muramidase and Autolysis

Autolysis of Enterococcus hirae ATCC 9790 is the result of the action of endogenous enzymes that hydrolyze bonds in the protective and shape-maintaining cell wall peptidoglycan. It is thought that these potentially suicidal enzymes play a positive role(s) in wall growth and division and are expressed as autolysins when cell wall assembly and/or repair are inhibited. E. hirae possesses two potentially autolytic enzymes, both of which are muramidases. Although they hydrolyze the same bond as hen egg-white lysozyme, both are high-molecular-mass, complex enzymes. Muramidase-1 is synthesized as a zymogen, requiring protease activation. It is a glucoenzyme that is also multiply nucleotidylated with an unusual nucleotide, 5-mercaptouridine monophosphate. Muramidase-2 is almost certainly a product of a separate gene. The deduced amino acid sequence of a cloned gene for extracellular muramidase-2 showed several unusual features. It appears to be a two-, or perhaps three-domain protein with a putative glycosidase-active site near the N-terminal end and six 45-amino-acid-long repeats at the C-terminal end which are presumed to be involved with high-affinity binding to the insoluble peptidoglycan substrate. Muramidase-2 binds penicillin with low affinity. The presence of several amino acid groupings characteristic of serine-active site beta-lactam-interactive proteins is consistent with the possible presence of a penicillin-binding, third domain. Indirect evidence consistent with a role(s) for these enzymes in cell wall growth and division has been obtained. However, proof of such role(s) awaits modern genetic, molecular, and biochemical analyses.

Topics: Amino Acid Sequence; Autolysis; Binding Sites; Enterococcus; Genes, Bacterial; Molecular Sequence Data; Muramidase; N-Acetylmuramoyl-L-alanine Amidase

Topics: Animals; Autolysis; Bacterial Infections; Cephalosporins; Clostridium; Crystallography; Drug Stability; Escherichia coli; Guinea Pigs; History, 20th Century; Humans; Mice; Microbial Sensitivity Tests; Micrococcus; Muramidase; Penicillanic Acid; Penicillins; Penicillium; Rabbits; Salmonella; Streptococcal Infections

Peptidoglycan (PG) and wall teichoic acid (WTA) are the major staphylococcal cell wall components, and WTA biosynthesis has recently been explored for drug development. Targocil is a novel agent that targets the TarG subunit of the WTA translocase (TarGH) that transports WTA across the membrane to the wall. Previously we showed that targocil treatment of a methicillin-susceptible

Topics: Autolysis; Bacterial Translocation; Lysostaphin; Muramidase; N-Acetylmuramoyl-L-alanine Amidase; Protein Transport; Quinazolines; Staphylococcus aureus; Teichoic Acids; Triazoles

When Lactococcus lactis subsp. lactis IL1403 or L. lactis subsp. cremoris MG1363 is grown in a medium with galactose as the carbon source, the culture lyses to a lesser extent in stationary phase than when the bacteria are grown in a medium containing glucose. Expression of AcmA, the major autolysin of L. lactis, is not influenced by the carbon source. Binding studies with a fusion protein consisting of the MSA2 protein of Plasmodium falciparum and the C-terminal peptidoglycan-binding domain of AcmA revealed that cell walls of cells from both subspecies grown on galactose bind less AcmA than cell walls of cells grown on glucose. Cells grown on glucose or galactose and treated with trichloroacetic acid prior to AcmA binding bind similar amounts of AcmA. Analysis of the composition of the lipoteichoic acids (LTAs) of L. lactis IL1403 cells grown on glucose or galactose showed that the LTA composition is influenced by the carbon source: cells grown on galactose contain LTA with less galactose than cells grown on glucose. In conclusion, growth of L. lactis on galactose changes the LTA composition in the cell wall in such a way that less AcmA is able to bind to the peptidoglycan, resulting in a decrease in autolysis.

