Autolytic Enzyme System of Streptococcus faecalis III. Localization of the Autolysin at the Sites of Cell Wall Synthesis

Cell walls from exponential-phase cultures of Streptococcus faecalis ATCC 9790 autolyzed in dilute buffers. Walls were isolated from cultures grown in the presence of (14)C-lysine for about 10 generations and then on (12)C-lysine for 0.1 to 0.8 of a generation (prelabeled). These walls released (14)C to the soluble fraction more slowly than they lost turbidity during the initial stages of autolysis. Walls isolated from cultures grown in the presence of (14)C-lysine for only the last 0.1 to 0.4 of a generation (postlabeled) released (14)C to the supernatant fluid more rapidly than they lost turbidity. Autolysin in both pre- and postlabeled walls was inactivated, and such walls were then incubated in the presence of unlabeled walls containing active autolysin. The inactivated walls lost their (14)C label only very slowly until autolysis of the unlabeled walls was virtually complete and release of soluble autolysin was expected. When this experiment was done in the presence of trypsin, a fourfold increase in the autolysis rate resulted, but the same pattern of (14)C release was observed. A parallel release of (14)C and loss of turbidity from pre- or postlabeled walls was observed upon trypsin "activation" and by addition of isolated soluble autolysin to inactivated walls. We conclude that the wall-bound autolysin acts first on the more recently synthesized portion of the wall. Trypsin appears to speed wall autolysis by activating additional latent autolysin in situ at sites in the older portion of the wall.     

Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells. 3. Interplay between limited cell-wall autolysis, pectin methyl esterase activity and electrostatic effects in soybean cell walls

Soybean cell walls display a process of autolysis which results in the release of reducing sugars from the walls. Loosening and autolysis of cell wall are involved in the cell-wall growth process, for autolysis is maximum during both cell extension and cell-wall synthesis. Autolysis goes to completion within about 50 h and is an enzymatic process that results from the activity of cell wall exo- and endo-glycosyltransferases. The optimum pH of autolysis is about 5. Increasing the ionic strength of the bulk phase where cell-wall fragments are suspended, results in a shift of the pH profile towards low pH. This is consistent with the view that at 'low' ionic strength, the local pH in the cell wall is lower than in the bulk phase. One of the main ideas of the model proposed in a preceding paper, is that pectin methyl esterase reaction, by building up a high fixed charge density, results in proton attraction in the wall. Low pH must then activate the wall loosening enzymes involved in autolysis and cell growth. This view may be directly confirmed experimentally. The pH of a cell-wall suspension, initially equal to 5, was brought to 8 for 20 min, then back to 5. Under these conditions, the rate of cell-wall autolysis was enhanced with respect to the rate of autolysis obtained with cell-wall fragments kept at pH 5. The pH response of the multienzyme plant cell-wall system basically relies on opposite pH sensitivities of the two types of enzymes involved in the growth process. Pectin methyl esterase, which generates the cell-wall Donnan potential, is inhibited by protons, whereas the wall-loosening enzymes involved in cell growth are activated by protons.     

Autolytic Enzyme System of Streptococcus faecalis IV. Electron Microscopic Observations of Autolysin and Lysozyme Action

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.     

Cell wall degradation in the autolysis of filamentous fungi

A systematic study on autolysis of the cell walls of fungi has been made on Neurospora crassa, Botrytis cinerea, Polystictus versicolor, Aspergillus nidulans, Schizophyllum commune, Aspergillus niger, and Mucor mucedo. During autolysis each fungus produces the necessary lytic enzymes for its autodegradation. From autolyzed cultures of each fungus enzymatic precipitates were obtained. The degree of lysis of the cell walls, obtained from non-autolyzed mycelia, was studied by incubating these cell walls with and without a supply of their own lytic enzymes. The degree of lysis increased with the incubation time and generally was higher with a supply of lytic enzymes.Cell walls from mycelia of different ages were obtained. A higher degree of lysis was always found, in young cell walls than in older cell walls, when exogenous lytic enzymes were present.In all the fungi studied, there is lysis of the cell walls during autolysis. This is confirmed by the change of the cell wall structure as well as by the degree of lysis reached by the cell wall and the release of substances, principally glucose and N-acetylglucosamine in the medium.     

Relationship between the Latent Form and the Active Form of the Autolytic Enzyme of Streptococcus faecalis

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.     

