The exudate of fully germinated spores of Clostridium perfringens S40 in 0.15 M KCl-50 mM potassium phosphate (pH 7.0) was found to contain another spore-lytic enzyme in addition to the germination-specific amidase previously characterized (S. Miyata, R. Moriyama, N. Miyahara, and S. Makino, Microbiology 141:2643-2650, 1995). The lytic enzyme was purified to homogeneity by anion-exchange chromatography and shown to be a muramidase which requires divalent cations (Ca ) for its activity. The enzyme was inactivated by sulfhydryl reagents, and sodium thioglycolate reversed the inactivation by Hg 2؉. The muramidase hydrolyzed isolated spore cortical fragments from a variety of wild-type organisms but had minimal activity on decoated spores and isolated cell walls. However, the enzyme was not capable of digesting isolated cortical fragments from spores of Bacillus subtilis ADD1, which lacks muramic acid ␦-lactam in its cortical peptidoglycan. This indicates that the enzyme recognizes the ␦-lactam residue peculiar to spore peptidoglycan, suggesting an involvement of the enzyme in spore germination. Immunochemical studies indicated that the muramidase in its mature form is localized on the exterior of the cortex layer in the dormant spore. A gene encoding the muramidase, sleM, was cloned into Escherichia coli, and the nucleotide sequence was determined. The gene encoded a protein of 321 amino acids with a deduced molecular weight of 36,358. The deduced amino acid sequence of the sleM gene indicated that the enzyme is produced in a mature form. It was suggested that the muramidase belongs to a separate group within the lysozyme family typified by the fungus Chalaropsis lysozyme. A possible mechanism for cortex degradation in C. perfringens S40 spores is discussed.Bacterial spore germination, defined as the irreversible loss of spore characteristics, is triggered by specific germinants and proceeds through a set of sequential steps. The later stage of germination involves decomposition of the spore peptidoglycan by a spore cortex-lytic enzyme(s) (8,24). The enzymes involved in the reaction have been identified from spores of Bacillus megaterium (7), Bacillus cereus (20, 26), Bacillus subtilis (25), and Clostridium perfringens (22, 23) and characterized as germination-specific amidases. A 30-kDa amidase of B. subtilis contributes to germination of the organism mediated by Lalanine but not to germination triggered by an unspecified germinant(s) present in culture medium such as Schaffer's medium (25). This suggests the presence of multiple pathways for spore germination, and it is likely that a germination-specific enzyme(s) other than the amidases identified to date is also involved in hydrolysis of spore peptidoglycan.Gombas and Labbe reported (14) that spore cortex-lytic enzymes differing in their substrate specificity are released during germination of C. perfringens spores, though it was not clear whether the enzymes are actually involved in germination; one such enzyme lyses decoated spores, and the other hydrolyzes i...
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