Lytic enzymes in sporulating Bacillus subtilis

Guinand, Micheline; Michel, Georges; Balassa, G

doi:10.1016/0006-291x(76)90336-3

Cited by 34 publications

(16 citation statements)

References 19 publications

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“…Such enzymes have been identified in Bacillus thuringiensis to be an N-acetylmuramyl-L-alanine amidase and an endopeptidase (24). Similar enzymes have also been reported in sporulating cells of Bacillus sphaericus (20) and Bacillus subtilis (19). However, in the work described here we found no association of extractable enzyme activity and sporulation.…”

Section: Discussionsupporting

confidence: 91%

Extraction, purification and characterization of a peptidoglycan hydrolase from vegetative cells of Clostridium perfringens type A.

Labbé¹,

Lu²,

Tang³

1990

J. Gen. Appl. Microbiol.

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A peptidoglycan hydrolase (PGH), which causes the germination and lysis of the coat-stripped spores of Clostridium perfringens, was extracted from perfringens vegetative cells. The enzyme was partially purified and showed an MW of 125,000 and a pI of 7.6. It was sensitive to a variety of monovalent and divalent cations and was inhibited by sulfhydryl-active agents. The sites of PGH action, the cell wall and the spore-cortex, were determined by the release of free amino groups from cell wall fragments and the release of soluble fluorescent products from fluorescamine-labelled cortical fragments of the C. perfringens spores. The enzyme appears distinct from the extracellular initiation protein produced by C. perfringens. PGH may be an autolysin with corticolytic activity.Clostridium perfringens is a common cause of human food poisoning in Western countries. Illness results from the ingestion of large numbers of vegetative cells which sporulate andd produce an enterotoxin in the intestine (30). The organism is commonly found in raw meat and poultry, frequently in the spore state. Temperature abuse of food allows germination and subsequent multiplication of vegetative cells.In previous papers we have described the extraction and isolation of a spore-lytic enzyme (SLE) from whole spores of C. perfringens (15, 16). And we have previously isolated and characterized an extracellular spore-lytic (germination) enzyme from vegetative cells of this organism (12,25,27,34,35). The latter enzyme causes the germination of coatless spores and hydrolysis of cortical fragments of this organism. The enzyme was termed initiation protein (IP) by Cassier and Sebald (6) who first detected it by its ability to promote recovery of heat-injured spores of this organism, presumably by replacing the inactivated, native, spore-germination enzyme.

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Section: Discussionsupporting

confidence: 91%

Extraction, purification and characterization of a peptidoglycan hydrolase from vegetative cells of Clostridium perfringens type A.

Labbé¹,

Lu²,

Tang³

1990

J. Gen. Appl. Microbiol.

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“…Those enzymes were found in particulate preparations from sporulating cells of B. sphaericus [3] and of B. subtilis [4] and, in a soluble state, in the culture media of sporulating B. sphaericus [5]. A strict specificity of the enzymes towards the diamino acid, L-lysine or meso-diaminopimelic acid, present in the peptide was shown in an earlier work [3].…”

Section: X-l-ala-d-glu L L -L~s -D -A~~ and (B) A Y-d-glutamyl-(l)mesmentioning

confidence: 85%

Specificity Profiles of the Membrane‐Bound γ‐d‐Glutamyl‐(l)meso‐diaminopimelate Endopeptidase and ld‐Carboxypeptidase from Bacillus sphaericus 9602

Arminjon

Guinand

Vacheron

et al. 1977

European Journal of Biochemistry

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Membrane-bound peptidases from Bacillus sphaericus 9602 were tested upon various peptides in order to determine the substrate requirements of each enzyme. LD-Carboxypeptidase and y-Dglutamyl-meso-diaminopimelate endopeptidase were never both found to be active on the same substrate. LD-Carboxypeptidase splits the L-Lys-D-Ala linkage of lysine-containing substrates and the msAzprn-~-Ala linkage of meso-diaminopimelic-acid-containing substrates which have an amide group on the o-carboxyl. The endopeptidase hydrolyses the D -G~ linkage of meso-diaminopimelic-acid-containing peptides and derivatives with free o-NH2 and w-COOH groups. The presence or the absence of an a-amide group on glutamic acid, the second residue of peptides, has no influence on the specificities of either enzyme. Likewise, N-substitution of N-terminal L-alanine does not modify the enzymic specificities.

