly illustrated by Ghosh et al. (2008), who showed that B. subtilis spores lacking most coat layers owing to mutations in the cotE and gerE genes (coding for a morphogenetic protein essential for formation of the outer coat, and a DNA-binding protein that itself regulates several genes coding for coat proteins (Driks 1999), respectively) became sensitive to hypochlorite to a level similar to that of vegetative cells. Despite the clear protective role of the spore coat, and an increasingly detailed understanding of the mechanisms , components and genetic controls involved in spore coat assembly (Driks 1999; Takamatsu and Watabe 2002; Henriques and Moran 2007; McKenney and Eichenberger 2012), no individual coat proteins have been identified as an essential protective component. "
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    ABSTRACT: The structure and chemical composition of bacterial spores differ considerably from those of vegetative cells. These differences largely account for the unique resistance properties of the spore to environmental stresses, including disinfectants and sterilants, resulting in the emergence of spore-forming bacteria such as Clostridium difficile as major hospital pathogens. Although there has been considerable work investigating the mechanisms of action of many sporicidal biocides against Bacillus subtilis spores, there is far less information available for other species and particularly for various Clostridia. This paucity of information represents a major gap in our knowledge given the importance of Clostridia as human pathogens. This review considers the main spore structures, highlighting their relevance to spore resistance properties and detailing their chemical composition, with a particular emphasis on the differences between various spore formers. Such information will be vital for the rational design and development of novel sporicidal chemistries with enhanced activity in the future.
    Journal of Applied Microbiology 05/2012; 113(3):485-98. DOI:10.1111/j.1365-2672.2012.05336.x · 2.48 Impact Factor
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    • "In fact, spores of a B. subtilis cotE gerE double mutant that almost entirely lack a spore coat germinated essentially normally and were resistant to wet heat, to mechanical disruption, and to treatment with detergents at an elevated temperature and pH (Ghosh, et al., 2008). One prospect in characterizing phenotypes is to expand the phenotypic assays, as has been done recently with spore digestion by the protozoan Tetrahymena thermophila (Klobutcher, et al., 2006) and the nematode Caenorhabditis elegans (Laaberki & Dworkin, 2008). "
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    ABSTRACT: The Gram-positive bacterium Bacillus subtilis can initiate the process of sporulation under conditions of nutrient limitation. Here, we review some of the last 5 years of work in this area, with a particular focus on the decision to initiate sporulation, DNA translocation, cell-cell communication, protein localization and spore morphogenesis. The progress we describe has implications not only just for the study of sporulation but also for other biological systems where homologs of sporulation-specific proteins are involved in vegetative growth.
    FEMS microbiology reviews 10/2011; 36(1):131-48. DOI:10.1111/j.1574-6976.2011.00310.x · 13.24 Impact Factor
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