Contributions of crust proteins to spore surface properties in <i>Bacillus subtilis</i>

Shuster, Bentley; Khemmani, Mark; Abe, Kimihiro; Huang, Xiaoyu; Nakaya, Yusei; Maryn, Nina; Buttar, Sally; Gonzalez, Adriana N.; Driks, Adam; Sato, Tsutomu; Eichenberger, Patrick

doi:10.1111/mmi.14194

Cited by 44 publications

(80 citation statements)

References 58 publications

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“…The percentage of spores remaining in the aqueous layer is obtained by comparing the optical densities of the spore suspension before and after vortexing. All three methods were used recently to determine the roles played by crust structural proteins in these properties (22). Previous work has revealed that the last four genes in the spsA-L operon encode enzymes involved in rhamnose biosynthesis during the late stages of sporulation (8).…”

Section: Resultsmentioning

confidence: 99%

“…In this article, we continue a line of research initiated when we investigated how spore surface properties were influenced by the structural proteins responsible for proper crust assembly, focusing on cotO and cotVWXYZ (22). The deletion of these genes greatly disrupted the crust structure, resulted in perturbations of the PS distribution around the spore, and caused modifications in the hydrophobicity of the spore surface.…”

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confidence: 99%

“…It remains unclear if the outer coat is an anchor site by default, i.e., solely used in the absence of the crust, or if wild-type spores bear more than one site for PS addition. Our previous work also looked at the cgeAB operon because of its potential role in PS anchoring in B. subtilis spores (22). Roels and Losick originally showed that cgeAB and cgeCDE are two adjacent operons expressed under the control of K and GerE that confer specific properties to the outermost layer of the spore, possibly by glycosylation of coat proteins (40).…”

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confidence: 99%

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Expansion of the Spore Surface Polysaccharide Layer in Bacillus subtilis by Deletion of Genes Encoding Glycosyltransferases and Glucose Modification Enzymes

et al. 2019

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Polysaccharides (PS) decorate the surface of dormant endospores (spores). In the model organism for sporulation, Bacillus subtilis, 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, spsABCDEFGHIJKL, yfnHGF-yfnED, ytdA-ytcABC, and cgeAB-cgeCDE, on the morphology and properties of the crust, the outermost spore layer. Our results show that all mutations in the sps 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 cgeD and the yfnH–D cluster noticeably expand the PS layer. In addition, yfnH–D mutant spores exhibit a crust with an unusual weblike morphology. The hydrophobic phenotype from sps mutant spores was partially rescued by a second mutation inactivating any gene in the yfnHGF operon. While spsI, yfnH, and ytdA 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. IMPORTANCE 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 Bacillus subtilis (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.

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Expansion of the Spore Surface Polysaccharide Layer in Bacillus subtilis by Deletion of Genes Encoding Glycosyltransferases and Glucose Modification Enzymes

et al. 2019

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“…It is known that the outermost B. subtilis spore layer, termed the crust, contains both proteins and carbohydrate, giving a matrix outside the coat protein layer (27). However, the precise structure and composition of the crust layer are not known, and the spore crust would be expected to be greatly reduced in severely coat-defective spores such as PS4150, which lacks the coat morphogenic protein CotE, as well as the GerE transcription factor for many coat protein genes (19,27). In principle, Tb 3ϩ /Dy 3ϩ could bind to either carboxylate groups or phosphate groups on either a sugar or a protein backbone.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, four isogenic B. subtilis strains were used with the PY79 genetic background, including PS3483 (the wild-type strain), PE620 lacking the cotXYZ operon, PE2763 lacking spsI, and PE2916 lacking cgeB. The latter three strains were obtained from Patrick Eichenberger, and each lacks various components of the spores' outermost crust layer (27). The B. cereus strain T was originally obtained from H. O. Halvorson.…”

Section: Methodsmentioning

confidence: 99%

Accumulation and Release of Rare Earth Ions by Spores of Bacillus Species and the Location of These Ions in Spores