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“…Bacillus subtilis 168 has two major vegetative autolysins, a 50-kDa amidase and a 90-kDa glucosaminidase, which have been purified and characterized (17,31). During sporulation, two distinct lytic enzymes, an amidase and an endopeptidase, have been identified but not studied in detail (14). A 30-kDa amidase has also been identified, and the gene has been cloned and studied at the molecular level (9,22).…”

mentioning

confidence: 99%

Analysis of the autolysins of Bacillus subtilis 168 during vegetative growth and differentiation by using renaturing polyacrylamide gel electrophoresis

1992

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The autolysins of Bacillus subtilis 168 were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with substrate-containing gels. Four bands of vegetative autolytic activity of 90, 50, 34, and 30 kDa (bands Al to A4) were detected in SDS and LiCl extracts and in native cell walls by using B. subtilis 168 vegetative cell walls as the substrate incorporated in the gel. The four enzyme activities showed different substrate specificities and sensitivities to various chemical treatments. The autolysin proffle was not medium dependent and remained constant during vegetative growth. During sporulation, band A4 greatly increased in activity just prior to mother-cell lysis. No germination-associated changes in the profile were observed, although a soluble 41-kDa endospore-associated cortex-lytic enzyme was found. By using insertionally inactivated mutants, bands Al and A2 were positively identified as the previously characterized 90-kDa glucosaminidase and 50-kDa amidase, respectively. The common filamentous phenotype of various regulatory mutants could not be correlated to specific changes in the autolysin profile.Despite abundant speculation, the role of bacterial autolysins during cell growth and division has remained elusive. All bacteria apparently possess a complement of potentially lethal autolysins capable of hydrolyzing the cell wall peptidoglycan (13). Bacillus subtilis 168 has two major vegetative autolysins, a 50-kDa amidase and a 90-kDa glucosaminidase, which have been purified and characterized (17, 31). During sporulation, two distinct lytic enzymes, an amidase and an endopeptidase, have been identified but not studied in detail (14). A 30-kDa amidase has also been identified, and the gene has been cloned and studied at the molecular level (9, 22). The specific function of all of these enzymes is at present unknown, as is the total number of B. subtilis 168 autolytic enzymes. Work mainly using regulatory mutants with reduced autolysin levels (lyt) has implicated them in several important cellular functions, including cell wall turnover, cell separation, flagellation, competence, and the lytic action of penicillin (8,28,30). The regulatory mutants sigD and sin share a common filamentous and Lyt-phenotype, and the lack of cell separation has been proposed to be due to reduced expression of autolysin genes (12,16,25,32). In some lyt mutants, however, the rate of wall turnover was dependent on the salt concentration, and the mutants showed levels of wall turnover comparable to that of the wild type when they were grown in high salt concentrations (38). Because of the possible functional redundancy (9) and compensatory effect of multiple autolysins, it is important to identify and characterize the total set of such enzymes present during growth and development in order to be able to assess their involvement in any given cellular process.The ability of autolysins to renature after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has allowed their activity to be studied ...

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Lytic enzymes in sporulating Bacillus subtilis

Cited by 34 publications

References 19 publications

Extraction, purification and characterization of a peptidoglycan hydrolase from vegetative cells of Clostridium perfringens type A.

Extraction, purification and characterization of a peptidoglycan hydrolase from vegetative cells of Clostridium perfringens type A.

Specificity Profiles of the Membrane‐Bound γ‐d‐Glutamyl‐(l)meso‐diaminopimelate Endopeptidase and ld‐Carboxypeptidase from Bacillus sphaericus 9602

Analysis of the autolysins of Bacillus subtilis 168 during vegetative growth and differentiation by using renaturing polyacrylamide gel electrophoresis

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