Proteins Involved in Formation of the Outermost Layer of Bacillus subtilis Spores

Abstract

To investigate the outermost structure of the Bacillus subtilis spore, we analyzed the accessibility of antibodies to proteins on spores of B. subtilis. Anti-green fluorescent protein (GFP) antibodies efficiently accessed GFP fused to CgeA or CotZ, which were previously assigned to the outermost layer termed the spore crust. However, anti-GFP antibodies did not bind to spores of strains expressing GFP fused to 14 outer coat, inner coat, or cortex proteins. Anti-CgeA antibodies bound to spores of wild-type and CgeA-GFP strains but not cgeA mutant spores. These results suggest that the spore crust covers the spore coat and is the externally exposed, outermost layer of the B. subtilis spore. We found that CotZ was essential for the spore crust to surround the spore but not for spore coat formation, indicating that CotZ plays a critical role in spore crust formation. In addition, we found that CotY-GFP was exposed on the surface of the spore, suggesting that CotY is an additional component of the spore crust. Moreover, the localization of CotY-GFP around the spore depended on CotZ, and CotY and CotZ depended on each other for spore assembly. Furthermore, a disruption of cotW affected the assembly of CotV-GFP, and a disruption of cotX affected the assembly of both CotV-GFP and CgeA-GFP. These results suggest that cgeA and genes in the cotVWXYZ cluster are involved in spore crust formation.

Water and Small-Molecule Permeation of Dormant Bacillus subtilis Spores

“…Although this trend suggests that proteins residing between the outside of the spore and the inner coat have increasing effects on the rate of water exchange, cotXYZ spores, which lack the outermost crust layer, display an exchange rate equivalent to that of cotE spores. Consistent with this result, it has been suggested that the spore crust may contribute to the structure of the outer coat, as this layer is easily disrupted in spores lacking CotXYZ (41). All of the other mutations tested exchange water faster than the current limit of detection of this assay.…”

The Exosporium Layer of Bacterial Spores: a Connection to the Environment and the Infected Host

“…In an analysis of spore coat proteins involving fluorescent protein fusions, the B. subtilis spore coat was predicted to be comprised of four distinct layers, including an outermost glycoprotein layer, named the "crust," that is separated from the outer coat by a small space (53). Genetic and localization studies have shown that cgeA and genes in the cotVWXYZ locus are involved in spore crust formation (53,54). Interestingly, the CotZ protein, which plays a role in the assembly of the crust layer of B. subtilis, displays sequence similarity to the ExsY protein (35% identity and 46% similarity over 141 residues), a protein involved in exosporium assembly in B. anthracis (42).…”

In vitro and in vivo analyses of the Bacillus anthracis spore cortex lytic protein SleL

“…Previous studies show that sleL is expressed under the control of s E , which is active in the mother cell during early stages of sporulation (Chen et al, 2000;Kodama et al, 1999). SleL localizes to the outer layers of the developing forespore and is associated with spore coat proteins (Chen et al, 2000;Imamura et al, 2010;McKenney et al, 2010). On the basis of sequence homologies, SleL is composed of three conserved domains: two N-terminal LysM domains and a C-terminal glycosyl hydrolase family 18 domain (Terwisscha van Scheltinga et al, 1995;van Aalten et al, 2001).…”