MassIVE MSV000086681 - Identification of Native Cross-links in Bacillus subtilis Spore Coat Proteins

The resistance properties of the bacterial spores are partially due to spore surface proteins, ~30% of which are said to form an insoluble protein fraction. Previous research has also identified a group of spore coat proteins affected by spore maturation, which exhibit an increased level of inter protein cross-linking. However, the proteins and the types of cross-links involved, previously proposed based on indirect evidence, have yet to be confirmed experimentally. To obtain more insight into the structural basis the proteinaceous component of the spore coat we attempted to identify coat cross-links and the proteins involved using new peptide fractionation and bioinformatic methods. Young (day 1) and matured (day 5) Bacillus subtilis spores of wild type and transglutaminase mutant strains were digested with formic acid and trypsin, and cross-linked peptides were enriched using strong cation exchange chromatography. The enriched cross-linked peptide fractions were subjected to FT-ICR MS/MS and the high-quality fragmentation data obtained were analysed using two specialized software tools, pLink2 and XiSearch, to identify cross-links. This analysis identified specific disulfide bonds between coat proteins CotE-CotE and CotJA-CotJC, obtained evidence for disulfide bonds in the spore crust proteins CotX, CotY and CotZ, and identified dityrosine and eplison-(gamma)-glutamyl-lysine cross-linked coat proteins. The findings in this report are the first direct biochemical data on protein cross-linking in the spore coat and the first direct evidence for the crosslinked building blocks of the highly ordered and resistant structure called the spore coat.