A Novel Spore Peptidoglycan Hydrolase of Bacillus cereus : Biochemical Characterization and Nucleotide Sequence of the Corresponding Gene, sleL

Abstract

The exudate of germinated spores of B. cereus IFO 13597 in 0.15 M KCl-50 mM potassium phosphate (pH 7.0) contained a spore-lytic enzyme which has substrate specificity for fragmented spore cortex from wild-type organisms (cortical-fragment-lytic enzyme [CFLE]), in addition to a previously characterized germinationspecific hydrolase which acts on intact spore cortex (spore cortex-lytic enzyme [SCLE]) (R. Moriyama, S. Kudoh, S. Miyata, S. Nonobe, A. Hattori, and S. Makino, J. Bacteriol. 178:5330-5332, 1996). CFLE was not capable of degrading isolated cortical fragments from spores of Bacillus subtilis ADD1, which lacks muramic acid ␦-lactam. This suggests that CFLE cooperates with SCLE in cortex hydrolysis during germination. CFLE was purified in an active form and identified as a 48-kDa protein which functions as an N-acetylglucosaminidase. Immunochemical studies suggested that the mature enzyme is localized on a rather peripheral region of the dormant spore, probably the exterior of the cortex layer. A gene encoding the enzyme, sleL, was cloned in Escherichia coli, and the nucleotide sequence was determined. The gene encodes a protein of 430 amino acids with a deduced molecular weight of 48,136. The N-terminal region contains a repeated motif common to several peptidoglycan binding proteins. Inspection of the data banks showed no similarity of CFLE with N-acetylglucosaminidases found so far, suggesting that CFLE is a novel type of N-acetylglucosaminidase. The B. subtilis genome sequence contains genes, yaaH and ydhD, which encode putative proteins showing similarity to SleL.The bacterial endospore cortex is responsible for the maintenance of spore dormancy and heat resistance. The cortex peptidoglycan has a unique spore-specific structure which allows it to fulfill its role, and cortex hydrolysis during spore germination is essential to allow spore outgrowth and the formation of a new vegetative cell (9, 21). The enzymes involved in these hydrolytic reactions have been identified from spores of several species.A germination-specific cortex-lytic enzyme (GSLE) from Bacillus megaterium spores (8) and a spore cortex-lytic enzyme (SCLE) from Bacillus cereus spores (16, 24), a mature form of SleB, were shown to hydrolyze intact spore peptidoglycan. A counterpart of B. cereus SCLE was also found to exist in Bacillus subtilis spores (23), though attempts to solubilize it were unsuccessful. The B. subtilis SCLE was involved in the response of spores to L-alanine-stimulated germination, and spores lacking this enzyme were unable to complete germination but showed limited cortex degradation (23). This suggests the contribution of a cortex-lytic enzyme(s) in addition to SCLE in the germination of B. cereus and B. subtilis spores. Indeed, analysis of muropeptide dynamics during germination of B. megaterium and B. subtilis spores unequivocally revealed that multiple enzymes are implicated in cortex degradation (2, 4). In Clostridium perfringens S40 spores, a cortical fragment-lytic enzyme (CFLE) which attacks disrupted corte...

Analysis of spore cortex lytic enzymes and related proteins in Bacillus subtilis endospore germination

“…YaaH is 49 % identical to SleL of B. cereus, an enzyme demonstrated to have N-acetylglucosaminidase activity (Chen et al, 2000a). Although Chen et al (2000a) reported a germination defect, spore preparations of a yaaH mutant constructed by transfer of their null mutation into our standard laboratory strain (HR) background showed wild-type germination kinetics in -alanine, and outgrew normally (data not shown). The composition of dormant-spore peptidoglycan, as revealed by RP-HPLC analysis of muropeptides after Cellosyl digestion, was identical to wild-type traces published previously (Atrih et al, 1998).…”

“…A ypeB mutant, defective in the gene downstream of sleB, shows the same germination defect as an sleB mutant (Boland et al, 2000). A cwlJ single mutant is blocked in late stages of germination, failing to phase-darken completely ; Chen et al (2000a) described a cortex-lytic enzyme of B. cereus (SleL) that is found in an active form in the germination exudate. It requires disrupted rather than intact spore peptidoglycan for its activity, and is therefore likely to act on a substrate that has already been cleaved by another lytic enzyme, such as SleB.…”

In vitro and in vivo analyses of the Bacillus anthracis spore cortex lytic protein SleL

“…SleL is an N-acetylglucosaminidase that breaks b-1,4 glycosidic bonds found in cortical PG (Chen et al, 2000;Lambert & Popham, 2008). Previous studies show that sleL is expressed under the control of s E , which is active in the mother cell during early stages of sporulation (Chen et al, 2000;Kodama et al, 1999).…”

“…Germination has been studied in an array of Gram-positive spore-forming species including Clostridium perfringens (Chen et al, 1997;Miyata et al, 1995), Bacillus thuringiensis (Hu et al, 2007), Bacillus subtilis (Ishikawa et al, 1998;Kodama et al, 1999;Moriyama et al, 1996a), Bacillus megaterium (Foster & Johnstone, 1987;Setlow et al, 2009), Bacillus cereus (Chen et al, 2000;Makino et al, 1994;Moriyama et al, 1996b) and B. anthracis (Heffron et al, 2009(Heffron et al, , 2010Lambert & Popham, 2008;Liu et al, 2004). The B. anthracis germination cascade begins when nutrient germinants contact receptors within the spore.…”

“…Shortly after, germination-specific lytic enzymes (GSLEs) are activated (Moir & Smith, 1990;Paidhungat et al, 2002;Popham et al, 1996a;Setlow et al, 2001). The first subset of these enzymes, termed spore cortex lytic enzymes (SCLEs), initiate the degradation of a modified layer of peptidoglycan (PG) known as the cortex, which is involved in the maintenance of spore core dehydration and resistance (Atrih et al, 1996;Chen et al, 2000;Gerhardt & Marquis, 1989;Makino & Moriyama, 2002;Popham et al, 1996a). B. anthracis contains three SCLEs: CwlJ1, CwlJ2 and SleB.…”