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Journal of Bacteriology, November 2005, p. 7753-7764, Vol. 187, No. 22
0021-9193/05/$08.00+0     doi:10.1128/JB.187.22.7753-7764.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Rita Zilhão,1,2,{dagger} Rachele Isticato,1,3,{dagger} Lígia O. Martins,1,4 Leif Steil,5 Uwe Völker,5 Ezio Ricca,3 Charles P. Moran Jr,6 and Adriano O. Henriques1*

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, Apartado 127, 2781-901 Oeiras Codex, Portugal,1 Universidade de Lisboa, Faculdade de Ciências, Campo Grande C2, 1700 Lisboa, Portugal,2 Dipartimento di Fisiologia Generale ed Ambientale, Università Federico II, Napoli, Italy,3 Universidade Lusófona de Humanidades e Tecnologias, Departamento de Engenharias e Tecnologias, Av. do Campo Grande, 376, 1749-024 Lisboa, Portugal,4 Ernst-Moritz-Arndt-University, Medical Faculty, Laboratory for Functional Genomics, D-17487 Greifswald, Germany,5 Emory University School of Medicine, Department of Microbiology and Immunology, Atlanta, Georgia 303226

Received 17 January 2005/ Accepted 26 August 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 {varepsilon}-({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.

* Corresponding author. Mailing address: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, Apartado 127, 2781-901 Oeiras Codex, Portugal. Phone: 351-21-4469521. Fax: 351-21-4411277. E-mail: aoh{at}itqb.unl.pt.

{dagger} These authors contributed equally to the work.

Journal of Bacteriology, November 2005, p. 7753-7764, Vol. 187, No. 22
0021-9193/05/$08.00+0     doi:10.1128/JB.187.22.7753-7764.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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