The Bacillus subtilis spore is encased in a multilayered protein coat that confers resistance to peptidoglycan-breaking enzymes and noxious chemicals and also influences the germination response. Biogenesis of the spore coat relies on the ordered synthesis and assembly of over 40 polypeptides (5, 10, 11). The assembly process is controlled at various levels. First, the time and degree of expression of the genes encoding the various coat components are under complex regulation. Most, if not all, components of the coat are produced in the mother cell chamber of the sporulating cell (5, 10, 11, 34). Some coat proteins are synthesized prior to engulfment of the prespore by the mother cell, and those are under the control of σE, whose activity is influenced by the GerR and SpoIIID ancillary transcription factors (5, 6, 9, 10, 11). However, the synthesis of many other coat proteins and the deposition of most of the coat structural components are governed by σK, which replaces σE following engulfment completion (5, 9, 10, 11). The activity of σK is modulated by the GerE transcription factor, itself the product of a σK-controlled gene, which can act both as an activator or a repressor (5, 6, 10, 11, 41). Second, the formation of the spore coat is controlled by a class of unique morphogenetic proteins which are required for the assembly of several structural components at the surface of the developing spore (5, 10, 11). Third, the process is also controlled by posttranscriptional and posttranslational mechanisms which include proteolysis, glycosylation, and cross-linking. Together, these mechanisms are thought to contribute to the proper structural organization and properties of the coat structure (5, 10, 11). The roles of several coat morphogenetic proteins have been examined in some detail (5, 10, 11, 22). However, our knowledge of the contribution of mechanisms such as protein cross-linking to the assembly process is still very limited. Nevertheless, about 30% of the total spore coat protein is refractory to extraction, forming an insoluble highly cross-linked fraction (5, 10, 30). Several coat proteins appear to be cross-linked, and the available evidence suggests that both reversible and irreversible cross-links occur within the coat (5, 10, 11, 12, 14, 20, 21, 42). In a classical study, Pandey and Aronson (30) have suggested the existence of o,o-dityrosine cross-links in purified coat material, an observation confirmed by more recent work (5). CotG and three other tyrosine-rich coat components, CotB, CotC, and CotU, have been recently shown to be present as multimers within the spore coat (12, 21, 14, 42). However, the nature of the cross-links in CotB, CotC, CotU, or CotG has not been reported, and even though other enzymes could, in principle, promote oxidative cross-linking of the coat structural components (see references 7 and 25 and references therein), a peroxidase activity associated with the B. subtilis spore has not yet been found. In contrast, a transglutaminase activity is detected in sporulating cells and spores (16, 18, 37), and ɛ-(γ-glutamyl)lysyl cross-links have been detected in B. subtilis spores and in purified coat material (17). Moreover, the gene (tgl) for a transglutaminase was cloned following the purification of a spore-associated activity (16, 37). The enzyme could cross-link α-casein and bovine serum albumin in vitro, and a cysteine residue at position 116 was found to be essential for the reaction (16). Tgl-like polypeptides are present in other Bacillus and related spore-forming species but absent from the genomes of other bacteria, suggesting a specific role in sporulation (Fig. (Fig.1).1). Recently, it was shown that the deletion of tgl prevents cross-linking of GerQ, a coat protein that is also involved in spore germination (29, 31, 32). However, it is not known whether GerQ is directly cross-linked by Tgl (31). Other polypeptides have been suggested to be cross-linked by a transglutaminase in the coats, including the lysine- and glutamine-rich CotX protein, a component of the coat insoluble fraction and CotM, a member of the α-crystallin family of small heat shock proteins (12, 39). FIG. 1. Tgl is present in other spore formers. The figure represents a Clustal W alignment (www.ebi.ac.uk) of the Tgl proteins found in the following sporeformers: Bs, B. subtilis 168 (NP-391005); Bl, Bacillus licheniformis DSM 13 (YP-080406); Gk, Geobacillus ... Transglutaminases intervene in key biological processes, such as cytoskeletal scaffolding, the control of protein-protein interactions, blood clotting, wound healing, apoptosis, development and morphogenesis, or the keratinization of skin (8, 23). Because the ɛ-(γ-glutamyl)lysyl isopeptide is stable and proteolysis resistant, it contributes to the rigidity and resistance to chemical, enzymatic, physical, and mechanical decay of various tissues and cellular structures (8, 23). Here, we have investigated the assembly and function of Tgl. Our results suggest that Tgl may be present at various levels within the coats and that its assembly is independent of its activity. We also found that in addition to GerQ, three other polypeptides are more extractable from spores of a tgl deletion mutant or from those with an inactive Tgl. The complete insolubilization of at least two of the Tgl-controlled polypeptides occurs several hours later than when the assembly of Tgl is first detected. Tgl is required for the structural integrity of the outer coat layers, and increased production of Tgl interferes with the assembly and structure of the coat and alters its germination properties.