An exosporium-like outer layer in Bacillus subtilis spores

Sousa, Joana; Silva, M. T.; Balassa, G

doi:10.1038/263053a0

Cited by 38 publications

(25 citation statements)

References 9 publications

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“…However, an exosporium has not been observed surrounding B. subtilis spores, with the exception of spores of strains isolated from the human gastrointestinal tract (14). Sousa et al observed an exosporium-like layer of B. subtilis after partial chemical extraction of the spore coats (39). More recently, an external glycoprotein layer surrounding spores of B. subtilis, which is less apparent with traditional staining methods used for electron microscopy, was found by ruthenium red staining (49).…”

Section: Discussionmentioning

confidence: 98%

Localization of Proteins to Different Layers and Regions ofBacillus subtilisSpore Coats

et al. 2010

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Bacterial spores are encased in a multilayered proteinaceous shell known as the coat. In Bacillus subtilis, over 50 proteins are involved in spore coat assembly but the locations of these proteins in the spore coat are poorly understood. Here, we describe methods to estimate the positions of protein fusions to fluorescent proteins in the spore coat by using fluorescence microscopy. Our investigation suggested that CotD, CotF, CotT, GerQ, YaaH, YeeK, YmaG, YsnD, and YxeE are present in the inner coat and that CotA, CotB, CotC, and YtxO reside in the outer coat. In addition, CotZ and CgeA appeared in the outermost layer of the spore coat and were more abundant at the mother cell proximal pole of the forespore, whereas CotA and CotC were more abundant at the mother cell distal pole of the forespore. These polar localizations were observed both in sporangia prior to the release of the forespore from the mother cell and in mature spores after release. Moreover, CotB was observed at the middle of the spore as a ring-or spiral-like structure. Formation of this structure required cotG expression. Thus, we conclude not only that the spore coat is a multilayered assembly but also that it exhibits uneven spatial distribution of particular proteins.Proper localization and assembly of proteins in cells and subcellular structures are essential features of living organisms. Complex protein assemblies, including ribosomes, flagella, and the cytokinetic machinery, play important roles in bacteria (26,27,40). Studying how these complex structures are formed is a fundamental theme in molecular biology. In this work, we developed a method to analyze one of the most complex bacterial protein assemblies: the spore coat of Bacillus subtilis.Sporulation of B. subtilis is initiated in response to nutrient limitation, and it involves a highly ordered program of gene expression and morphological change (33, 42). The first morphological change of sporulation is the appearance of an asymmetrically positioned septum that divides the cell into a larger mother cell and a smaller forespore. Next, the mother cell membrane migrates around the forespore membrane during a phagocytosislike process called engulfment. The completion of engulfment involves fusion of the mother cell membrane to pinch off the forespore within the mother cell. Compartment-specific gene expression brings about maturation of the spore and its release upon lysis of the mother cell (reviewed in reference 19). Mature spores remain viable during long periods of starvation and are resistant to heat, toxic chemicals, lytic enzymes, and other factors capable of damaging vegetative cells (30). Spores germinate and resume growth when nutrients become available (32).The outer portions of Bacillus spores consist of a cortex, a spore coat layer, and in some cases, an exosporium. The cortex, a thick layer of peptidoglycan, is deposited between the inner and the outer membranes of the forespore, and it is responsible for maintaining the highly dehydrated state of the core, thereby contri...

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Section: Discussionmentioning

confidence: 98%

Localization of Proteins to Different Layers and Regions ofBacillus subtilisSpore Coats

et al. 2010

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“…The existence of a corresponding outer layer in B. subtilis is uncertain, however. Nevertheless, a tight-fitting exosporium-like envelope that is visible by electron microscopy after partial extraction of coat proteins has been described (55). The effects of cge mutations, then, may reflect a role in biosynthesis or modification of this exosporium layer, presumably affecting its polysaccharide component.…”

Section: Discussionmentioning

confidence: 99%

Adjacent and divergently oriented operons under the control of the sporulation regulatory protein GerE in Bacillus subtilis

Roels

Losick

1995

J Bacteriol

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The DNA-binding protein GerE is the latest-acting regulatory protein in the mother cell line of gene expression during sporulation in Bacillus subtilis. GerE directs the transcription of several genes that encode structural components of the protein coat that encases the mature spore. We report on the identification and characterization of a cluster of additional genes whose transcription is dependent on GerE. These genes, which are located in the replication terminus region of the chromosome (181؇ on the genetic map), are arranged in adjacent and divergently oriented operons called cgeAB and cgeCDE, which consist of two and at least three genes, respectively. CgeD, the product of the second member of the cgeCDE operon, is strikingly similar to the product of a B. subtilis gene (ipa-63d) of unknown function and is similar at its amino terminus to certain glycosyl transferases involved in polysaccharide biosynthesis. Strains with mutations in the cgeAB and cgeCDE operons produce spores with altered surface properties, on which basis we propose that proteins encoded by these operons influence maturation of the outermost layer of the spore, perhaps by glycosylation of coat proteins at the spore surface.

