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.     

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.     

Autolytic formation of protoplasts (autoplasts) of Streptococcus faecalis; location of active and latent autolysin.

Over 80% of the active and porteinase-activatable, latent forms of the autolytic N-acetylmuramide glycanhydrolase of Streptococcus faecalis ATCC 9790 were released to the supernatant buffer during the autolytic formation of protoplasts (autoplasts) in the presence of absence of trypsin. Autolysin activity was not found in association with released mesosomal vesicles and had little affinity for binding to membranes or to the outer surface of the wall. Isolated walls were able to bind over four times as much autolysin activity as that present on wall exponential-phase cells. Using a rapid technique for wall isolation, evidence was obtained that the latent form (as well as the active form) was wall bound in intact cells. In addition, isotope labeling and ultrastructural studies were able to show that latent autolysin was concentrated in the newer, septally associated portion of the wall.     

Some Properties of Two Autolytic-Defective Mutants of Streptococcus faecalis ATCC 9790

The isolation and some properties of two mutants of Streptococcus faecalis ATCC 9790 (S. faecium) which autolyze at a much slower rate than the wild type are described. Compared with the wild type, mutant E71 autolyzed more slowly, contained less active but more latent autolysin in the isolated wall fraction, and possessed a wall of very similar chemical composition and degree of cross-bridging. Ultrastructural studies of exponential phase cells showed that cells of E71 were on the average slightly longer and had slightly thickened walls compared to the wild type. Mutant E81 autolyzed much more slowly, grew exponentially in long chains (8 to 40 cells compared with mainly diplococci), contained much less active and latent autolysin in the wall, and possessed a wall of very similar chemical composition but with about twice the content of N-terminal groups. Mutant E81 walls were more susceptible to isolated autolysin but possessed an autolysin of the same specificity as the wild type. Ultrastructurally E81 cells were, on the average, significantly longer and had thicker walls than the wild type. Mutant E71 may be partially blocked at either transport of autolysin to the wall or in conversion of latent to active autolysin. The pleitropic effects noted in mutant E81 have been taken to suggest a possible membrane defect and to support the role of the autolysin in cell separation.     

Relationship Between the Location of Autolysin, Cell Wall Synthesis, and the Development of Resistance to Cellular Autolysis in Streptococcus faecalis After Inhibition of Protein Synthesis

Ten minutes after inhibition of protein synthesis with chloramphenicol (CAP) the ability of cells of Streptococcus faecalis (ATCC 9790) to autolyze decreased to less than 20% of the rate for exponential-phase cells. After threonine exhaustion, the time for a 50% drop in the rate of cellular autolysis was about 20 min. These rapid increases in resistance to cellular autolysis could not be accounted for by: (i) the relatively slow and small overall decrease in susceptibility of isolated cell walls to added autolysin, or (ii) a decreased content of either the active or latent (proteinase activatable) form of the autolysin in the wall fraction. Continued wall synthesis resulted in dilution of preexisting autolysin in the isolated wall fraction. The release of labeled "old" relative to "new" wall from CAP-treated cultures showed that wall synthesis shifted away from the areas of wall previously shown to be associated with wall synthesis (extension) in exponential-phase cells. A corresponding dispersal of active autolysin activity was not observed. By using actinomycin D and CAP, a requirement for ribonucleic acid and protein synthesis early in the recovery of cells from amino acid starvation was demonstrated for the recovery in the ability of cells to autolyze. Evidence was obtained which suggests that a protein is involved in the conversion of latent to active autolysin. During recovery from amino acid starvation, increase in wall synthesis and content of active autolysin was delayed (25 to 35 min), whereas an increase in turbidity and latent enzyme content began within 10 min. After treatment with CAP at 22 or 52 min of recovery, a further increase in levels of both active and latent autolysin was severely inhibited; however, the increase in rate of wall synthesis was indistinguishable from that of an untreated control. This suggests that an increase in rate of wall synthesis does not depend on an increase in level of active autolysin.     

Morphological and physiological study of autolytic-defective Streptococcus faecium strains.

Three autolytic-defective mutants of Streptococcus faecium (S. faecalis ATCC 9790) were isolated. All three autolytic-defective mutants exhibited the following properties relative to the parental strain: (i) slower growth rates, especially in chemically defined medium; (ii) decreased rates of cellular autolysis and increased survival after exposure to antibiotics which block cell wall biosynthesis; (iii) decreased rates of cellular autolysis when treated with detergents, suspended in autolysis buffers, or grown in medium lacking essential cell wall precursors; (iv) a reduction in the total level of cellular autolytic enzyme (active plus latent forms of the enzyme); (v) an increased ratio of latent to active forms of autolysin; and (vi) increased levels of both cellular lipoteichoic acid and lipids.     

