Partial characterization of an enzyme fraction with protease activity which converts the spore peptidoglycan hydrolase (SleC) precursor to an active enzyme during germination of Clostridium perfringens S40 spores and analysis of a gene cluster involved in the activity

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 con sisting of four domains, After cleavage of an N-terminal presequence and a C-terminal prosequence during spore maturation, inactive proenzyme is conve rted to active enzyme by processing of an N-terminal prosequence with germi nation-specific protease (GSP) during germination. The present study was un dertaken to characterize GSP, In the presence of 3-[(3-chotamidopropyl)dime thylammonio] -1-propanesulfonic acid (CHAPS), a nondenaturing detergent whi ch 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 ma sses 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 treat ment at 55 degreesC for 40 min. GSP specifically cleaved the peptide bond b etween Val-149 and Val-150 of SleC to generate mature enzyme. Inactivation of GSP by phenylmethylsutfonyl fluoride and HgCl2 indicated that the protea se is a cysteine-dependent serine protease, Several pieces of evidence demo nstrated that three protein components of the enzyme fraction are processed forms of products of cspA, cspB, and cspC, which are positioned in a tande m array just upstream of the 5 ' end of sleC, The amino acid sequences dedu ced from the nucleotide sequences of the csp genes showed significant simil arity and showed a high degree of homology with those of the catalytic doma in and the oxyanion binding region of subtilisin-like serine proteases, Imm unochemical studies suggested that active GSP likely is localized with majo r cortex-lytic enzymes on the exterior of the cortex layer in the dormant s pore, a location relevant to the pursuit of a cascade of cortex hydrolytic reactions.