Some Properties of the Autolytic N-Acetylmuramidase of Lactobacillus acidophilus

The autolytic N-acetylmuramidase present in Lactobacillus acidophilus strain 63 AM Gasser has an optimal pH between 5 and 6 when lysing intact cells or isolated cell walls. Cellular lysis at pH 5 is two to four times more rapid in citrate buffer of 0.01 M and 0.5 M or higher than in 0.1 M acetate buffer. It seems that sulfhydryl groups are required for both cell and wall autolysis. Heavy metal ions and p-chloro-mercuribenzoate, at low concentrations, are powerful inhibitors. Ethylenediaminetetraacetic acid stimulates cellular but not wall autolysis in acetate buffer to the level obtained in citrate buffer. The possible involvement of sulfhydryl groups in a mechanism of control of cellular autolytic activity is discussed. The autolytic enzyme, although unstable in solution at 37 C, can be extracted from walls by the use of solutions of bovine serum albumin (100 mug/ml) in 0.01 N NaOH. Soluble enzyme extracted from walls rebinds on to sodium decylsulfate-treated walls, but three times as much of the wall material is required to completely re-adsorb the activity.     

Autolysis in Staphylococcus aureus: Preferential Release of Old Cell Walls

The kinetics of release of old versus new cell wall in two strains of Staphylococcus aureus were studied during autolysis. In both strains the autolytic enzyme is an amidase. Cells were double labeled with (3)H and (14)C, and the distribution of radioactivity in the cell walls was monitored during autolysis. In all cases the rate of release of steady-state lable from peptidoglycan was significantly higher than that of pulse label. Identical results were obtained with whole cells or isolated cell walls. The results suggest that in S. aureus the old cell wall is preferentially released during autolysis.     

Auxin-enhanced glucan autohydrolysis in maize coleoptile cell walls

Cell walls isolated from auxin-pretreated maize (Zea mays L.) coleoptile segments were assayed to disclose evidence for the existence of enhanced autolysis. To improve the sensitivity of the measurements and to facilitate kinetic analysis, isolated cell walls were consolidated within a small column, and the autolysis rate was directly determined from the sugar content of the effluent. This protocol revealed that the maximum rate of autohydrolysis of walls prepared from segments occurs within the first 2 hours and a steady decline commences almost immediately. Walls from indoleacetic acid pretreated segments (0.5-4 hours) released sugar at a higher rate initially (110-125% of controls) and the enhanced rate of autolysis continued for 6 to 8 hours, but then it became equivalent to that of the controls. Pretreatment of the segments at acidic pH had no effect on the measurable rates of autolysis. The (1→3), (1→4)-β-d-glucan content of the walls and the extractable glucanase activities support the hypothesis that temporal enhancement of autohydrolysis is a function of auxin on enzyme activity. The progressive decline in autolysis during prolonged incubations is consistent with the decrease in the quantity of the β-d-glucan in the wall. The relationship between glucan content and autolysis rate is supported by the observation that while glucose pretreatment of segments had only a small effect on initial autolysis rates, the presence of the sugar during pretreatment served to extend the interval over which higher rates of autolysis could be sustained. The results demonstrate that autolysis is related to auxin-induced wall metabolism in maize coleoptiles.     

Changes in the Autolytic Activities of Maize Coleoptile Cell Walls during Coleoptile Growth

Autolytic activities of coleoptile cell walls were measuredin developing maize seedlings. The major neutral sugar componentsof the cell wall polysaccharides were arabinose, xylose andglucose. The quantities of all these components per coleoptileincreased for 5 d after germination, suggesting that levelsare augmented by biosynthetic processes during coleoptile growth.However, cell wall preparations isolated from the coleoptilesalso revealed increasing rates of autolytic activity directedtoward each of the sugar components. This result suggests thatthe constitutive hydrolytic activities expressed by cell wallsalso increase as a function of coleoptile age. The proportionof glucose in autolysis products relative to that present inthe cell walls specifically increased with coleoptile age, whilethe ratios for arabinose and xylose decreased. Kinetic analysesof autolysis demonstrated that the reactions specific for pentosesat the early growth stage are transient events and that initiallow rates of glucan autolysis increased sharply and persistedlonger. In these experiments the products of glucan autolysiswere largely monomeric while those of the pentose-specific reactionsconsisted of both monomeric and polymeric sugars. Based on theseresults, we concluded that two distinct phases of autolyticactivities are expressed in the mediation of cell wall polysaccharidemetabolism in situ. (Received July 17, 1996; Accepted November 25, 1996)     

