Molecular cloning, sequence analysis, and characterization of a new cell wall hydrolase, CwIL, of Bacillus licheniformis

Oda, Yasushi; Nakayama, Ryuji; Kuroda, Akio; Sekiguchi, Junichi

doi:10.1007/bf00284691

Cited by 21 publications

(18 citation statements)

References 23 publications

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“…Western blot analysis with an antibody against the N-terminal region of AtlA confirmed that the deletion oc- curred in this region. The degradation of cell wall hydrolases without a loss of enzymatic activity has been observed previously for Bacillus licheniformis (23), Bacillus subtilis (13,26), and Lactococcus lactis (3). These results suggest that the atlA gene cloned for this study encodes the major autolysin of S. mutans.…”

Section: Discussionsupporting

confidence: 65%

Identification and Characterization of an Autolysin-Encoding Gene of Streptococcus mutans

et al. 2005

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We identified a gene (atlA) encoding autolytic activity from Streptococcus mutans Xc. The AtlA protein predicted to be encoded by atlA is composed of 979 amino acids with a molecular weight of 107,279 and has a conserved ␤-1,4-N-acetylmuramidase (lysozyme) domain in the C-terminal portion. Sodium dodecyl sulfate extracts of strain Xc showed two major bacteriolytic bands with molecular masses of 107 and 79 kDa, both of which were absent from a mutant with inactivated atlA. Western blot analysis revealed that the 79-kDa band was derived from the 107-kDa peptide by cleavage of its N-terminal portion. The inactivation of atlA resulted in a marked decrease in autolysis and the formation of very long chains of cells compared to the case for the parent strain. Although both the parent and mutant strains formed biofilms in the presence of sucrose, the biofilms formed by the mutant had a sponge-like architecture with large gaps and contained 30% less biomass than those formed by the parent strain. Furthermore, strain Xc formed glucose-dependent, loose biofilms in the absence of sucrose, but the mutant lost this ability. These results suggest that AtlA may play an important role in biofilm formation by S. mutans. The antibody produced against the C-terminal peptide containing the ␤-1,4-N-acetylmuramidase domain drastically inhibited the autolytic activity of strain Xc. This inhibition was specific among the oral streptococci to S. mutans. These results indicate that the catalytic domain of AtlA is located at the C terminus, suggesting that further characterization of this domain may provide a means to control cariogenic dental plaque formation.

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Section: Discussionsupporting

confidence: 65%

Identification and Characterization of an Autolysin-Encoding Gene of Streptococcus mutans

et al. 2005

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“…The motif is composed of three -helices (Ghuysen et al, 1994) and classified in the putative peptidoglycan binding domain-1 [Pfam database]. This motif is conserved among many peptidoglycan hydrolases (e.g., SleC from C. perfringens (Miyata et al, 1995a), CwlA from B. subtilis (Kuroda and Sekiguchi, 1990), CwlL from B. licheniformis (Oda et al, 1993), DDcarboxypeptidase from Streptmyces albus (Joris et al, 1983), and autolytic lysozyme from C. acetobutylicum (Croux and Garcia, 1991)). Interestingly, when the importance of the motif in SleB localization was examined, we could discriminate the localization patterns of two fusion proteins in the gerE mutant ( Fig.…”

Section: Discussionmentioning

confidence: 99%

Subcellular Localization of a Germiantion-specific Cortex-lytic Enzyme, SleB, of Bacilli during Sporulation

et al. 2006

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The subcellular localization of a germination-specific cortex-lytic enzyme, SleB, of Bacillus subtilis during sporulation was observed by using fusions of N-terminal region of SleB to the green fluorescent protein (GFP). A fusion with a putative peptidoglycan-binding motif (SleB 1-108 -GFP) formed a fluorescent ring around the forespore of the wild type strain, as expected from the known location of the intact SleB in the dormant spore. SleB 1-108 -GFP formed a similar fluorescent ring around the forespore of the gerE mutant which has a severe defect in the coat structure, and of the cwlD mutant which lacks a muramic δ -lactam unique to the spore peptidoglycan (cortex), whereas the fusion could not attach to the spore of the cwlDgerE mutant. By contrast, a fusion without the motif (SleB 1-45 -GFP) could not be recruited around the forespore of the gerE mutant though it appeared to be accumulated on the outside of the spore of the wild type strain. Since SleB was shown to degrade only the cortex with muramic δ -lactam, these results suggested that a proper localization of SleB requires a strict interaction between the motif of the enzyme and the δ -lactam structure of the cortex, not the formation of normal coat layer.

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“…Cell wall-lytic amidases in the genus Bacillus are classified into two groups; class I contains CwlA (8,22), CwlL (36), and a lytic enzyme from PBSX (XlyA) (29), and class II contains CwlB (LytC) (23,28) and CwlM (27). Furthermore, amino acid sequence homology among other autolytic enzymes suggests that a lytic enzyme of a Bacillus species (39) and a sporulation phase-specific lytic enzyme, CwlC (20), belong to class I and class II, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…In E. coli and Salmonella typhimurium, the gene product (amidase) of amiB upstream of the DNA repair gene (mutL) (50) and the deduced protein of orf32 upstream of hemF (an oxygen-dependent coproporphyrinogen oxidase gene) (48,49,52) belong to the class II group. Recent reports suggest that the class I family may be lytic enzymes derived originally from phages, because the genes neighboring the lytic enzyme gene are functionally similar to phage genes (8,10,29,36). However, in spite of extensive research on autolysins, their role in cell differentiation is almost unknown.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 1995

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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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Molecular cloning, sequence analysis, and characterization of a new cell wall hydrolase, CwIL, of Bacillus licheniformis

Cited by 21 publications

References 23 publications

Identification and Characterization of an Autolysin-Encoding Gene of Streptococcus mutans

Identification and Characterization of an Autolysin-Encoding Gene of Streptococcus mutans

Subcellular Localization of a Germiantion-specific Cortex-lytic Enzyme, SleB, of Bacilli during Sporulation

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

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