Topics: Autolysis; Bacteriolysis; Base Sequence; Cell Wall; DNA, Bacterial; Galactose; Kinetics; Lactococcus lactis; Molecular Sequence Data; Muramidase; Peptidoglycan; Plasmids; Protein Binding; Restriction Mapping

The insertional inactivation of the dlt operon from Lactobacillus plantarum NCIMB8826 had a strong impact on lipoteichoic acid (LTA) composition, resulting in a major reduction in D-alanyl ester content. Unexpectedly, mutant LTA showed high levels of glucosylation and were threefold longer than wild-type LTA. The dlt mutation resulted in a reduced growth rate and increased cell lysis during the exponential and stationary growth phases. Microscopy analysis revealed increased cell length, damaged dividing cells, and perforations of the envelope in the septal region. The observed defects in the separation process, cell envelope perforation, and autolysis of the dlt mutant could be partially attributed to the L. plantarum Acm2 peptidoglycan hydrolase.

Topics: Alanine; Autolysis; Base Sequence; Cell Wall; DNA Primers; Esters; Kinetics; Lactobacillus plantarum; Lipopolysaccharides; Microscopy, Electron, Scanning; Muramidase; N-Acetylmuramoyl-L-alanine Amidase; Operon; Polymerase Chain Reaction; Restriction Mapping; Teichoic Acids

Comparison of several cell wall-related properties of the ATCC 9790 strain and the R40 strain, a penicillin-resistant, PBP5 overproducing strain, and Rev14, a penicillin-hypersensitive, PBP5-deficient strain, is consistent with a role of the genetic element, psr, in the global regulation of lysozyme sensitivity, autolytic capacity, and wall-rhamnose-containing polysaccharide content. These parameters appear to be independently regulated by a system that involves psr in a currently unknown manner.

Topics: Autolysis; Bacterial Proteins; Carrier Proteins; Cell Wall; Enterococcus; Hexosyltransferases; Isoenzymes; Muramidase; Muramoylpentapeptide Carboxypeptidase; Penicillin Resistance; Penicillin-Binding Proteins; Penicillins; Peptidyl Transferases; Protoplasts

Cyclodextrin glycosyltransferase (CGTase) was released into the culture fluid by Bacillus macerans predominantly in the late stationary phase of growth and during autolysis in the presence of either glucose or starch as a carbon source. In both cases significant soluble intracellular enzyme activity could be observed in the early stationary phase, and a low non-soluble intracellular CGTase activity could be demonstrated also in the exponential growth phase in the presence of starch. At the end of the exponential phase the non-soluble specific intracellular enzyme activity was found to be constant with a value of 0.63 +/- 0.06 nkat/10(9) viable cells. Since amylase activity could not be detected in any intracellular or extracellular sample taken at any culture time, we conclude that cellbound CGTase is the only starch-digesting enzyme in growing B. macerans and, hence, may be fully responsible for the degradation of starch in the culture fluid.

Topics: Amylases; Autolysis; Bacillus; Cells, Cultured; Culture Media; Endopeptidases; Glucose; Glucosyltransferases; Muramidase; Starch; Time Factors

The genes hbl3, cpl1 and cpl7 coding for the pneumococcal phage lytic enzymes HBL3, CPL1 and CPL7, respectively, have been cloned into shuttle plasmids that can replicate in Streptococcus pneumoniae and Escherichia coli. All these genes were expressed in E. coli under the control of either the lytP promoter of the lytA gene, which codes for the major pneumococcal autolysin, or the promoter of the tetracycline-resistance gene (tetP). In contrast, cpl1 and cpl7 genes that code for lysozymes were expressed in pneumococcus only under the control of tetP, whereas the hbl3 gene that codes for an amidase can be expressed using either promoter. The phage lysozymes or amidase expressed in S. pneumoniae M31, a mutant deleted in the lytA gene coding for short chains, were placed under physiological control since these transformed bacteria grew as normal 'diplo' cells during the exponential phase and underwent autolysis only after long incubation at 37 degrees C. The lysis genes appear to be expressed constitutively in the transformed pneumococci, since sharply defined lysis of these cultures could be induced prematurely during the exponential phase of growth by addition of sodium deoxycholate.