Autolysis of cell walls in pea epicotyls during growth. Enzymatic activities involved

Pisum sativum L. (cv. Lincoln) epicotyl cell walls show autohydrolysis and release into the incubation medium up to 120 μg of sugar per mg of cell wall dry weight in 30 h. Cell walls from younger epicotyls with high growth capacity showed higher auto-lytic capacity than older epicotyls. This suggests that both processes, growth and au-tolysis, are related. The proteins responsible for autolysis were extracted from the wall fraction with high saline solution (3 M LiCl) and enzymatic activities associated with the proteins were studied. The highest activity corresponded to α-galactosidase; lower activities were found for β-galactosidase, a-arabinosidase and exoglucanase. Changes in enzymatic activities and changes in the proportion of sugars released in autolysis by cell walls during the growth of epicotyls support the notion that α-galac-tosidase is one of the enzymes involved in the process of autolysis, and that the liberation of arabinose and galactose in this process occurs as arabinogalactan.     

Cell wall structure regulates the autolytic process throughout growth of Cicer arietinum epicotyls

The autolytic process in epicotyl cell walls of Cicer arietinum L. cv. Castellana, and also the hydrolysis of heat-inactivated cell walls as mediated by a cell wall β-galactosidase (EC 3.2.1.23) (named βIII and previously characterized as responsible for the autolysis), are maximal on the fourth day of germination and coincide with the maximal growth capacity. They decrease during the following days, in which the growth rate diminishes. In both cases, no differences were observed in the percentages of the different sugars released, galactose being the principal one. The βIII fraction from aged epicotyl cell walls hydrolyzed young walls in proportion to its specific activity, and more efficient than when cell walls from aged material were used as the substrate. The βIII fraction from 4 day-old epicotyls (the time for maximal autolysis) was incapable of hydrolyzing aged epicotyl cell walls to the same extent as young ones. These results, together with the levels and activity of the enzyme throughout growth, allow the assumption that the variations in the autolysis and hydrolysis caused by βIII during growth processes are due to structural modifications in the cells walls, modifications that would limit access of the enzyme to its substrate, thus impeding the release of galactose, even though the enzyme is present.     

Fractionation and partial characterization of the products of autolysis of cell walls of Bacillus subtilis

Young, Frank E. (Western Reserve University, Cleveland, Ohio). Fractionation and partial characterization of the products of autolysis of cell walls of Bacillus subtilis. J. Bacteriol. 92:839-846. 1966.-Autolysis of the cell wall of Bacillus subtilis by an indigenous autolytic enzyme results in solubilization of 90% of the cell wall. The solubilized cell wall (supernatant fraction) was fractionated by the combination of ion-exchange chromatography on diethylaminoethyl cellulose and gel filtration on Sephadex G-25 into polysaccharides (composed of N-acyl glucosamine and N-acyl muramic acid), mucopeptides, peptides, and teichoic acid. The chemical composition of the products of autolysis confirms the proposed mechanism of autolysis and establishes the autolytic enzyme as an N-acyl muramyl-l-alanine amidase. The heteropolymers in the cell wall are linked by peptide bridges. Two peptides which account for 70% of the peptides of the cell wall have a molar ratio of 1.0:0.9:1.3 for diaminopimelic acid, glutamic acid, and alanine, respectively. Other minor peptides contain diaminopimelic acid, glutamic acid, and alanine in molar ratios of 1.0:0.9:1.5, 1.0:0.5:1.0, and 1.0:1.5:1.7, respectively. The procedures employed in this study should be applicable to the fractionation of heteropolymers in cell walls of other gram-positive organisms and thereby aid in the study of the structure of antigenic determinants and endotoxins.     

Derepression of beta-1,3-glucanases in Penicillium italicum: localization of the various enzymes and correlation with cell wall glucan mobilization and autolysis.

