Sporulation in Bacillus subtilis involves an asymmetric cell division followed by differentiation into two cell types, the endospore and the mother cell. The endospore coat is a multilayered shell ...that protects the bacterial genome during stress conditions and is composed of dozens of proteins. Recently, fluorescence microscopy coupled with high-resolution image analysis has been applied to the dynamic process of coat assembly and has shown that the coat is organized into at least four distinct layers. In this Review, we provide a brief summary of B. subtilis sporulation, describe the function of the spore surface layers and discuss the recent progress that has improved our understanding of the structure of the endospore coat and the mechanisms of coat assembly.
The Spore Coat Driks, Adam; Eichenberger, Patrick
Microbiology spectrum,
04/2016, Letnik:
4, Številka:
2
Journal Article
Recenzirano
Spores of Clostridiales and Bacillales are encased in a complex series of concentric shells that provide protection, facilitate germination, and mediate interactions with the environment. Analysis of ...diverse spore-forming species by thin-section transmission electron microscopy reveals that the number and morphology of these encasing shells vary greatly. In some species, they appear to be composed of a small number of discrete layers. In other species, they can comprise multiple, morphologically complex layers. In addition, spore surfaces can possess elaborate appendages. For all their variability, there is a consistent architecture to the layers encasing the spore. A hallmark of all Clostridiales and Bacillales spores is the cortex, a layer made of peptidoglycan. In close association with the cortex, all species examined possess, at a minimum, a series of proteinaceous layers, called the coat. In some species, including Bacillus subtilis, only the coat is present. In other species, including Bacillus anthracis, an additional layer, called the exosporium, surrounds the coat. Our goals here are to review the present understanding of the structure, composition, assembly, and functions of the coat, primarily in the model organism B. subtilis, but also in the small but growing number of other spore-forming species where new data are showing that there is much to be learned beyond the relatively well-developed basis of knowledge in B. subtilis. To help summarize this large field and define future directions for research, we will focus on key findings in recent years.
Spores produced by bacilli and clostridia are surrounded by a multilayered protein shell called the coat. As the armor-like appearance of the coat suggests, this structure, along with others within ...the spore, confers the remarkable resistance properties that make
Bacillus anthracis spores such potent biological weapons. Here, I review recent studies of coat assembly in the model organism
Bacillus subtilis, and explore the implications of these findings for coat assembly in
B. anthracis and for defense against biological weapons.
The protein armor surrounding bacterial spores, called the coat, has a complex architecture and assembly program. Analysis of coat composition and morphogenesis reveals important lessons for basic cell biology and defense against biological warfare.
Bacilli and Clostridia generate dormant, highly resistant cells, called spores, in response to stress. Spores of many species are decorated by morphologically diverse structures of unknown function ...called appendages that have yet to be studied at the molecular level. In this issue, Walker et al. employ reverse genetics to identify genes encoding protein components of the ornate ribbon-like appendages of the spores of Clostridium taeniosporum. Their results reveal striking commonalities between these genes and those encoding outer structures in phylogenetically and taxonomically distinct spore-forming species. The insights gained from this work demonstrate the value of analysis of non-model spore formers.
The Dynamic Spore Driks, Adam
Proceedings of the National Academy of Sciences - PNAS,
03/2003, Letnik:
100, Številka:
6
Journal Article
Recenzirano
Odprti dostop
Most bacteria can, at least to some degree, hunker down during periods of stress and wait for good times to return. No cells, however, do this as effectively as those Bacilli and Clostridia that form ...spores.
Summary
Surface properties, such as adhesion and hydrophobicity, constrain dispersal of bacterial spores in the environment. In Bacillus subtilis, these properties are influenced by the outermost ...layer of the spore, the crust. Previous work has shown that two clusters, cotVWXYZ and cgeAB, encode the protein components of the crust. Here, we characterize the respective roles of these genes in surface properties using Bacterial Adherence to Hydrocarbons assays, negative staining of polysaccharides by India ink and Transmission Electron Microscopy. We showed that inactivation of crust genes caused increases in spore relative hydrophobicity, disrupted the spore polysaccharide layer, and impaired crust structure and attachment to the rest of the coat. We also found that cotO, previously identified for its role in outer coat formation, is necessary for proper encasement of the spore by the crust. In parallel, we conducted fluorescence microscopy experiments to determine the full network of genetic dependencies for subcellular localization of crust proteins. We determined that CotZ is required for the localization of most crust proteins, while CgeA is at the bottom of the genetic interaction hierarchy.
A group of Bacillus subtilis coat proteins, encoded by cotO, cotVW, cotXYZ and cgeAB, influence spore crust structure, relative surface hydrophobicity and anchoring of a polysaccharide layer. Crust assembly is controlled by a complex set of interactions, with CotZ acting as the principal morphogenetic crust protein and cgeA at the bottom of the genetic hierarchy. CotO promotes encasement of the spore by the crust.
