Bacillus anthracis is a spore-forming bacterium that is included on the list of select agents compiled by the Centers for Disease Control. When a B. anthracis spore germinates, a protective layer of peptidoglycan known as the cortex must be depolymerized by germination-specific lytic enzymes (GSLEs) before the bacterium can become a metabolically active vegetative cell. By exploiting cortex lytic enzymes it may be possible to control germination. This could be beneficial in elucidating ways to enhance current decontamination methods. In this work we created in-frame deletion mutants to study not only the role of one GSLE, SleL, but by creating multi-deletion mutants, we were able to analyze how the protein cooperates with other lytic enzymes to efficiently hydrolyze the cortical PG. We determined that SleL plays an auxiliary role in complete peptidoglycan hydrolysis, secondary to cortex lytic enzymes CwlJ1, CwlJ2, and SleB. The loss of sleL results in a delay in the loss of optical density during germination. However, spores are capable of completing germination as long as CwlJ1 or SleB remains active. HPLC analysis of muropeptides collected from B. anthracis sleL strains indicates that SleL is an N-acetylglucosamidase that acts on cortical PG to produce small muropeptides which are quickly released from the germinating spore. By analyzing the in vitro and in vivo activities of SleL we confirmed the enzymatic activity of the protein, characterized its substrates, and studied the roles of its putative LysM domains in substrate binding and spore-protein association. We were able to show that purified SleL is capable of depolymerizing partially digested spore PG resulting in the production of N-acetylglucosaminidase products that are readily released as small muropeptides. In vitro, loss of the LysM domain(s) decreases hydrolysis effectiveness. The reduction in hydrolysis is likely due to LysM domains being involved in substrate recognition and PG binding. When the SleL derivatives are expressed in vivo those proteins lacking one or both LysM domains do not associate with the spore, suggesting that LysM is involved in directing protein localization. / Ph. D.

Quando le condizioni sono sfavorevoli alla crescita, membri dei generi Bacillus e Clostridia (incluso Clostridium botulinum, l’agente eziologico del botulismo) formano endospore, forme cellulari estremamente resistenti, metabolicamente dormienti e difficili da distruggere. Tuttavia, le spore attraverso il processo di germinazione, riattivano il ciclo vegetativo non appena le condizioni tornano favorevoli. Questa capacità di “riattivazione” delle spore è causa di “food spoilage” e di intossicazioni alimentari. Considerando che le specie Clostridium botulinum e Clostridium sporogenes sono filogeneticamente correlate, in questo lavoro, il ceppo Clostridium sporogenes UC9000, isolato da latte crudo, è stato utilizzato come modello non-patogeno di Clostridium botulinum per studiare la germinazione. Studi fisiologici hanno rivelato che le spore del ceppo UC9000 germinano in presenza di L-alanina/ L-cisteina in combinazione con L-lattato, mentre un analisi in silico ha permesso di identificare omologhi dei recettori coinvolti nella risposta all’L-alanina in Bacillus. Attraverso l’analisi del genoma sono stati identificati gli enzimi SleB, CwlJ e SleL, responsabili della degradazione del cortex. CwlJ è stato localizzato nel coat della spora grazie ad uno studio di proteomica, è stato espresso in forma solubile in E. coli ed un test di attività in vitro ha evidenziato la sua capacità di indurre la germinazione di spore “decoated” / When environmental conditions are unfavorable to the growth, Bacillus and Clostridium bacteria (including Clostridium botulinum, the causative agent of foodborne botulism) form endospores, metabolically dormant cell types resistant to several adverse conditions and difficult to kill. However, under suitable conditions, spores resume the vegetative life by triggering the germination process. Thus, spores are dangerous agents of human foodborne disease and food spoilage. In this work, the strain Clostridium sporogenes UC9000, isolated from raw milk, was used like not-pathogenic model of Clostridium botulinum to better understand the mechanisms underpinning the Clostridium germination. Clostridium sporogenes is a species phylogenetically related to Clostridium botulinum and often used like its surrogate. Physiological studies revealed that UC9000 spores germinate in presence of L-alanine/L-cysteine in combination with L-lactate, while in silico analyses allowed the identification of homologues of the Bacillus germinant receptors responsive to L-alanine. The genome screening also detected genes coding for SleB, CwlJ and SleL, enzymes participating to the cortex degradation. CwlJ was found resident in the spore coat by performing a proteomic analysis, it was expressed in soluble form in E. coli and an in vitro assay of activity revealed its capability to induce germination when added exogenously to decoated spores