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

Abstract

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...

Germination of spores of Clostridium difficile strains, including isolates from a hospital outbreak of Clostridium difficile-associated disease (CDAD)

“…Similar, albeit not identical, CLEs have been identified in other endospore-forming species (Foster & Johnstone, 1987;Ishikawa et al, 1998;Makino et al, 1994;Miyata et al, 1995Miyata et al, , 1997Shimamoto et al, 2001), and bioinformatic analyses reveal that similar CLEs are also present in C. difficile (Sebaihia et al, 2006). When spores of strains JIR8094 and Pitt301 without prior heat activation were incubated with Ca-DPA and germination was measured, there were no significant changes in OD 600 or spore refractility, and no release of DPA (data not shown).…”

“…One possible explanation is that K + ions and P i directly activate a CLE and/or Ca-DPA release. Ongoing work is thus oriented towards investigating the roles played by a number of proteins, including CLEs that may be involved in cortex PG hydrolysis (Foster & Johnstone, 1987;Ishikawa et al, 1998;Makino et al, 1994;Miyata et al, 1995Miyata et al, , 1997Shimamoto et al, 2001) and SpoVA proteins implicated in Ca-DPA release (Paredes-Sabja et al, 2008b), in the germination of C. difficile spores with K + and P i . The resolution of the paradox noted above as well as a more thorough understanding of the germination of C. difficile spores may well have applications in the areas of public health and food safety.…”

Characterization of Clostridium perfringens Spores That Lack SpoVA Proteins and Dipicolinic Acid

“…This suggests that CLEs in C. perfringens spores are located only in or adjacent to the outer edge of the cortex. The CLEs SleC and SleM, one of which (SleC) is activated by proteolysis early in germination, have been identified in and purified from C. perfringens spores, and their enzymatic activity has been characterized (7,23,31,39,65,69). However, the roles of these proteins in vivo during spore germination have not been established, and they show little homology with the CLEs CwlJ and SleB that catalyze cortex hydrolysis during B. subtilis spore germination.…”

Germination of Spores of Bacillus Species: What We Know and Do Not Know

“…Like its Bacillus spore counterparts, SleC has lytic transglycosylase activity and is also in the spores' outer layers (4,70,72,73). However, SleC is present in spores as an inactive zymogen, proSleC, which is activated by protease cleavage in the first minutes of germination (4,(74)(75)(76)(77). One or multiple subtilisin-like serine proteases, termed Csp proteases, cleave and activate proSleC in the germination of spores of clostridia (74)(75)(76)(77).…”