Wei

Camilleri

et al. 2019

Appl Environ Microbiol

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Two rare earth ions, Tb3+ and Dy3+, were incorporated into spores of Bacillus species in ≤5 min at neutral pH to 100 to 200 nmol per mg of dry spores, which is equivalent to 2 to 3% of the spore dry weight. The uptake of these ions had, at most, minimal effects on spore wet heat resistance or germination, and the ions were all released upon germination, probably by complex formation with the huge depot of dipicolinic acid (DPA) released when spores germinate. Adsorbed Tb3+/Dy3+ were also released by exogenous DPA within a few minutes and faster than in spore germination. The accumulation of Tb3+/Dy3+ was not reduced in Bacillus subtilis spores by several types of coat defects, significant modification of the spore cortex peptidoglycan structure, specific loss of components of the outer spore crust layer, or the absence of DPA in the spore core. All of these findings are consistent with Tb3+/Dy3+ being accumulated in spores’ outer layers, and this was confirmed by transmission electron microscopy. However, the identity of the outer spore components binding the Tb3+/Dy3+ is not clear. These findings provide new information on the adsorption of rare earth ions by Bacillus spores and suggest this adsorption might have applications in capturing rare earth ions from the environment. IMPORTANCE Biosorption of rare earth ions by growing cells of Bacillus species has been well studied and has attracted attention for possible hydrometallurgy applications. However, the interaction of spores from Bacillus species with rare earth ions has not been well studied. We investigated here the adsorption and/or desorption of two rare earth ions, Tb3+ and Dy3+, by Bacillus spores, the location of the adsorbed ions, and the spore properties after ion accumulation. The significant adsorption of rare earth ions on the surfaces of Bacillus spores and the ions’ rapid release by a chelator could allow the development of these spores as a biosorbent to recover rare earth ions from the environment.

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Localization of the CotY and ExsY proteins to the exosporium basal layer of Bacillus anthracis

Durand-Heredia

Stewart

2022

MicrobiologyOpen

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Spores are an infectious form of the zoonotic bacterial pathogen, Bacillus anthracis . The outermost spore layer is the exosporium, comprised of a basal layer and an external glycoprotein nap layer. The major structural proteins of the inner basal layer are CotY (at the mother cell central pole or bottlecap) and ExsY around the rest of the spore. The basis for the cap or noncap specificity of the CotY and ExsY proteins is currently unknown. We investigated the role of sequence differences between these proteins in localization during exosporium assembly. We found that sequence differences were less important than the timing of expression of the respective genes in the positioning of these inner basal layer structural proteins. Fusion constructs with the fluorescent protein fused at the N‐terminus resulted in poor incorporation whereas fusions at the carboxy terminus of CotY or ExsY resulted in good incorporation. However, complementation studies revealed that fusion constructs, although accurate indicators of protein localization, were not fully functional. A model is presented that explains the localization patterns observed. Bacterial two‐hybrid studies in Escherichia coli hosts were used to examine protein–protein interactions with full‐length and truncated proteins. The N‐terminus amino acid sequences of ExsY and CotY appear to be recognized by spore proteins located in the spore interspace, consistent with interactions seen with ExsY and CotY with the interspace proteins CotE and CotO, known to be involved with exosporium attachment.

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Contributions of crust proteins to spore surface properties in Bacillus subtilis

Cited by 44 publications

References 58 publications

Expansion of the Spore Surface Polysaccharide Layer in Bacillus subtilis by Deletion of Genes Encoding Glycosyltransferases and Glucose Modification Enzymes

Expansion of the Spore Surface Polysaccharide Layer in Bacillus subtilis by Deletion of Genes Encoding Glycosyltransferases and Glucose Modification Enzymes

Accumulation and Release of Rare Earth Ions by Spores of Bacillus Species and the Location of These Ions in Spores

Localization of the CotY and ExsY proteins to the exosporium basal layer of Bacillus anthracis

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