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Studies on the relationship between assembly of the spore coat and the biofilm
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Resumo(s)
In the model organism Bacillus subtilis, the surface of the spore consists of a protein coat that affords protection against noxious chemicals and lytic enzymes and modulates spore germination and binding to cells and abiotic surfaces. The coat, formed by over 80 proteins, is divided into an inner layer, an outer layer and a crust. The coat proteins are synthesized in the mother cell and are recruited to the surface of the developing spores through specific protein-protein interactions and also according to successive waves of gene expression. SafA is a morphogenetic protein responsible for the assembly of the inner coat layer. SafA exists in three forms, the full-length protein, SafAFL, SafAC30 which corresponds to the C-terminal moiety of the protein, and SafAN21, corresponding to the N-terminal moiety of the protein. The C30 region is responsible for recruiting the inner coat proteins, whereas the N-terminal region carries the signals for localization at the spore surface. Among the proteins that SafA recruits to the inner coat is a transglutaminase, called Tgl, in a process termed substrate-driven localization. Assembly of SafA is auto-regulatory in that once assembled, Tgl introduces ε-(γ glutamyl)lysil bonds into SafA. SafAC30 forms an oblong hexamer, (SafAC30)6 and this species is cross linked in vitro by Tgl. Strikingly, the activity of Tgl decreased with increasing SafAC30 concentration, suggesting both that Tgl cross-linked itself to SafAC30 and a mechanism for controlling the activity of the enzyme in vivo. Here we overproduced and purified SafAFL and SafAN21 and tested for cross-linking by purified Tgl. Although Tgl proved to be much less efficient in cross-linking SafAN21 than SafAFL or SafAC30, its activity was inversely proportional to the concentration of all three substrates. Thus, in vitro, all three forms of SafA have the potential of controlling the activity of Tgl in a concentration-dependent manner. That SafAN21 is a poor substrate for Tgl, in spite of the fact that it contains several glutamine and lysine residues, suggests that this region of SafA is not involved in cross-linking to itself or to Tgl in vivo and raises the possibility that it may be involved in cross-linking to another protein or directly to the cortex peptidoglycan. Moreover, we found that formation of (SafAC30)6 involves disulfide bonds and that Tgl exerts a “spotwelding” activity on this species, i.e., not all the monomers in (SafAC30)6 are cross-linked by Tgl. Two lysine residues in SafAC30, K177 and K318 are important for this activity but K318 makes a more important contribution. Since K318 is close to two of the four cysteine residues in SafAC30, C323 and C325, and also in the close vicinity of several glutamine residues, a model is proposed in which disulfide bond formation at this position nucleates further cross-linking of (SafAC30)6 by Tgl. Veg, an 86 amino acid protein, was known to influence biofilm formation and spore germination. The veg gene is downstream of yabG, a gene that is part of a genomic signature for sporulation. yabG codes for a cysteine protease required for processing of the Tgl substrates prior to their cross-linking by Tgl. YabG also functions in the assembly of the spore surface layers in the human pathogen Clostridioides difficile. Here, we have shown that the overexpression of veg during the late stages of sporulation in B. subtilis impairs assembly of the abundant CotB and CotG, the latter a critical determinant of the structural organization of the outer coat. The assembly of other proteins, such as the peptidoglycan hydrolase YdhD and of a putative L-cysteine binding protein was also affected. Veg bears a Sm-like motif characteristic of proteins that regulate mRNA stability and translation in all domains of life. Molecular modelling studies suggest that, like the Sm proteins, Veg forms an heptamer with a central channel and a positively charged surface that may mediate interactions with single-stranded nuclei acids. Veg may thus define a novel level of control in spore coat assembly, influencing mRNA stability and/or translation and we propose that it functions in the same way in biofilm development. We overproduced and purified the C. difficile Veg protein, an 11 kDa protein with significant similarity to B. subtilis Veg. The structural and biochemical characterization of Veg will provide important insights into its function and its suspected functional relationship to YabG.
Descrição
Tese de mestrado, Microbiologia Aplicada, Universidade de Lisboa, Faculdade de Ciências, 2021
Palavras-chave
manto montagem SafA Tgl Veg Teses de mestrado - 2021