A polysaccharide deacetylase homologue, PdaA, was determined to act as an N-acetylmuramic acid deacetylase in vitro. Histidine-tagged truncated PdaA (with the putative signal sequence removed) was overexpressed in Escherichia coli cells and purified. Measurement of deacetylase activity showed that PdaA could deacetylate peptidoglycan treated with N-acetylmuramoyl-L-alanine amidase CwlH but could not deacetylate peptidoglycan treated with or without DL-endopeptidase LytF (CwlE). Reverse-phase high-performance liquid chromatography and mass spectrometry (MS) and MS-MS analyses indicated that PdaA could deacetylate the N-acetylmuramic acid residues of purified glycan strands derived from Bacillus subtilis peptidoglycan.It is thought that the spore cell wall peptidoglycan of Bacillus subtilis plays a role in maintenance of heat resistance and dormancy. The B. subtilis spore cell wall is composed of two layers, the germ cell wall (inner layer) and the cortex (outer layer). In the outer layer (cortex) the muramic acid residues are replaced by single L-alanine residues and muramic ␦-lactam (1, 13). The muramic ␦-lactam structure is important for spore germination in B. subtilis because CwlD (L-alanine amidase homologue)-or PdaA (polysaccharide deacetylase homologue)-deficient spores have no muramic ␦-lactam structure in the cortex and cannot germinate (6,12,15). Previously, we predicted that the first, second, and third steps of muramic ␦-lactam formation are the cleavage of muropeptide side chains by CwlD, de-N-acetylation of muramic acid residues by PdaA, and transpeptidation of muramic acid residues, respectively (6).In this study we found that PdaA of B. subtilis can individually deacetylate the N-acetylmuramic acid residues of purified glycan strands derived from B. subtilis peptidoglycan in vitro. MATERIALS AND METHODSPlasmid construction for h-⌬PdaA overexpression. To construct a truncated PdaA expression plasmid, pUC⌬SfjS (6) was digested with SacI and HindIII, and then a fragment containing the pdaA gene with a deletion of the putative signal sequence was prepared with a GeneCleanII kit (Funakoshi). The fragment was ligated to the corresponding site of pQE-30 (Ap r ; QIAGEN), resulting in pQE⌬SfjS. The pQE⌬SfjS plasmid was used for production of h-⌬PdaA, which is a truncated PdaA protein with a histidine tag at its N terminus.Overexpression and purification of h-⌬PdaA. To overexpress h-⌬PdaA, pQE⌬SfjS was used for the transformation of Escherichia coli JM109 [recA1 ⌬(lac-proAB) endA1 gyrA96 thi-1 hsdR17 relA1 supE44 (FЈ traD36 proAB ϩ lacI q lacZ ⌬M15)]. The resultant transformant, JM109(pQE⌬SfjS), was cultured in Luria-Bertani medium (14) or 2ϫ YT medium (16 g of Bacto Tryptone [Difco] per liter, 10 g of yeast extract per liter, 5 g of NaCl per liter; pH 7.3) at 37°C. When an optical density at 600 nm of 1.0 was reached, isopropyl-␤-D-thiogalactopyranoside (IPTG) (final concentration, 1 mM) was added to the culture. After 1 h of incubation, the cells were harvested by centrifugation. Purification of h-⌬PdaA...

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...

Bacillus subtilis has various cell wall hydrolases, however, the functions and