Regulation and Characterization of a Newly Deduced Cell Wall Hydrolase Gene (cwlJ) Which Affects Germination of Bacillus subtilis Spores

The predicted amino acid sequence of Bacillus subtilis ycbQ (renamed cwlJ) exhibits high similarity to those of the deduced C-terminal catalytic domain of SleBs, the specific cortex-hydrolyzing enzyme of B. cereus and the deduced one of B. subtilis. We constructed a cwlJ::lacZ fusion in the B. subtilis chromosome. The β-galactosidase activity and results of Northern hybridization and primer extension analyses of the cwlJ gene indicated that it is transcribed by Eς^E RNA polymerase. cwlJ-deficient spores responded to both l-alanine and AGFK, the A₅₈₀ values of spore suspensions decreased more slowly than in the case of the wild-type strain, and the mutant spores released less dipicolinic acid than did those of the wild-type strain during germination. However, the mutant spores released only slightly less hexosamine than did the wild-type spores. In contrast, B. subtilis sleB spores did not release hexosamine at a significant level. While cwlJ and sleB spores were able to germinate, CJSB (cwlJ sleB) spores could not germinate but exhibited initial germination reactions, e.g., partial decrease in A₅₈₀ and slow release of dipicolinic acid. CJSB spores became slightly gray after 6 h in the germinant, but their refractility was much greater than that of sleB mutant spores. The roles of the sleB and cwlJ mutations in germination and spore maturation are also discussed.During sporulation and germination of Bacillus subtilis, the action of autolysins is assumed to be required for asymmetric septum peptidoglycan hydrolysis, engulfment, cortex maturation, mother cell lysis, and cortex hydrolysis during germination (28, 33). Mother cell lysis depends on the compensatory effect of cell wall hydrolases CwlB (LytC) and CwlC (11, 13, 34). For cortex maturation, a defect in the cwlD gene leads to a lack of germination and blocking of the formation of muramic acid lactam structure in the cortex (2, 26, 31). Recently, Makino and colleagues reported that the B. cereus sleB gene encodes a 24-kDa mature germination-specific N-acetylmuramoyl-l-alanine amidase which degrades decoated spores from various organisms (18, 22). B. subtilis sleB is homologous to B. cereus sleB, and B. subtilis sleB mutant spores are able to germinate and form colonies. However, B. subtilis SleB showed no activity against degraded decoated spores or other substrates (21).Our work on the B. subtilis genome sequencing project has revealed the ycbQ gene, which is homologous with the cortex-hydrolyzing sleB genes (22, 25). In this study, we describe the regulation and function of the cwlJ (ycbQ) gene and the compensatory effect of the CwlJ and B. subtilis SleB proteins on germination.