Autolytic defective mutant of Streptococcus faecalis.

Properties of a variant of Streptococcus faecalis ATCC 9790 with defective cellular autolysis are described. The mutant strain was selected as a survivor from a mutagenized cell population simultaneously challenged with two antibiotics which inhibit cell wall biosynthesis, penicillin G and cycloserine. Compared to the parental strain, the mutant strain exhibited: (i) a thermosensitive pattern of cellular autolysis; (ii) an autolytic enzyme activity that had only a slightly increased thermolability when tested in solution in the absence of wall substrate; and (iii) an isolated autolysin that had hydrolytic activity on isolated S. faecalis wall substrate indistinguishable from that of the parental strain, but that was inactive when tested on walls of Micrococcus lysodeikticus as a substrate. These data indicate an alteration in the substrate specificity of the autolytic enzyme of the mutant which appears to result from the synthesis of an altered form of autolytic enzyme.     

Autolytic enzyme system of Streptococcus faecalis. V. Nature of the autolysin-cell wall complex and its relationship to properties of the autolytic enzyme of Streptococcus faecalis

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.     

Electron microscopy of Staphylococcus aureus cell wall lysis

Virgilio, Rafael (Escuela de Química y Farmacia, Universidad de Chile, Santiago, Chile), C. González, Nubia Mu?oz, and Silvia Mendoza. Electron microscopy of Staphylococcus aureus cell wall lysis. J. Bacteriol. 91:2018-2024. 1966.-A crude suspension of Staphylococcus aureus cell walls (strain Cowan III) in buffer solution was shown by electron microscopy to lyse slightly after 16 hr, probably owing to the action of autolysin. The lysis was considerably faster and more intense after the addition of lysozyme. A remarkable reduction in thickness and rigidity of the cell walls, together with the appearance of many irregular protrusions in their outlines, was observed after 2 hr; after 16 hr, there remained only a few recognizable cell wall fragments but many residual particulate remnants. When autolysin was previously inactivated by trypsin, there was a complete inhibition of the lytic action of lysozyme; on the other hand, when autolysin was inactivated by heat and lysozyme was added, a distinct decrease in the thickness of the cell walls was observed, but there was no destruction of the walls. The lytic action of lysozyme, after treatment with hot 5% trichloroacetic acid, gave rise to a marked dissolution of the structure of the cell walls, which became lost against the background, without, however, showing ostensible alteration of wall outlines. From a morphological point of view, the lytic action of autolysin plus lysozyme was quite different from that of trichloroacetic acid plus lysozyme, as shown by electron micrographs, but in both cases it was very intense. This would suggest different mechanisms of action for these agents.     

Autolytic Enzyme System of Clostridium botulinum

Isolated cell walls of Clostridium botulinum type A strain 190L released an autolysin during autolysis of the cell walls. The autolysin was isolated from the cell walls, and partially purified 18.6-fold by ammonium sulfate precipitation, chromatography on DEAE-cellulose and gel filtration through Sephadex G-100. The purified preparation of the autolysin showed 2 major and 2 minor protein bands on Polyacrylamide gel electrophoresis. Some properties of the autolysin were examined using SDS-treated cell walls of the organisms as a substrate. The autolysin was active over a pH range of 6 to 8, with a maximum near pH 6.8. The lytic activity was stimulated by 10^?4 M each of Co⁺⁺, Mg⁺⁺ and Ca⁺⁺ in the order, whereas it was inhibited markedly by Cu⁺⁺. Mercaptoethanol (10^?4–10^?3 M) significantly activated the lytic action. Trypsin and nagarse (10 μg/ml) also stimulated the lytic activity. The lytic spectrum of the autolysin toward the SDS-treated cell walls obtained from various types of C. botulinum and C. perfringens indicated a relatively high specificity. After treatment with hot formamide the cell walls of C. botulinum increased in susceptibility to the autolysin.     