Topics: Amidohydrolases; Autolysis; Cloning, Molecular; Escherichia coli; Gene Expression; Genes, Viral; Genetic Vectors; Muramidase; Plasmids; Streptococcus Phages; Streptococcus pneumoniae

The optimum conditions for autolysis of Clostridium acetobutylicum ATCC 824 were determined. Autolysis was optimal at pH 6.3 and 55 degrees C in 0.1 M-sodium acetate/phosphate buffer. The ability of cells to autolyse decreased sharply at the end of the exponential phase of growth. Lysis was stimulated by monovalent cations and compounds that complex divalent cations, and inhibited by divalent cations. The autolysin of C. acetobutylicum, which was mainly cytoplasmic, was purified to homogeneity and characterized as a muramidase. The enzyme was identical to the extracellular muramidase in terms of M(r), isoelectric point and NH2-terminal amino acid sequence. The autolysin was inhibited by lipoteichoic acids and cardiolipin but not by phosphatidylethanolamine and phosphatidylglycerol. A mechanism of regulation and fixation involving lipoteichoic acid, cardiolipin and divalent cations is proposed.

Topics: Amino Acid Sequence; Amino Acids; Amino Alcohols; Amino Sugars; Autolysis; Cell Division; Cell Wall; Chelating Agents; Clostridium; Hot Temperature; Hydrogen-Ion Concentration; Ions; Lipopolysaccharides; Molecular Sequence Data; Mucoproteins; Muramidase; Phospholipids; Sequence Homology, Nucleic Acid; Subcellular Fractions; Teichoic Acids

The surfactants tested in this study lysed Bacillus subtilis 168 cells at the logarithmic growth phase. Results obtained with inhibitors and a mutant that had defective autolytic enzymes suggested that cell lysis resulted from the deregulation of autolysin activity. The addition of surfactants at sublytic concentrations produced twisted cells, filamented cells, or both. Autolysins extracted with 5 M LiCl from the cell wall fraction and lysozyme added to cells that were treated with surfactants restored the apparently normal cell rod morphology, suggesting that surfactants interfere with the role of autolysins in normal construction of the cell envelope. The rates of cellular autolysis and autolysin activity remaining in growing cells after exposure to a surfactant at a sublytic concentration decreased, although the rate of turnover of cell wall peptidoglycan was the same as that of control cells. Surfactants were suggested to interact with the regulatory system of autolysins and, thus, to affect the activities of autolysins in B. subtilis cells and to cause either morphological changes or cell autolysis, depending on the concentration of surfactants.

Topics: Autolysis; Bacillus subtilis; Cell Wall; Muramidase; Peptidoglycan; Potassium; Sodium; Surface-Active Agents

The log phase cells of autolytic Microccus lysodeikticus (luteus) IFO 3333 did not autolyze when grown in the presence of trypsin although the growth curve and morphology of the cells were not influenced. A non-autolytic mutant was obtained by subculture of the wild-type strain IFO 3333 on an agar slant containing 1% glucose. The mutant (strain MT) was wild-type IFO 3333 which occurred singly or in irregular masses. The mutant MT grown in a culture medium containing trypsin caused remarkable alteration in cell morphology: large cell packets consisting of a number of "unit tetrads" arranged regularly in three dimensions were formed by the addition of trypsin to the medium. The findings suggest that inhibition of the separation of divided cells is brought about by inactivation or suppression of a cell wall autolytic enzyme which plays an important role in the separation step and is accessible to externally added trypsin in the mutant cells but not in the wild-type cells. The possibility that there are two kinds or phases of autolytic enzymes "a physiological autolytic enzyme" and "a useless autolytic enzyme", is discussed.

