Nucleotide sequence and regulation of a new putative cell wall hydrolase gene, cwlD, which affects germination in Bacillus subtilis.

Abstract

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...

Development of natto with germination-defective mutants of Bacillus subtilis (natto)

“…The mutation of sleB was more effective in NAFM5 than in 168. Sekiguchi et al (1995) reported that the germination rate of the cwlD mutant in B. subtilis was less than 3.7 x 10 -8 , but our results were 1.9x 10 -4 and 1.7 x 10 -3 for 168 and NAFM5, respectively. Popham et al (1996) showed that the germination rate of the cwlD mutant in B. subtilis was 5 x 10 -4 (with the respect to the wild type).…”

“…These results suggest that an early germination event (i.e., dipicolinic acid release) was not affected by the double mutations in either strain. These data were supported by the results for each single mutation in B. subtilis (Sekiguchi et al 1995;Moriyama et al 1996). Phase-contrast microscopy of the wild type and mutants is shown in Fig.…”

“…SleB is a cortex-lytic enzyme (Moriyama et al 1996) and is assumed to digest the cortex containing muramic delta-lactam, a unique peptidoglycan component (Warth and Strominger 1969). The delta-lactam structure is produced by CwlD and PdaA (Sekiguchi et al 1995;Fukushima et al 2002;Gilmore et al 2004;Fukushima et al 2005). Since the genome organizations of B. subtilis 168 and B. subtilis (natto) are very similar (Qiu et al 2004), we attempted amplification of the upstream and downstream regions of sleB, cwlD, and pdaA of B. subtilis (natto) with B. subtilis (natto) DNA as a template and primers based on the sequences of B. subtilis 168.…”

“…These mutants are also inadequate, because the mutants are not clear germination-defective mutants and may affect the quality of natto. The fourth group is related to cortex-lysis or modification, and it contains cwlD, sleB, cwlJ, and pdaA (Sekiguchi et al 1995;Moriyama et al 1996;Ishikawa et al 1998). CwlD, SleB, and CwlJ were considered to be cortex-lytic enzymes.…”

“…Muramic-delta-lactam is produced by PdaA (polysaccharide deacetylase) and CwlD (N-acetylmuramoyl-L-alanine amidase homologue) (Sekiguchi et al 1995;Fukushima et al 2002;Gilmore et al 2004;Fukushima et al 2005). The mutations in the fourth group affect neither vegetative growth nor sporulation, but severely affect germination.…”