Sporangila autolysis in Clostridium perfringens type A

The autolytic system functioning in the release of mature spores and enterotoxin from sporangia of Clostridium prefringens was partially characterized. After sporangial autolysis in buffer, the supernatant fluid of the suspension contained autolysin active against purified sporangial walls. The autolysin was most active at pH 8 and 37°C, in the presence of Co²⁺ (0.3 · 10⁻³ M CoCl₂) and trypsin (48 μg/ml). Sodium dodecyl sulfate-treated sporangial walls further extracted with trichloroacetic acid to remove teichoic acid were a better enzyme substrate than walls treated only with sodium dodecyl sulfate. N-Acetylmuramyl-l-alanine amidase activity which released N-terminal alanine, and endopeptidase activity which hydrolysed the d-alanyl-glycine linkage liberating N-terminal glycine and C-terminal alanine, were both functional at pH 8. It is not known if one or two enzyme are involved. Autolysin appeared in cells as early as 2 h after inoculation into sporulation medium. Two asporogenic Stage 0 mutants grown in sporulation medium also produced autolysin identical in mode of action to that of the sporogenic wild type. Although the active cellular autolysin concentration subsequently decreased as cells sporilated, the walls of 8-h-old sporangia containing refractile heat-resistant spores were more susceptible to digestion by autolysin, than those of 2-, 4-, or 6-h-old cells grown in sporulation medium or of 4- or 14-h vegetative cells from growth medium. The results suggest that a progressive change may occur in the structure of the sporangial wall during spore morphogenesis, thus increasing its susceptibility to autolysis.     

Autolytic Activity and an Autolysis-Deficient Mutant of Clostridium acetobutylicum

The optimum conditions for autolysis and autoplast formation in Clostridium acetobutylicum P262 have been defined. Autolysis was optimal at pH 6.3 in 0.04 M sodium phosphate buffer, and the bacterium produced latent and active forms of an autolytic enzyme. The ability of cells to autolyze decreased sharply when cultures entered the stationary phase. Autoplasts were induced by 0.25 to 0.5 M sucrose and were stable in media containing sucrose, CaCl₂, and MgCl₂. A pleiotropic autolysis-deficient mutant (lyt-1) was isolated. The mutant produced less autolysin than did the parent P262 strain, and it had an altered cell wall which was more resistant to both its own and P262 autolysins. The mutant formed long chains of cells, and lysozyme was required for the production of autoplasts. Growth of the P262 strain or the lyt-1 mutant was inhibited by the same concentrations of penicillin, ampicillin, and vancomycin. The lyt-1 mutant strain treated with the minimum growth-inhibitory concentration of penicillin autolyzed upon the addition of wild-type autolysin to the autolysis buffer at the same rate as did the untreated P262 strain. Chloramphenicol did not protect the penicillin-treated lyt-1 cells against autolysis enhanced by exogenous wild-type autolysin.     

Inhibition of wall autolysis in Streptococcus faecalis by lipoteichoic acid and lipids.

Fully acylated lipoteichoic acid (LTA) isolated from Streptococcus faecalis ATCC9790 (S. faecium) inhibited autolysis of walls from the same organism at concentrations (1.0 to 1.5 nmol of LTA per mg of wall) comparable to those found in intact cells. Partially deacylated LTA isolated from S. faecalis or chemically deacylated LTA failed to inhibit significantly in the same concentration range. Beef heart cardiolipin and commercially obtained dipalmitoyl phosphatidyl glycerol were also found to inhibit wall autolysis in S. faecalis. Chemical deacylation of beef heart cardiolipin also removed the inhibitory activity of this molecule. Lipid fractions isolated from S. faecalis that inhibited wall autolysis were: diphosphatidyl glycerol (cardiolipin), phosphatidyl glycerol, aminoacyl phosphatidyl glycerol, and a neutral lipid fraction. Glycolipids were not found to be effective inhibitors. The possible role of LTA and/or certain lipids as regulators of cellular autolytic activity is discussed.     

Isolation, and morphological and chemical properties of an autolysis-deficient mutant of Clostridium botulinum type A.

An autolysis-deficient mutant was isolated from Clostridium botulinum type A 190L by treatment with ethyl methanesulfonate. The cell wall prepared from the mutant autolyzed at much slower rate than that from the parent strain, accompanying with much less liberation of both amino terminals and reducing groups. Electron microscopic observation revealed that the mutant strain was converted to short rod or curved spherical form with thickened cell walls when the growth temperature was shifted from 37 to 45 C. The mutant had a significantly larger amount of non-peptidoglycan-carbohydrate complexes than did the parent strain and became markedly resistant to the autolysin partially purified from the parent, compared with the parent strain. Furthermore, the mutant was fairly tolerant to killing by penicillin. These results suggest that the autolysis deficiency of the mutant was due not only to the deficient production of autolysin but also to the excess accumulation of carbohydrate in the cell wall.     

Distribution of teichoic acid in the cell wall of Bacillus subtilis.