Topics: Agglutination; Autolysis; Cell Count; Cell Wall; Culture Media; Immune Sera; Micrococcus; Muramidase; Mutation; Peptide Hydrolases; Trypsin

A system for the formation of apparently wall-free protoplasts from exponential-phase cells of Streptococcus faecalis ATCC 9790 in the absence of added lytic enzymes was developed. Exponential-phase cells suspended in 0.04 M ammonium acetate, pH 6.7, 1 mM magnesium acetate, and 0.5 M sucrose become osmotically fragile within 1 to 1.5 h due to the action of the native, autolytic enzyme on the cell wall peptidoglycan. However, maximal cell wall loss occurred much more slowly, being complete only after 3 to 6 h. Under these conditions, the autolytically formed protoplasts (autoplasts) remained intact for prolonged periods (up to 24 h) with less than 5% of their deoxyribonucleic acid, ribonucleic acid, and protein lost during the first 6 h. During dissolution of the cell wall, release of autolytic enzyme to the supernatant fluid began after 60% of the wall was lost. The addition of trypsin to the incubation mixture increased the rate of attainment of osmotic fragility and cell wall loss two- to threefold, apparently due to the activation of the latent form of the autolysin. Electron microscopy was used to confirm cell wall loss and the presence of intact protoplasts at the end of the incubation periods.

Topics: Acetates; Autolysis; Carbon Radioisotopes; Cell Wall; Enterococcus faecalis; Hydrolysis; Magnesium; Microscopy, Electron; Muramidase; Osmotic Fragility; Peptidoglycan; Phosphates; Phosphotungstic Acid; Protoplasts; Quaternary Ammonium Compounds; Staining and Labeling; Sucrose; Time Factors; Tritium; Trypsin

Minicells produced by Bacillus subtilis strains carrying the div IV-B1 mutation, (CU 403 div IV-B1 and CU 403 div IV-B1, tag-1), were purified by a procedure which destroys parental cells with ultrasound, but spares minicells. Such preparations generally contain 10(9) or more minicells/ml and less than 10(4) colony-forming units/ml. Purified minicells are resistant to autolysis in tris(hydroxymethyl)aminomethane buffer, pH 7.5, at 30 C, conditions which result in total lysis of parental cells. Minicells are not completely devoid of autolytic activity, however. The medium in which minicells are produced, the temperature at which purified minicells are incubated, and the genotype of cells from which the minicells are derived all influence the rate of autolysis of purified minicells. These parameters are demonstrated by using minicells obtained from div IV-B1 and div IV-B1, tag-1 strains. Ultrastructural differences have been observed in the products of autolysis of these two minicell strains. Minicells are sensitive to low levels of lysozyme and yield miniprotoplasts when the wall is removed in an osmotically protective environment. Although minicells are unable to grow, they can maintain their integrity over long periods of time, which suggests functional energy metabolism in minicells. Direct measurements of adenosine 5'-triphosphate (ATP) levels by the luciferase assay indicated that minicells can produce ATP. Oxygen consumption, measured by standard respirometry techniques, also indicates functional metabolism in minicells. These findings demonstrate that minicells purified by ultrasound are suitable material for study of physiological processes in anucleate cells.

Topics: Adenosine Triphosphate; Autolysis; Bacillus subtilis; DNA, Bacterial; Drug Resistance, Microbial; Glucose; Microscopy, Electron; Muramidase; Mutation; Oxygen Consumption; Temperature; Ultrasonics

Topics: Autolysis; Bacillus subtilis; Cell Survival; Cell Wall; Culture Media; DNA, Bacterial; Genetics, Microbial; Muramidase; Mutation; Transformation, Genetic

Topics: Amino Acids; Autolysis; Bacteriolysis; Calcium; Cations, Divalent; Cell Division; Cell Wall; Chymotrypsin; Edetic Acid; Freezing; Hydrogen-Ion Concentration; Klebsiella pneumoniae; Lipoproteins; Magnesium; Microscopy, Electron; Molecular Conformation; Mucoproteins; Muramidase; Mutation; Phosphorus; Pronase; Sodium Dodecyl Sulfate; Trypsin

Topics: Autolysis; Bacteriolysis; Cell Wall; Culture Media; Enterococcus faecalis; Muramidase; Mutation; Osmolar Concentration; Sodium Dodecyl Sulfate; Time Factors

