Identification of CgeA as a glycoprotein that anchors polysaccharides to the spore surface in Bacillus subtilis.

The Bacillus subtilis spore is composed of a core, containing chromosomal DNA, surrounded by a cortex layer made of peptidoglycan, and a coat composed of concentric proteinaceous layers. A polysaccharide layer is added to the spore surface, and likely anchored to the crust, the coat outermost layer. However, the identity of the coat protein(s) to which the spore polysaccharides (SPS) are attached is uncertain. First, we showed that the crust proteins CotVWXYZ and CgeA were all contained in the peeled SPS layer obtained from a strain missing CotE, the outer coat morphogenetic protein, suggesting that the SPS is indeed bound to at least one of the spore surface proteins. Second, CgeA is known to be located at the most downstream position in the crust assembly pathway. An analysis of truncated variants of CgeA suggested that its N-terminal half is required for localization to the spore surface, while its C-terminal half is necessary for SPS addition. Third, an amino acid substitution strategy revealed that SPS was anchored at threonine 112 (T112), which constitutes a probable O-glycosylation site on CgeA. Our results indicated that CgeA is a glycoprotein required to initiate SPS assembly and serves as an anchor protein linking the crust and SPS layers.

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

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.

Contributions of crust proteins to spore surface properties in Bacillus subtilis.

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.