DNA sequencing of a region upstream of the mms223 gene of Bacillus subtilis showed the presence of two open reading frames, orf1 and orf2, which may encode 18-and 27-kDa polypeptides, respectively. The predicted amino acid sequence of the latter shows high similarity to a major autolysin of B. subtilis, CwlB, with 35% identity over 191 residues, as well as to other autolysins (CwlC, CwlM, and AmiB). The gene was tentatively named cwlD. Bright spores produced by a B. subtilis mutant with an insertionally inactivated cwlD gene were committed to germination by the addition of L-alanine, and spore darkening, a slow and partial decrease in A 580 , and 72% dipicolinic acid release compared with that of the wild-type strain were observed. However, degradation of the cortex was completely blocked. Spore germination of the cwlD mutant measured by colony formation after heat treatment was less than 3.7 ؋ 10 Bacillus subtilis produces several autolysins (9), including two major autolysins (CwlB [LytC] and CwlG [LytD]) (23,28,31,40). CwlB is a 50-kDa N-acetylmuramoyl-L-alanine amidase (amidase) which cleaves the amide bond between the lactyl group of muramic acid and the ␣-amino group of Lalanine (23,28), and CwlG is a 90-kDa endo-␤-N-acetylglucosaminidase which cleaves the glycosyl bond between glucosamine and muramic acid (31,40).During sporulation and germination, the action of autolysins is assumed to be required for asymmetric septum peptidoglycan hydrolysis, which is a morphogenic transition between sporulation stages II and III, cortex maturation, mother cell lysis, and cortex hydrolysis during germination (5,6,13,42,45). The spore cortex, with a chemical structure slightly distinct from that of vegetative cell wall peptidoglycan, is apparently responsible for the maintenance of spore dormancy (6, 9). At the onset of germination, the cortex is selectively hydrolyzed, leaving a thin layer of vegetative cell peptidoglycan which forms the basis of the new vegetative cell wall (11). Germination-specific cortex-lytic enzymes which are apparently responsible for hydrolysis of the spore cortex during the germination response have been purified from spores of Bacillus megaterium KM (11, 12) and Bacillus cereus (30). It has proved difficult to solubilize autolysins from spores of B. subtilis (5, 9), although several sporulation-specific lytic activities have been identified by means of synthetic substrates (16) or by using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels (9). We recently cloned a sporulation-specific cell wall hydrolase gene (cwlC) from B. subtilis (20). CwlC degraded spore cortex peptidoglycan, but its function is still obscure.We report here that a new gene exhibiting sporulation phase-specific gene expression, cwlD, encodes a putative cell wall hydrolase and that spores from a mutant having an insertionally inactivated cwlD gene are deficient in germination. MATERIALS AND METHODSBacterial strains, phages, and plasmids. The strains of B. subtilis used in this s...

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