Topics: Autolysis; Bacillus subtilis; Bacterial Proteins; Cell Wall; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Isoelectric Focusing; Lithium; Molecular Weight; Muramidase; Mutation; Osmolar Concentration; Peptidoglycan; Protein Conformation; Sodium Dodecyl Sulfate; Teichoic Acids; Temperature; Urea

Topics: Animals; Autolysis; Chickens; Chromatography, Paper; Chymotrypsin; Copper; Dithiothreitol; Female; Hydrolysis; Muramidase; Ovum; Oxidation-Reduction; Pepsin A; Protein Conformation; Sulfhydryl Compounds; Urea

Topics: Amidohydrolases; Amines; Autolysis; Borohydrides; Cell Division; Cell Wall; Chromatography, Paper; Chromatography, Thin Layer; Hexosamines; Hot Temperature; Hydrogen-Ion Concentration; Lactobacillus; Muramidase; Osmolar Concentration; Protein Denaturation; Solubility; Spectrophotometry; Subtilisins; Sucrose; Time Factors; Tritium; Trypsin

Topics: Ammonium Sulfate; Autolysis; Bacteria; Cell Fractionation; Cell Wall; Cell-Free System; Chemical Precipitation; Ecology; Esterases; Glucosidases; Hydrolysis; Marine Biology; Muramidase; Peptide Hydrolases; Phosphoric Monoester Hydrolases; Phytoplankton; Polysaccharides; Pseudomonas; Seawater; Succinate Dehydrogenase; Vibration; Water Microbiology

Interactions between concanavalin A and cell wall digests of Bacillus subtilis 168 resulted in insoluble complexes as observed by double gel diffusion, turbidity, and analysis of the precipitate. The macromolecular constituent of the cell walls complexing with concanavalin A was the polyglucosylglycerol phosphate teichoic acid. The complex exhibited two pH optima: 3.1 and 7.4. The complex could be dissociated by saccharides which bind to concanavalin A. In contrast to concanavalin A-neutral polysaccharide complexes, formation of the concanavalin A-wall complex was inhibited by salts. It was subsequently shown that salts induce conformational changes in cell wall digests. The data suggested that for complex formation to occur a rigid rod conformation in the glucosylated teichoic acid is probably necessary. Concanavalin A can be used as a probe to study structural features of bacterial cell walls.

Topics: Autolysis; Bacillus subtilis; Binding Sites; Cell Wall; Chemical Precipitation; Concanavalin A; Culture Media; Densitometry; Glycogen; Hydrogen-Ion Concentration; Immunodiffusion; Iodides; Lectins; Muramidase; Potassium Chloride; Sodium; Sodium Chloride; Solubility; Teichoic Acids; Trichloroacetic Acid; Ultrasonics; Vibration; Viscosity

The growth of Bacillus subtilis mutant betaA177 can be inhibited under special conditions in which not enough autolytic enzymes are produced for optimal growth. Electron microscopy studies show that during growth inhibition there is localized thickening of the cell wall at positions where cells bend. A model is proposed to explain this result. Rapid growth can be restored by adding lysozyme or a B. subtilis autolysin mixture to a growth-inhibited betaA177 culture. Such addition reduces the localized wall thickening and causes other changes in surface morphology which are described and discussed. Septum formation seems to be relatively less inhibited than cell elongation when lytic enzyme levels are reduced. Measurements were made demonstrating that walls at ends of cells are morphologically different from walls at sides of cells in cultures of betaA177 growing at 51 C.