The localization of the derepressible beta-1,3-glucanases of Penicillium italicum and the cell wall autolysis under conditions of beta-1,3-glucanase derepression (24 h in a low-glucose medium) were studied. About 15% of the total activity was secreted into the culture medium during the 24-h period and consisted of similar amounts of each of the three beta-1,3-glucanases (I, II, III) produced by this species. Treatment of derepressed mycelia with periplasmic enzyme-inactivating agents resulted in a loss of 45% of the mycelium-bound beta-1,3-glucanase. Analysis of periplasmic enzymes solubilized by 2 M NaCl or by autolysis of isolated cell walls revealed that only beta-1,3-glucanases II and III were bound to the cell wall. These two enzymes were capable of releasing in vitro reducing sugars from cell walls, whereas beta-1,3-glucanase I was not. In addition, the autolytic activity of cell walls isolated from derepressed mycelium was greater than that of cell walls isolated from repressed mycelium. The incubation of the fungus in the low-glucose medium also resulted in the in vivo mobilization of 34% of the cell wall beta-1,3-glucan, and this mobilization was fully prevented by cycloheximide, which also blocked derepression of beta-1,3-glucanases. Derepression of beta-1,3-glucanase seems to be coupled to the mobilization of cell wall glucan.     

Autolysis of the cell wall. Its possible role in endogenous and IAA-induced growth in epicotyls of Cicer arietinum

Indoleacetic acid (IAA), a factor that induces growth in epicotyls of cicer arietinum L. cv. Castellana, increases the autolytic capacity of the cell walls by 50%, suggesting that autolysis is related to the processes of cell wall loosening that accompany growth. IAA promotes an increase in the specific activities of the enzymes involved in autolysis, mainly α-galactosidase (EC 3.2.1.22). This relationship autolysis-growth. was also observed in a study of the autolytic capacity of cell walls from regions of the epicotyl with different growth capacity. The sugars released and the level of enzymatic protein were higher in the subapical region that towards the base.     

Effect of β-Lysin on Isolated Cell Walls and Protoplasts of Bacillus subtilis

The cytoplasmic membrane is the site of beta-lysin action. Protoplasts lysed rapidly in its presence, whereas cell walls and wall autolysis were unaffected.     

In vitro autolysis of plant cell walls

Primary cell walls of Zea mays prepared in a glycerol medium are capable of autolysis in vitro. Autolysis results in solubilization of about 10% of the wall substance during an 8 hour incubation period. Approximately 10% of the solubilized material is glucose and the remainder consists of an unidentified polymer which yields only glucose upon hydrolysis. Cell wall autolysis is a linear function of time of incubation and of wall concentration. The autolytic process occurs optimally over the pH range of 5.5 to 6.5. The possible relationship between autolytic capacity and capacity for elongation is discussed.     

Identification of polysaccharide hydrolases involved in autolytic degradation of zea cell walls

Cell walls of Zea mays (cv L.G.11) seedlings labeled with ¹⁴C were treated with α-amylase from Bacillus subtilis to remove starch and mixed linkage glucans. These walls released arabinose, xylose, galactose, and galacturonic acid in addition to glucose when they were allowed to autolyze. Methylation analysis was performed on samples of wall which had been incubated autolytically and the results indicated that degradation of the major polymer of the wall, the glucoarabinoxylan, had occurred. A number of glycanases could be dissociated from the wall by use of 3 m LiCL. The proteins which were released were found to contain a number of exoglycosidase activities in addition to being effective in degrading the polysaccharide substrates, araban, xylan, galactan, laminarin, mannan, and polygalacturonic acid. The effects of these enzymes on the wall during autolysis appear to result from endo-activity in addition to exo-activity. The structural changes that occurred in the cell walls during autolysis were found to be related to the changes previously found to occur in cell walls during auxin induced extension.     

Role of teichuronic acid in Bacillus licheniformis: defective autolysis due to deficiency of teichuronic acid in a novobiocin-resistant mutant.

