Analysis of Bacillus subtilis 168 prophage-associated lytic enzymes; identification and characterization of CWLA-related prophage proteins

Foster, Simon J.

doi:10.1099/00221287-139-12-3177

Cited by 22 publications

(19 citation statements)

References 26 publications

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“…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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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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“…Like the majority of low-molecular-mass (Յ50 kDa) peptidoglycan hydrolases associated with gram-positive bacteria for which data are available (7,9,14,19,23,26,33,36), CwlC is an N-acetylmuramoyl-L-alanine amidase. In particular, its substrate specificity is consistent with its amino acid sequence similarity to the CwlM amidase of B. licheniformis and LytC of B. subtilis (18,23).…”

Section: Discussionmentioning

confidence: 99%

“…The mode of action of purified CwlC was determined as previously described (7,9). Purified 30-kDa lytic enzyme was dialyzed against 100 volumes of 1 mM Tris-HCl buffer (pH 7.5).…”

Section: Methodsmentioning

confidence: 99%

Characterization of the involvement of two compensatory autolysins in mother cell lysis during sporulation of Bacillus subtilis 168

Smith

Foster

1995

J Bacteriol

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The 30-kDa sporulation-specific peptidoglycan hydrolase CwlC of Bacillus subtilis 168 was purified and characterized. It is an N-acetylmuramoyl-L-alanine amidase (amidase) that is associated with the mother cell wall of sporulating cells, and although it is secreted, it undergoes no N-terminal processing except removal of the initial methionine. It was found that mother cells of a strain insertionally inactivated in cwlC and lytC (the major vegetative amidase gene) did not lyse at the end of sporulation. Mutants with single mutations in cwlC or lytC lysed, and so the two autolysins must have mutually compensatory roles in mother cell lysis. Active CwlC and LytC are present at the time of mother cell lysis; however, reporter gene analysis revealed that lytC transcription ceases early in sporulation, and therefore the function that LytC has in mother cell lysis is performed by material remaining from presporulation expression. Autolytic enzymes similar in molecular mass to CwlC were detected in two other Bacillus species by their cross-reactivity with anti-CwlC antiserum.Autolysins, enzymes that hydrolyze bacterial cell wall peptidoglycan, are ubiquitous among bacteria (11), although their precise roles remain uncertain. In the spore-forming grampositive bacterium Bacillus subtilis, there may be 10 or more peptidoglycan hydrolases (8, 10). Such enzymes have been implicated in several functions during vegetative growth and sporulation (34,41).The differentiation process of sporulation involves a number of events of cell wall rearrangement, in which peptidoglycandegradative enzymes evidently play a fulcral role (10). These include digestion of the asymmetric septum to permit prespore engulfment (5), cortex maturation (1, 42), mother cell lysis, and cortex autolysis during germination (5). Mother cell lysis to release the mature endospore is the final characteristic morphological event that occurs during sporulation. A 30-kDa mother cell-associated autolysin appears during sporulation and has been postulated to be responsible for mother cell lysis (8). A gene, cwlC, which appears to encode this protein has been cloned and sequenced; the deduced gene product is a 255-amino-acid, 27-kDa peptide. Its expression is controlled by K (18), the mother cell-specific, late-sporulation sigma factor (6, 27). However, its role in sporulation was unknown, as the mother cells of a cwlC mutant still lyse (18).LytC is the major vegetative cell amidase of B. subtilis 168 and is also present during sporulation (8). The lytC gene is part of a three-gene operon, lytABC (25). lytR, which is transcribed divergently from lytABC, encodes a putative DNA-binding protein, which represses expression of itself and lytABC (25). lytA encodes an acidic protein that is thought to be membrane associated (20,25). lytB encodes a cell wall-associated modifier protein, which binds to the LytC amidase and enhances its activity (14). A study with a plasmid clone carrying a lytA::lacZ translational fusion suggested that, during vegetative growth, expression ...

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“…In addition, Foster (1993) reported that c w l A was never expressed under the conditions tested, whereas this gene seemed to be expressed from its own promoter in E. coli and so is in itself functional. Importantly, PBSX, a B. snbtilis defective prophage, encodes two lytic enzymes which cross-react with CwlA antisera (Foster, 1993). Presumably, CwlA is a phageencoded lytic enzyme, suggesting that the skin element is derived from an ancestral temperate phage.…”

Section: K K M ( L S E K F T P H K E V I I R M K~k~a K S T H Imentioning

confidence: 99%

Complete nucleotide sequence of a skin element excised by DNA rearrangement during sporulation in Bacillus subtilis

Takemaru

Mizuno²,

Sato³

et al. 1995

Microbiology

102

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~~As part of the Bacillus subtilis genome sequencing project, we have determined the complete nucleotide sequence of a skin element which is located between spolVCB and spol//C. The entire sequence of this element is 48032 bp in length, and contains 57 ORFs with putative ribosome-binding sites. Two of them correspond to previously sequenced and characterized genes, cwlA and spolVcA. Furthermore, seven ORF products identified in this element show interesting similarities with known proteins present in data banks, including the 4105 immunity repressor, the 4105 Cro-like protein and the SPPl terminase. These results indicate the possibility that the skin element is a cryptic remnant of an ancestral temperate phage.

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Analysis of Bacillus subtilis 168 prophage-associated lytic enzymes; identification and characterization of CWLA-related prophage proteins

Cited by 22 publications

References 26 publications

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

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

Characterization of the involvement of two compensatory autolysins in mother cell lysis during sporulation of Bacillus subtilis 168

Complete nucleotide sequence of a skin element excised by DNA rearrangement during sporulation in Bacillus subtilis

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