Topics: Autolysis; Bacillus subtilis; Bacteriological Techniques; Cell Wall; Enzymes; Genetics, Microbial; Microscopy, Electron; Microscopy, Phase-Contrast; Models, Biological; Muramidase; Mutation

Topics: Adsorption; Autolysis; Bacteriolysis; Bacteriophages; Cell Wall; Citrates; Culture Media; Cytosine; DNA, Bacterial; Enzymes; Guanine; Lead; Microscopy, Electron; Microscopy, Phase-Contrast; Muramidase; Staining and Labeling; Streptococcus; Trypsin

Topics: Autolysis; Bacillus cereus; Bacillus megaterium; Carbon Isotopes; Cell Wall; Chloramphenicol; Chromatography; Culture Media; Decarboxylation; Enzymes; Glycosaminoglycans; Leucine; Lysine; Muramidase; Penicillins; Peptidoglycan; Pimelic Acids; Polysaccharides, Bacterial; Solvents; Spectrophotometry; Spores, Bacterial; Trichloroacetic Acid; Tritium; Trypsin

Pneumococci in which the choline component of the cell wall teichoic acid was replaced by ethanolamine contain an abnormal autolytic enzyme that has a low molecular weight and low activity in contrast to the enzyme typical of choline-containing bacteria that has a high molecular weight and high activity. The abnormal autolysin can be converted to the normal (cholinetype) enzyme by incubation in vitro with choline-containing cell walls.

Topics: Amino Alcohols; Autolysis; Biodegradation, Environmental; Carbon Isotopes; Cell Wall; Choline; Culture Media; Glycosides; Hydrogen-Ion Concentration; Muramidase; Phosphoric Acids; Streptococcus pneumoniae; Teichoic Acids; Tritium

Topics: Amidohydrolases; Autolysis; Bacillus; Bacteriolysis; Cell Wall; Genetics, Microbial; Glucose; Muramidase; Mutation; Pimelic Acids; Uronic Acids

Topics: Amino Acids; Autolysis; Chromatography, DEAE-Cellulose; Chromatography, Gel; Chromatography, Ion Exchange; Chromatography, Paper; Clostridium botulinum; Electrophoresis; Glycoside Hydrolases; Glycosides; Hexosamines; Hydrolases; Muramidase; Paper; Peptides; Polysaccharides

Preparations of purified cell walls from Staphylococcus aureus were shown to contain small amounts of phospholipid and glycerol teichoic acid. Since these are components of the cell membrane, it is probable that the wall itself contains no lipid, but does retain fragments of membrane because of physical connections between wall and membrane. In walls of S. aureus strain 52A5, which completely lacks ribitol teichoic acid, the only phosphorylated compound identified as a genuine wall component was a phosphorylated derivative of murein that gave rise to muramic acid phosphate on acid hydrolysis. Muramic acid phosphate was also identified in hydrolysates of walls from S. aureus H and strain 52A2.

Topics: Acyltransferases; Autolysis; Bacterial Proteins; Carbon Isotopes; Cell Membrane; Cell Wall; Chromatography, Gas; Chromatography, Ion Exchange; Chromatography, Paper; Chromatography, Thin Layer; Electrophoresis; Fatty Acids; Glycerol; Glycerophosphates; Glycosides; Hot Temperature; Hydrolysis; Indicators and Reagents; Lipopolysaccharides; Muramidase; Nucleic Acids; Paper; Pentosephosphates; Phospholipids; Phosphorus Isotopes; Polymers; Ribose; Staphylococcus; Transferases

Cell walls from exponential-phase cultures of Streptococcus faecalis ATCC 9790 contain an autolysin (a beta-N-acetylmuramide glycanhydrolase, E.C. 3.2.1.17) which has been isolated from trypsin-speeded wall autolysates. The autolysin, which was excluded from Bio-Gel P-60, was further fractionated by diethylaminoethyl (DEAE)-cellulose chromatography or filtration on Bio-Gel P-200. After DEAE-cellulose chromatography, which removed most of the wall polysaccharide, autolysin activity was extremely labile and was rapidly lost at -20 C, even in the presence of albumin. The P-60-excluded enzyme was rapidly bound by walls at both 37 C (50% bound in about 1 min) and 0 C (50% bound in less than 4 min). Wall-bound autolysin could not be removed by 1.0 m ammonium acetate (pH 6.9). Autolysin was also bound by walls that had been extracted with 10% trichloroacetic acid or treated with 0.01 n periodate, suggesting that the nonpeptidoglycan wall polymers are not important for binding. Wall-bound autolysin was more stable than the soluble enzyme to proteinase digestion, acetone (40%), 8 m urea (at 0 C), or to inactivation at 56 C. Two bacterial neutral proteinases (which do not hydrolyze ester bonds) activated latent wall-bound autolysin, suggesting that activation results from the cleavage of one or more peptide bonds. The group A streptococcal proteinase activated latent autolysin but differed from the other proteinases in that it did not inactivate soluble autolysin. The results suggest that the autolysin is not covalently linked to the wall. The high affinity of the walls for the autolysin appears to be responsible for the firm, not easily reversed binding.