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“…B. subtilis, the paradigm sporeformer, does not have this distinct layer, although the B. subtilis spore coat may have a tight-fitting outermost layer that can be visualized after extraction of coat material with urea or mercaptoethanol (23). Scanning electron microscopy has revealed that the exosporium is composed of two layers-a paracrystalline basal layer with hexagonal periodicity and a "hairy nap" outer layer (11).…”

Section: Endospores Of the Bacillus Cereus Family Which Includesmentioning

confidence: 99%

ExsY and CotY Are Required for the Correct Assembly of the Exosporium and Spore Coat of Bacillus cereus

et al. 2006

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The exosporium-defective phenotype of a transposon insertion mutant of Bacillus cereus implicated ExsY, a homologue of B. subtilis cysteine-rich spore coat proteins CotY and CotZ, in assembly of an intact exosporium. Single and double mutants of B. cereus lacking ExsY and its paralogue, CotY, were constructed. The exsY mutant spores are not surrounded by an intact exosporium, though they often carry attached exosporium fragments. In contrast, the cotY mutant spores have an intact exosporium, although its overall shape is altered. The single mutants show altered, but different, spore coat properties. The exsY mutant spore coat is permeable to lysozyme, whereas the cotY mutant spores are less resistant to several organic solvents than is the case for the wild type. The exsY cotY double-mutant spores lack exosporium and have very thin coats that are permeable to lysozyme and are sensitive to chloroform, toluene, and phenol. These spore coat as well as exosporium defects suggest that ExsY and CotY are important to correct formation of both the exosporium and the spore coat in B. cereus. Both ExsY and CotY proteins were detected in Western blots of purified wild-type exosporium, in complexes of high molecular weight, and as monomers. Both exsY and cotY genes are expressed at late stages of sporulation. Endospores of the Bacillus cereus family, which includesBacillus anthracis and Bacillus thuringiensis, are enveloped by a large, balloon-like layer known as the exosporium (11,19). B. subtilis, the paradigm sporeformer, does not have this distinct layer, although the B. subtilis spore coat may have a tight-fitting outermost layer that can be visualized after extraction of coat material with urea or mercaptoethanol (23). Scanning electron microscopy has revealed that the exosporium is composed of two layers-a paracrystalline basal layer with hexagonal periodicity and a "hairy nap" outer layer (11). The exosporium is chemically complex and is composed of 53% protein, 20% amino and neutral polysaccharides, 18% lipids, and ϳ4% ash (17).Multiple proteins from the exosporium of both B. cereus and B. anthracis have been identified (22,24,31); the majority do not have homologues in B. subtilis. For example, the surface layers of B. cereus, B. anthracis, and B. thuringiensis spores all contain glycoproteins with characteristic collagen-like repeat regions (5,10,27). Of these, the B. anthracis glycoprotein BclA is an essential component of the hairy nap layer of the exosporium (27, 29).Analysis of genome sequence information from B. cereus ATCC 14579 and B. anthracis Ames identified a cluster of genes near bclA implicated in exosporium production, including the region whose genes were designated yjbX-exsYyjcA-yjcB-exsFA-cotY (31). The ExsY and CotY proteins are homologues (ca. 35% amino acid identity) of the B. subtilis coat proteins CotY and CotZ (37). ExsY and CotY proteins have both been detected in purified exosporium from B. anthracis endospores by N-terminal sequencing of separated peptides (22). ExsY and CotY are very sim...

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An exosporium-like outer layer in Bacillus subtilis spores

Cited by 38 publications

References 9 publications

Localization of Proteins to Different Layers and Regions ofBacillus subtilisSpore Coats

Localization of Proteins to Different Layers and Regions ofBacillus subtilisSpore Coats

Adjacent and divergently oriented operons under the control of the sporulation regulatory protein GerE in Bacillus subtilis

ExsY and CotY Are Required for the Correct Assembly of the Exosporium and Spore Coat of Bacillus cereus

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