No abstract

The endospore of Bacillus subtilis is formed intracellularly upon nutrient starvation and is encased by proteinaceous shells. The outermost layer, the crust, is a postulated glycoprotein layer that is composed of six proteins: CotV, W, X, Y, Z and CgeA. Despite some insight into protein interactions and the identification of players in glycosylation, a clear picture of its architecture is still missing. Here, we report a comprehensive mutational analysis that confirms CotZ as the anchor of the crust, while the crust structure is provided by CotV, CotX and CotY. CotY seems to be the major structural component, while CotV and CotX are polar and co-depend on each other and partially on CotW. CotW is independent of other crust proteins, instead depending on outer coat proteins, indicating a role at the interface of crust and coat. CgeA is coexpressed with putative glycosyltransferases (CgeB and CgeD) and implicated in crust glycosylation. In accordance, we provide evidence that CgeB, CgeCDE, SpsA-L, SpsM and SpsNOPQR (formerly YfnHGFED) contribute to the glycosylation state of the spore. The crust polysaccharide layer consists of functionally linked rhamnose-and galactose-related variants and could contain rare sugars. It may therefore protect the crust against biological degradation and scavenging. Protein interaction network and structure of the crustThe crust contains at least six different proteins: CotVWXYZ, which were long known as part of the insoluble fraction of the spore coat (Zhang et al.

The electronic structure of individual sheets of the bacterial surface protein layer (S layer) of Bacillus sphaericus NCTC 9602 was studied using a photoemission electron microscope (PEEM) operating in near-edge X-ray absorption fine structure spectroscopy mode. The laterally resolved measurements performed at the C 1s, N 1s, and O 1s thresholds on fresh samples revealed characteristic differences compared to the laterally integrated data, where substrate contributions were taken along with the protein signals. During the PEEM experiments an irradiation-induced increase of the C-C bond density at the cost of the densities of the C-O and C-N bonds related to a rearrangement of the contributing atoms of the S layer deposited onto a Si substrate was observed. The critical irradiation doses for the C-O and C-N bond breaking and formation of the new C-C bonds were derived.