Characterization of LytH, a Differentiation-Associated Peptidoglycan Hydrolase of Bacillus subtilis Involved in Endospore Cortex Maturation

Abstract

The cortex peptidoglycan from endospores of Bacillus subtilis is responsible for the maintenance of dormancy. LytH (YunA) has been identified as a novel sporulation-specific component with a role in cortex structure determination. The lytH gene was expressed only during sporulation, under the control of the mother cellspecific sigma factor K . Spores of a lytH mutant have slightly reduced heat resistance and altered staining when viewed by electron microscopy. Analysis of the peptidoglycan structure of lytH mutant spores shows the loss of muramic acid residues substituted with L-alanine and a corresponding increase in muramic acid residues substituted with tetrapeptide compared to those in the parent strain. In a lytH cwlD mutant, the lack of muramic acid residues substituted with L-alanine and ␦-lactam leaves 97% of residues substituted with tetrapeptide. These results suggest that lytH encodes an L-Ala-D-Glu peptidase involved in production of single L-alanine side chains from tetrapeptides in the spore cortex. The lack of di-or tripeptides in a lytH mutant reveals the enzyme is an endopeptidase.Bacterial endospores are characterized by their extreme dormancy and high-level resistance to a range of stresses, in particular heat (7). The specialized spore structure determines resistance properties, with the dehydrated spore core being crucial for heat resistance. The spore cell wall peptidoglycan, known as the spore cortex, is essential for the maintenance of the dehydrated core, heat resistance, and dormancy (7). The cortex has a unique, spore-specific structure, and recent analysis has begun to identify features important in its role during differentiation and to reveal components responsible for cortex synthesis, modification during sporulation, and hydrolysis during germination (1, 2). Spore cortex is characterized by an extremely low level of cross-linking, which is important for the maintenance of heat resistance (1, 15). A unique feature of the dormant spore cortex is the presence of muramic acid ␦-lactam residues. This modification has a specific role during germination, because the putative germination-specific lytic enzymes SleB and CwlJ, which are responsible for cortex degradation, require the presence of muramic acid ␦-lactam for substrate recognition, since cortex lacking this modification is unable to be hydrolyzed during germination (2).During cortex maturation, a sporulation-specific amidase, CwlD, and a recently described polysaccharide deacetylase, PdaA, are responsible for the formation of the characteristic ␦-lactam residues of bacterial endospores (1,8,15). Although cwlD or pdaA mutants and a cwlD pdaA double mutant can produce resistant endospores, no cortex hydrolysis is observed during germination, and the spores do not outgrow (1,2,8,16,18). The final characteristic feature of the cortex is that about 25% of the muramic acid side chains are substituted for with single L-alanine residues (1, 17). These may be formed by the action of an L-Ala-D-Glu endopeptidase on nascent stem peptides. ...

Characterization of new l,d-endopeptidase gene product CwlK (previous YcdD) that hydrolyzes peptidoglycan in Bacillus subtilis

“…We also tried to align CwlK and B. subtilis LytH, which was identified as an L-alanoyl-D-glutamate peptidase (Horsburgh et al 2003a). However, the similarity was very low.…”

“…However, the characterization of several hydrolases i.e., muramidase and lytic transglycosylase LytH is an L,D-endopeptidase (Horsburgh et al 2003a). However, there is a possibility that LytH is a carboxypeptidase cleaving the D-alanine-meso-diaminopimeric acid-D-alanine linkage in the spore cortex (Horsburgh et al 2003a). …”

“…Moreover, PgdS (YwtD) is a polyglutamic acid-degrading enzyme produced during the vegetative growth phase and it belongs to the "D,L-endopeptidase" family (Suzuki and Tahara 2003). However, the characterization of several hydrolases i.e., muramidase and lytic transglycosylase LytH is an L,D-endopeptidase (Horsburgh et al 2003a). However, there is a possibility that LytH is a carboxypeptidase cleaving the D-alanine-meso-diaminopimeric acid-D-alanine linkage in the spore cortex (Horsburgh et al 2003a).…”

Bacillus subtilis CwlP of the SP-β Prophage Has Two Novel Peptidoglycan Hydrolase Domains, Muramidase and Cross-linkage Digesting dd-Endopeptidase

“…It is also known that LytM in Staphylococcus aureus has a peptidase M23 domain, which has been identified as a glycylglycine endopeptidase (4). However, B. subtilis LytH also has a peptidase M23 domain and has been shown to digest the L-Ala-D-Glu bond of spore peptidoglycans in vivo (5). At present, the activity of the peptidase M23 domain of CwlP is unknown.…”

Identification and characterization of a novel spore-associated subtilase from Thermoactinomyces sp. CDF

“…Ultrathin sections of the spores were prepared according to the method of Horsburgh et al (2003). Specimens were viewed at a range of magnifications on an H-7000FA (HITACHI) transmission electron microscope operated at 75 kV.…”