nov-12, a novobiocin-resistant mutant of Bacillus licheniformis ATCC 9945, grows as long chains of cells, a characteristic of autolytic-deficient (Lyt-) mutants. Isolated walls from nov-12 autolyzed at a rate equal to 5% of that displayed by wild-type walls, thus confirming the Lyt- phenotype. Protein-free nov-12 walls displayed marked resistance to, and also failure to bind, added autolysin solubilized from wild-type walls. Comparison of isolated cell walls revealed a deficiency in teichuronic acid in the mutant. Lesser differences were observed in walls of this strain, including a reduction in galactose, an increase in the proportion of peptidoglycan, and small quantitative differences in peptidoglycan composition though the proportions of protein and teichoic acid were similar in walls of both strains. Autolytic sensitivity was studied in walls in which protein, teichoic acid, and teichuronic acid were removed successively by selective extraction procedures. Autolysis of wild-type walls was unaffected by removal or protein or teichoic acid, but teichuronic acid removal rendered wild-type walls as insensitive to autolysis as mutant walls had been throughout. Therefore, in this mutant, deficiency in teichuronic acid alone leads to the Lyt- phenotype and, hence, activity and binding of autolysin(s) are dependent upon teichuronic acid but not teichoic acid. Also, the potential rate of autolysis of cell walls in this organism was correlated with the proportion of teichuronic acid in the wall. The possible significance of these findings with respect to control of autolysis and cell separation is discussed.     

Autolysis of Bacillus cereus cell walls and isolation of structural components

Autolysis of Bacillus cereus N.R.R.L. 569 cell walls was accompanied by hydrolysis of the majority of the 4-O-beta-N-acetylglucosaminyl-N-acetylmuramic acid linkages in mucopeptide, presumably by an endo-beta-N-acetylglucosaminidase. Hydrolysis of the N-acetylmuramyl-l-alanine linkages by an amidase also occurred. Free d-alanine residues were detected in isolated cell walls and the proportion of these residues increased during autolysis, presumably due to d-alanine carboxypeptidase action. Fractionation and analysis of the products of autolysis confirmed these results. Among the products originating from mucopeptide were a disaccharide, N-acetylmuramyl-N-acetylglucosamine, and a tetrapeptide of sequence l-Ala-d-Glu-meso-Dap-d-Ala (Dap=diaminopimelate). A dimer fraction containing a d-Ala-meso-Dap cross-link was also isolated. Two polysaccharides were obtained from the products of autolysed cell walls and from walls made soluble by Chalaropsis B glycosidase. A neutral polysaccharide accounted for about 40% of the wall and contained N-acetylglucosamine, N-acetylgalactosamine and glucose. The neutral polysaccharide isolated from wall autolysates was attached to a part of the glycan moiety of mucopeptide. The molecular weight of the complex was approx. 28000. Stoicheiometric amounts of phosphorus were present, possibly in linkages between the polysaccharide and mucopeptide moieties. The second polysaccharide accounted for 12% of the wall and was very acidic. After acidic hydrolysis of the polysaccharide, glucosamine, galactosamine and unidentified acidic substances were detected. The acid polysaccharide isolated from wall autolysates contained only traces of mucopeptide constituents and no phosphorus.     

Cell Wall Composition and Associated Properties of Methicillin-Resistant Staphylococcus aureus Strains

Methicillin-resistant (MR) Staphylococcus aureus strains have previously been reported to be deficient in surface negative charge; this has been correlated with methicillin resistance and ascribed to a deficiency of teichoic acid at the cell surface (A. W. Hill and A. M. James, Microbios 6:157-167, 1972). Teichoic acid was present in walls of MR organisms as revealed by appreciable phosphate levels and detection of ribitol residues. Phosphate levels in walls from five MR strains (0.54 to 0.77 mumol/mg of wall) were lower than in three unrelated methicillin-sensitive (MS) strains (0.86 to 1.0 mumol/mg of wall). However, two MS strains derived from two of the MR strains had wall phosphate levels very similar to those of the MR strains. No evidence for unusual wall polymers was found. Simple deficiency of wall teichoic acid does not result in methicillin resistance since an independently isolated teichoic acid-deficient strain (0.1 mumol of phosphate per mg of wall) was not methicillin resistant. In studies of biological properties possibly related to wall teichoic acid, it was discovered that walls isolated from MR organisms grown in the presence of methicillin autolyzed more rapidly than those isolated from organisms grown in the absence of the drug. Since methicillin resistance is enhanced by NaCl and suppressed by ethylenediaminetetraacetate, the effects of these compounds on autolysis of isolated walls were studied. NaCl (1.0 M) and ethylenediaminetetraacetate (1.0 mM) inhibited the autolysis of walls isolated from MR and MS strains. An MR strain bound phage 47, 52A, and 3A only slightly less well than their respective propagating strains.