Finally, there are also disulfide links connecting the cysteine-rich outermost crust proteins CotX, CotY, and CotZ, which are encoded in the cotVWXYZ gene cluster. (5) Proteins GerQ and SafA of the inner coat are among the known substrates of Tgl, (6,7) but it is not known which proteins they are linked to. (4) The ε-(γ)-glutamyl-lysine links are formed by the enzyme transglutaminase (Tgl). (3) There are several tyrosine-rich proteins in the outer spore coat, of which proteins CotC and CotU have been shown to assemble into high mass forms. Dityrosine cross-links are likely formed by an unknown peroxidase, which is thought to be associated with multimerization of the outer coat protein CotG, and in the presence of the hydrogen-peroxide-producing enzyme SodA. However, although some spore coat proteins are rich in lysine, glutamine, tyrosine, and cysteine, estimating the proportions of proteins incorporating the cross-links is still a challenge. (1,2) Three types of cross-links are suggested to be present in the spore coat: dityrosine, ε-(γ)-glutamyl-lysine, and disulfide cross-links. (1) It has been reported that at least 30% of coat proteins are characterized by interprotein cross-linking. (1) The morphologically complex coat is a proteinaceous layer with ∼25% of total spore proteins and has posed a significant challenge in the extraction of coat proteins, many of which are insoluble. The resistance properties of Bacillus subtilis spores are partially due to the assembly of the coat during sporulation in addition to the spore core levels of Ca 2+-dipicolinic acid (DPA), small acid-soluble DNA-binding proteins, and water. The findings in this Letter are the first direct biochemical data on protein cross-linking in the spore coat and the first direct evidence of the cross-linked building blocks of the highly ordered and resistant structure called the spore coat. This analysis identified specific disulfide bonds between coat proteins CotE–CotE and CotJA–CotJC, obtained evidence of disulfide bonds in the spore crust proteins CotX, CotY, and CotZ, and identified dityrosine and ε-(γ)-glutamyl-lysine cross-linked coat proteins.
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The enriched cross-linked peptide fractions were subjected to Fourier-transform ion cyclotron resonance tandem mass spectrometry, and the high-quality fragmentation data obtained were analyzed using two specialized software tools, pLink2 and XiSearch, to identify cross-links. 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.
To obtain more insight into the structural basis of 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.
However, the proteins and the types of cross-links involved, previously proposed based on indirect evidence, have yet to be confirmed experimentally. Previous research has also identified a group of spore coat proteins affected by spore maturation, which exhibit an increased level of interprotein cross-linking. 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.