A spore cortex-lytic enzyme of Clostridium perfringens S40 which is encoded by sleC is synthesized at an early stage of sporulation as a precursor consisting of four domains. After cleavage of an N-terminal presequence and a C-terminal prosequence during spore maturation, inactive proenzyme is converted to active enzyme by processing of an N-terminal prosequence with germination-specific protease (GSP) during germination. The present study was undertaken to characterize GSP. In the presence of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), a nondenaturing detergent which was needed for the stabilization of GSP, GSP activity was extracted from germinated spores. The enzyme fraction, which was purified to 668-fold by column chromatography, contained three protein components with molecular masses of 60, 57, and 52 kDa. The protease showed optimum activity at pH 5.8 to 8.5 in the presence of 0.1% CHAPS and retained activity after heat treatment at 55°C for 40 min. GSP specifically cleaved the peptide bond between Val-149 and Val-150 of SleC to generate mature enzyme. Inactivation of GSP by phenylmethylsulfonyl fluoride and HgCl 2 indicated that the protease is a cysteine-dependent serine protease. Several pieces of evidence demonstrated that three protein components of the enzyme fraction are processed forms of products of cspA, cspB, and cspC, which are positioned in a tandem array just upstream of the 5 end of sleC. The amino acid sequences deduced from the nucleotide sequences of the csp genes showed significant similarity and showed a high degree of homology with those of the catalytic domain and the oxyanion binding region of subtilisin-like serine proteases. Immunochemical studies suggested that active GSP likely is localized with major cortex-lytic enzymes on the exterior of the cortex layer in the dormant spore, a location relevant to the pursuit of a cascade of cortex hydrolytic reactions.Bacterial spore germination, defined as the irreversible loss of spore characteristics, is triggered by specific germinants and proceeds through a set of sequential steps. Spore germination is essential to allow spore outgrowth and the formation of a new vegetative cell; once triggered, it proceeds in the absence of germinants and germinant-stimulated metabolism. This fact indicates that spore germination is a process controlled by the sequential activation of a set of preexisting germination-related enzymes but not by protein synthesis (10, 26).Among the key enzymes involved in the spore germination of Bacillus subtilis 168, Bacillus cereus IFO 13597, and Clostridium perfringens S40 are a group of cortex-lytic enzymes which degrade spore-specific cortex peptidoglycan. In the spores, at least two cortex hydrolases, spore cortex-lytic enzyme (SCLE) and cortical fragment-lytic enzyme (CFLE), are suggested to cooperatively function for cortex degradation. That is, cortex hydrolysis during germination is initiated by attack of SCLE on intact spore peptidoglycan, which likely leads to un-cross-linking of...