Identification and Characterization of Novel Cell Wall Hydrolase CwlT

Abstract

A cell wall hydrolase homologue, Bacillus subtilis YddH (renamed CwlT), was determined to be a novel cell wall lytic enzyme. The cwlT gene is located in the region of an integrative and conjugative element (ICEBs1), and a cwlT-lacZ fusion experiment revealed the significant expression when mitomycin C was added to the culture. Judging from the Pfam data base, CwlT (cell wall lytic enzyme T (Two-catalytic domains)) has two hydrolase domains that exhibit high amino acid sequence similarity to DL-endopeptidases and relatively low similarity to lytic transglycosylases at the C and N termini, respectively. The purified C-terminal domain of CwlT (CwlT-C-His) could hydrolyze the linkage of D-␥-glutamyl-meso-diaminopimelic acid in B. subtilis peptidoglycan, suggesting that the C-terminal domain acts as a DL-endopeptidase. On the other hand, the purified N-terminal domain (CwlT-N-His) could also hydrolyze the peptidoglycan of B. subtilis. However, on reverse-phase HPLC and mass spectrometry (MS) and MS-MS analyses of the reaction products by CwlT-N-His, this domain was determined to act as an N-acetylmuramidase and not a lytic transglycosylase. Moreover, the site-directed mutagenesis analysis revealed that Glu-87 and Asp-94 are sites related with the cell wall lytic activity. Because the amino acid sequence of the N-terminal domain of CwlT exhibits low similarity compared with those of the soluble lytic transglycosylase and muramidase (goose lysozyme), this domain represents "a new category of cell wall hydrolases."Many microorganisms have peptidoglycan as a major component of the cell wall, which consists of glycan strands crosslinked by peptides. Bacteria have various cell wall lytic enzymes. For Bacillus subtilis, more than 30 candidate peptidoglycan hydrolases are proposed on the basis of amino acid sequence similarity (1), and these enzymes are important in various cellular processes during vegetative growth, sporulation, and germination (1, 2). Several groups of peptidoglycan hydrolases are enzymatically identified as follows: N-acetylglucosaminidases (digesting GlcNAc-MurNAc 3 linkage), N-acetylmuramoyl-Lalanine amidases (digesting MurNAc-L-alanine linkage), DL-endopeptidases (digesting D-glutamic acid-meso-A 2 pm linkage) (1, 2), and LD-endopeptidase (digesting L-alanine-D-glutamic acid linkage) (3). However, the characterization of the group of DD-endopeptidase (digesting the cross-linked D-alanine-meso-A 2 pm linkage) has not been reported in B. subtilis. Interestingly, the groups of muramidase and lytic transglycosylase (digesting MurNAc-GlcNAc linkage) in B. subtilis are still not characterized, even many hydrolases in those groups, including hen egg white lysozyme, have already been identified.Previously Atrih et al. (4) described that the vegetative peptidoglycan in B. subtilis includes (136)-anhydro-N-acetylmuramic acid. Thus, it is possible that lytic transglycosylase, which digests MurNAc-GlcNAc linkage with synthesis of a 1,6-anhydro bond in the N-acetylmuramic acid (5), hydrolyzes the vegetative peptid...

The Bifunctional Cell Wall Hydrolase CwlT Is Needed for Conjugation of the Integrative and Conjugative Element ICEBs1in Bacillus subtilis and B. anthracis

“…Cell wall hydrolases from B. subtilis phage and conjugative elements typically have multiple domains (9,26). CwlT has two domains for peptidoglycan hydrolysis, and each has been characterized biochemically (9).…”

“…2) capable of degrading peptidoglycan (9). Peptidoglycan is the major component of the bacterial cell wall and is composed of long carbohydrate chains of alternating amino sugars, N-acetylglucosamine and N-acetylmuramic acid, cross-linked by short peptide chains (10)(11)(12).…”

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

“…Because the glutamic acid residue is conserved in all muramidases characterized to date, including CwlP and lytic transglycosylases (such as Slt70), it appears likely that it could be involved in the hydrolytic activity of the enzyme. Nevertheless, Asp 94 of CwlT also appears to be associated with hydrolytic activity, either directly or indirectly (19). However, Weaver et al (24) reported that goose egg-white lysozyme lacks a catalytic aspartic acid residue on the basis of their crystallographic analysis of the protein.…”

“…We previously reported that the N-terminal domain of CwlT exhibits muramidase activity, and that the critical amino acid residues comprised Glu 87 and Asp 94 (Fig. 4A) (19). In that report, we hypothesized that Glu 87 was the critical amino acid residue controlling hydrolytic activity, because other muramidases such as hen egg-white lysozyme (20,21), bacteriophage T4 lysozyme (22), and goose egg-white lysozyme (23), have a conserved glutamic acid residue in the corresponding position that controls their catalytic activity.…”

“…The characterized or predicted critical amino acid residues for the hydrolytic activity (Refs. 19,33,34,35) are shown in boxes. Amino acid residues identical to those in the CwlP sequence are shaded.…”