Zilhão Rita, Isticato Rachele, Martins Lígia O, Steil Leif, Völker Uwe, Ricca Ezio, Moran Charles P, Henriques Adriano O
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras Codex, Portugal.
J Bacteriol. 2005 Nov;187(22):7753-64. doi: 10.1128/JB.187.22.7753-7764.2005.
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 epsilon-(gamma-glutamyl)lysyl cross-links, and the expression of the gene (tgl) for a spore-associated 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 sigma(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.
枯草芽孢杆菌芽孢周围多蛋白外壳的组装赋予了对裂解酶和有害化学物质的抗性，并确保正常萌发。外壳的一部分是交联的，且对溶解具有抗性。外壳含有ε-（γ-谷氨酰）赖氨酰交联键，之前已证明与芽孢相关的转谷氨酰胺酶基因（tgl）的表达是外壳蛋白GerQ交联所必需的。在这里，我们研究了Tgl的组装和功能。我们发现，Tgl尽管水平有所降低，但仍与无法组装外层外壳（cotE）、缺少内层外壳且外层外壳有很大改变（gerE）或缺乏可辨别的内层和外层外壳结构（cotE gerE双突变体）的突变体的外壳相关联。这表明Tgl以不同水平存在于外壳晶格中。Tgl的组装独立于其自身活性，因为Tgl活性位点的一个半胱氨酸被丙氨酸单氨基酸取代（C116A）并不影响其积累或组装。然而，与tgl插入突变一样，tglC116A等位基因除了导致GerQ（20 kDa）外，还使约40、28和16 kDa的多肽的可提取性增加，并影响外壳的结构完整性。我们表明，大多数Tgl在母细胞中受σ（K）控制合成后不久就组装到芽孢表面，但至少两种受Tgl控制的多肽的完全不溶性在数小时后才发生。我们还表明，tgl的多拷贝等位基因会导致Tgl组装增加，并影响外壳的组装、结构和功能特性。