Topics: Autolysis; Cell Wall; Chromatography, Gel; Chromatography, Ion Exchange; Enterococcus faecalis; Muramidase; Peptide Hydrolases

A 10-hr starvation of Streptococcus faecalis ATCC 9790 for the amino acids methionine and threonine results in cells which are resistant to autolysis and which contain greatly reduced quantities of both active and latent (proteinase activable) forms of the autolytic enzyme (an N-acetyl-muramide glycanhydrolase). Cell walls were isolated from cells harvested at various times during the recovery from such starvation and were assayed for active and latent forms of the autolysin. Within 10 min of recovery the latent enzyme began to increase. Only after 30 to 60 min did the active enzyme begin to increase; after a similar lag, the cells' proneness to lysis markedly increased. The intracellular localization of both forms of the autolysin was examined, using as an experimental tool the ability of added cell wall to bind autolysin. (14)C-lysine-labeled, inactivated cell walls were added to exponential-phase cells, which were then disrupted, and the mixed wall population was isolated. Measurement of the (14)C release during wall autolysis indicated that the active enzyme in the cells was not available for binding to the added (14)C-labeled walls and was therefore wall-bound in vivo. In contrast, up to 85% of latent autolysin activity was found to have been efficiently bound to the added (14)C walls. The results obtained suggest (i) cellular autolysis is a reflection of the level of active enzyme and not of latent enzyme, and (ii) autolysin is synthesized and mainly located in the cytoplasm as an inactive latent precursor (proenzyme) which is transported to sites on the cell wall associated with wall biosynthesis, where it becomes activated.

Topics: Autolysis; Carbon Isotopes; Cell Wall; Centrifugation; Cytoplasm; Enterococcus faecalis; Enzyme Precursors; Methionine; Muramidase; Threonine

Cell walls (LOG walls) were isolated from cultures of Streptococcus faecalis ATCC 9790 in the exponential phase of growth. These walls were either allowed to undergo autolytic dissolution (in the presence or absence of trypsin) or wall autolysis was inactivated with sodium dodecylsulfate (SDS walls). Inactivated walls were treated either with lysozyme or with isolated, partially purified S. faecalis autolysin. During wall lysis, samples were removed, negatively stained with phosphotungstate, and examined in the electron microscope. Both lysozyme and isolated autolysin appeared to act over the entire surface of SDS walls. After partial dissolution, a fibrous network over the surface was revealed. Lysozyme digestion revealed the presence of prominent, highly-contrasted equatorial and subequatorial bands around the walls. After trichloroacetic acid extraction, the bands were seen less frequently and less distinctly in the partially lysozyme digested walls, suggesting that the bands contained nonpeptidoglycan polymers. In the absence of trypsin (which activates a latent form of the autolysin), autolysis of LOG walls appeared to start at the equatorial bands and to proceed back towards the apex of the coccus. Ribbons of wall material coming off the wide edge of the nearly hemispherical wall fragments were observed. Activation of latent autolysis resulted in lytic action over the entire wall surface. The results are consistent with the previously postulated location of active autolysin at the areas of new wall synthesis and the random location of latent autolysin in LOG walls.

Topics: Autolysis; Bacteriolysis; Cell Wall; Enterococcus faecalis; Microscopy, Electron; Muramidase