Hydrolysis of the cell wall of Bacillus subtilis 168 by autolysins or lysozyme resulted in the exposure of glucosylated teichoic acid molecules as evidenced by increased precipitation of [14C] concanavalin A. The number of concanavalin A-reactive sites increased significantly after only limited enzymatic digestion of the walls. Quantitative analyses of [14C] concanavalin A-treated wall or wall hydrolysate complexes indicate that approximately one-half of the teichoic acid molecules are surface-exposed, whereas the remainder are probably embedded within the peptidoglycan matrix. Treatment of the cell walls with sodium dodecyl sulfate or Triton X-100 did not result in new concanavalin A-reactive sites. Partial autolysis diminished the ability of the cell walls to adsorb bacteriophage phi25. Fluorescein-labeled concanavalin A bound intensely over the entire surface of growing B. subtilis 168 cells, suggesting that teichoic acid molecules are located on the total solvent-exposed surface area of the bacteria.     

Sites of Cellular Autolysis in Lactobacillus acidophilus

Ultrastructural changes which occur during cellular autolysis of Lactobacillus acidophilus strain 63AM Gasser in 0.05 M citrate buffer, pH 5.0, were examined. Early in the process, randomly distributed electron-dense patches were seen on the wall surface, along with an accompanying eversion of mesosomes. Later, after a loss of about 20% of the initial cellular turbidity, dissolution from the outside of nascent cross walls was seen. This observation was related to the normal process of cell separation. After this stage, short lengths of the cylindrical portion of the wall appeared to be completely removed in a random manner over the entire surface. This dissolution produced gaps in the wall which allowed the extrusion of membrane and cytoplasm. Although membrane was usually extruded through one major, polar, subpolar, or septal site, other secondary points of membrane extrusion were also frequently seen in the same cell section.     

Model for Cell Wall Growth of Streptococcus faecalis

In exponentially growing and dividing cells of Streptococcus faecalis, it is proposed that the leading edge of the annularly closing cross wall is the point of extension for both cross wall and peripheral wall. Peripheral wall extension is thought to be produced by the separation or splitting of the cross wall at its junction with peripheral wall. This results in the pushing of the equatorial wall bands, found on S. faecalis walls, to subsequatorial positions. These bands therefore mark the separation of old wall from new wall. Mesosomal formation was observed usually to precede cross wall initiation.     

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.     

Regulation of bacterial cell walls: correlation between autolytic activity and cell wall turnover in Staphylococcus aureus.

Cell wall turnover was examined in parent and mutant strains of Staphylococcus aureus. Peptidoglycan and teichoic acid were observed to undergo turnover in the wild-type strain during exponential growth; however, the rate of turnover did not decrease when the growth rate slowed, as the culture entered stationary phase. Isolated native cell walls and crude soluble autolytic enzyme were prepared from cells harvested during exponential and postexponential phases of growth. Native cell walls from both phases of growth autolyzed in buffer at identical rates; similarily, crude soluble enzyme from both preparations degraded radioactive cell walls at the same rate. Therefore, the activity of the autolysin in both exponential and postexponential cells was similar. The autolysis of whole cells of a mutant tar-1 was enhanced by 1.0 M NaCl. When 1.0 M NaCl was present under growing conditions, the rate of cell wall turnover was greatly increased. The presence of chloramphenicol, which inhibits whole-cell autolysis, also inhibited turnover. Analysis of the cell wall material recovered from spent medium revealed products consistent with the known mode of action of the endogenous autolysin. It is concluded that cell wall turnover in S. aureus is independent of the stage of culture growth but is dependent instead on the activity of the autolysin.     

Influence of concanavalin a on autolysis of gametes from Chlamydomonas reinhardii.

The phytohemagglutinin concanavalin A inhibited zygote formation of Chlamydomonas reinhardii. 15--50 mug lectin/ml not only interfered with the mating reaction, but also with cell wall lysis of gametes and zoospores in a crude autolysin preparation gained from copulating gametes. Further, the structure of cell walls shed into the medium after autolysis in the course of the mating reaction and after lysis "from without" in the crude autolysin preparation was stabilized by Con A. Therefore, it must be assumed that the lectin inhibited zygote formation of C. reinhardii by interfering with autolysis of the cell walls of the gametes. Though Con A inhibited the lytic processes of C. reinhardii, an activation of the autolytic system in theta gametes by the lectin was found to compete with its inhibitory reaction. Con A induced autolysis of theta gametes was dependent on adherence of the cells by their flagella to the surface of the culture vessel or the liquid medium and did not occur in cultures stirred by rotation. The interferences of Con A with the autolytic serum of C. rienhardii were inhibited by methyl-alpha-D-mannopyrano-side and to a lesser degree by glucose, indicating that the carbohydrate binding sites of the lectin were involved in its reactions with the cells.