Assembly and Function of a Spore Coat-Associated Transglutaminase of Bacillus subtilis

Abstract

The assembly of a multiprotein coat around the Bacillus subtilis spore confers resistance to lytic enzymes and noxious chemicals and ensures normal germination. Part of the coat is cross-linked and resistant to solubilization. The coat contains -(␥-glutamyl)lysyl cross-links, and the expression of the gene (tgl) for a sporeassociated transglutaminase was shown before to be required for the cross-linking of coat protein GerQ. Here, we have investigated the assembly and function of Tgl. We found that Tgl associates, albeit at somewhat reduced levels, with the coats of mutants that are unable to assemble the outer coat (cotE), that are missing the inner coat and with a greatly altered outer coat (gerE), or that are lacking discernible inner and outer coat structures (cotE gerE double mutant). This suggests that Tgl is present at various levels within the coat lattice. The assembly of Tgl occurs independently of its own activity, as a single amino acid substitution of a cysteine to an alanine (C116A) at the active site of Tgl does not affect its accumulation or assembly. However, like a tgl insertional mutation, the tglC116A allele causes increased extractability of polypeptides of about 40, 28, and 16 kDa in addition to GerQ (20 kDa) and affects the structural integrity of the coat. We show that most Tgl is assembled onto the spore surface soon after its synthesis in the mother cell under K control but that the complete insolubilization of at least two of the Tgl-controlled polypeptides occurs several hours later. We also show that a multicopy allele of tgl causes increased assembly of Tgl and affects the assembly, structure, and functional properties of the coat.

Localization of the Transglutaminase Cross-Linking Sites in the Bacillus subtilis Spore Coat Protein GerQ

“…However, to date, Tgl-dependent cross-linking has been shown only for GerQ, making it the only known Tgl substrate in B. subtilis (19,25). Recent work has also provided additional evidence for Tgl-dependent cross-linking of GerQ, although the number of Tgl-dependent cross-linked species present in spore extracts in this recent study was greatly reduced compared to that in our wild-type spores (13,19).…”

“…A number of coat proteins have been suggested to be involved in Tgl-mediated cross-linking (8,19,(24)(25)(26). However, to date, Tgl-dependent cross-linking has been shown only for GerQ, making it the only known Tgl substrate in B. subtilis (19,25).…”

“…Tgl activity has been detected in sporulating cells of B. subtilis, and a single tgl gene from this species has been identified (10,12). Purified B. subtilis Tgl is able to cross-link ␣-casein and bovine serum albumin in vitro (23), and recent evidence suggests that Tgl and the cross-links that it forms are present in the spore coat (11,12,25).…”

Use of transglutaminases in foods and potential utilization of plants as a transglutaminase source – Review

“…Iranzo et al (2002) suggested that transglutaminase may be involved in the formation of covalent cross-links between proteins during cell wall construction of Saccharomyces cerevisiae. In addition, some studies have shown that TGases are involved in the assembly of spore coat proteins of the genus Bacillus through epsilon-(gammaglutamyl)lysine bonds (KobAyAsHi et al, 1998;ZiLHão et al, 2005;PLáCIDo et al, 2008).…”

Purification and On-Column Activation of a Recombinant Histidine-Tagged Pro-Transglutaminase after Soluble Expression inEscherichia coli

“…5,6) In prokaryotes, calcium-independent TGs with only one subunit have been discovered in Streptomyces 7,8) and Bacillus. 9,10) A typical microbial transglutaminase (MTG) was produced by the wild-type Streptomyces mobaraensis strain, which is widely used as a protein crosslinking enzyme, especially in food processing. 11) To date, the main source of MTG is conventional fermentation at high cost.…”