The ability to grow as a biofilm can facilitate survival of bacteria in the environment and promote infection. To better characterize biofilm formation in the pathogen Clostridium difficile, we ...established a colony biofilm culture method for this organism on a polycarbonate filter, and analyzed the matrix and the cells in biofilms from a variety of clinical isolates over several days of biofilm culture. We found that biofilms readily formed in all strains analyzed, and that spores were abundant within about 6 days. We also found that extracellular DNA (eDNA), polysaccharide and protein was readily detected in the matrix of all strains, including the major toxins A and/or B, in toxigenic strains. All the strains we analyzed formed spores. Apart from strains 630 and VPI10463, which sporulated in the biofilm at relatively low frequencies, the frequencies of biofilm sporulation varied between 46 and 65%, suggesting that variations in sporulation levels among strains is unlikely to be a major factor in variation in the severity of disease. Spores in biofilms also had reduced germination efficiency compared to spores obtained by a conventional sporulation protocol. Transmission electron microscopy revealed that in 3 day-old biofilms, the outermost structure of the spore is a lightly staining coat. However, after 6 days, material that resembles cell debris in the matrix surrounds the spore, and darkly staining granules are closely associated with the spores surface. In 14 day-old biofilms, relatively few spores are surrounded by the apparent cell debris, and the surface-associated granules are present at higher density at the coat surface. Finally, we showed that biofilm cells possess 100-fold greater resistance to the antibiotic metronidazole then do cells cultured in liquid media. Taken together, our data suggest that C. difficile cells and spores in biofilms have specialized properties that may facilitate infection.
Polysaccharides (PS) decorate the surface of dormant endospores (spores). In the model organism for sporulation,
, the composition of the spore PS is not known in detail. Here, we have assessed how ...PS synthesis enzymes produced during the late stages of sporulation affect spore surface properties. Using four methods, bacterial adhesion to hydrocarbons (BATH) assays, India ink staining, transmission electron microscopy (TEM) with ruthenium red staining, and scanning electron microscopy (SEM), we characterized the contributions of four sporulation gene clusters,
,
,
-
, and
, on the morphology and properties of the crust, the outermost spore layer. Our results show that all mutations in the
operon result in the production of spores that are more hydrophobic and lack a visible crust, presumably because of reduced PS deposition, while mutations in
and the
cluster noticeably expand the PS layer. In addition,
mutant spores exhibit a crust with an unusual weblike morphology. The hydrophobic phenotype from
mutant spores was partially rescued by a second mutation inactivating any gene in the
operon. While
, and
are paralogous genes, all encoding glucose-1-phosphate nucleotidyltransferases, each paralog appears to contribute in a distinct manner to the spore PS. Our data are consistent with the possibility that each gene cluster is responsible for the production of its own respective deoxyhexose. In summary, we found that disruptions to the PS layer modify spore surface hydrophobicity and that there are multiple saccharide synthesis pathways involved in spore surface properties.
Many bacteria are characterized by their ability to form highly resistant spores. The dormant spore state allows these species to survive even the harshest treatments with antimicrobial agents. Spore surface properties are particularly relevant because they influence spore dispersal in various habitats from natural to human-made environments. The spore surface in
(crust) is composed of a combination of proteins and polysaccharides. By inactivating the enzymes responsible for the synthesis of spore polysaccharides, we can assess how spore surface properties such as hydrophobicity are modulated by the addition of specific carbohydrates. Our findings indicate that several sporulation gene clusters are responsible for the assembly and allocation of surface polysaccharides. Similar mechanisms could be modulating the dispersal of infectious spore-forming bacteria.
Bacillus subtilis spores are encased in a protein assembly called the spore coat that is made up of at least 70 different proteins. Conventional electron microscopy shows the coat to be organized ...into two distinct layers. Because the coat is about as wide as the theoretical limit of light microscopy, quantitatively measuring the localization of individual coat proteins within the coat is challenging. We used fusions of coat proteins to green fluorescent protein to map genetic dependencies for coat assembly and to define three independent subnetworks of coat proteins. To complement the genetic data, we measured coat protein localization at subpixel resolution and integrated these two data sets to produce a distance-weighted genetic interaction map. Using these data, we predict that the coat comprises at least four spatially distinct layers, including a previously uncharacterized glycoprotein outermost layer that we name the spore crust. We found that crust assembly depends on proteins we predicted to localize to the crust. The crust may be conserved in all
Bacillus spores and may play critical functions in the environment.
► The
B. subtilis spore coat is composed of at least four distinct layers ► SafA is the major organizer of the inner coat ► The spore crust is the outermost layer of the
B. subtilis coat ► The
cotXYZ gene cluster is necessary for crust assembly
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