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.     

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.     

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 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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Modification of autolysis by synthetic peptides derived from the presumptive binding domain of Staphylococcus aureus autolysin

The autolytic cell wall hydrolase of Staphylococcus aureus, Atl, contains three highly cationic repeats in the central region of the amino acid sequence, and the repeats are presumed to have the role of binding the enzyme to some components on the cell surface. To explain the possible function of the repeats, we synthesized a number of 10- to 30-mer oligopeptides based on the Atl amino acid sequence (Thr432-Lys610) containing repeat 1, and examined their effects on the autolysis of S. aureus cells. When the peptides were added to a cell suspension of S. aureus under low ionic strength conditions, five peptides, A10, A11, A14, A16 and B9, showed immediate increases in optical density (OD) of the cell suspension accompanied by decreases in viable cell counts. After the immediate increases, the ODs for A10 and A14 changed little in the first 2 hr. In contrast, the ODs for A11 and A16 decreased rapidly. When peptide A10 was added to suspensions of heat-killed whole cells, crude cell walls and a crude peptidoglycan preparation, their ODs were increased approximately 2-fold. In contrast, the OD was not increased when the peptide was added to a suspension of pure peptidoglycan from which anionic polymers had been removed. Light microscopic and transmission electron microscopic study showed that A10 and A14 inhibited autolysis and that A11 and A16 induced autolysis earlier than the control. These results suggest strongly that the peptides adsorb to and precipitate on the anionic cell surface polymers such as teichoic acid and lipoteichoic acid via ionic interaction. The effects of peptides on the autolysis may be the results of the modification of S. aureus autolysin activities. These peptides, especially the 10-mer peptide B9 (PGTKLYTVPW) that represents the C-terminal half of A10 and N-terminal half of A11, may be important segments for Atl to bind to the cell surface.     

Rapid Methods for Extracting Autolysins from Bacillus subtilis

Two procedures are described for the extraction of autolysins from whole cells. One method uses 5 M LiCl at 4 C. The amount of enzyme obtained by this method is six times more than that obtained by autolysis of cell walls and fourteen times more than that obtained by extracting cell walls with LiCl. With the other method, cells are extracted with 2% Triton X-100. This is less efficient than the LiCl method but yields about one-half the amount of enzyme obtained by cell wall autolysis and about the same amount as obtained by extracting cell walls with salt. Both procedures yield autolysin with multiple pH optima. Autolysins can be extracted from several bacterial species by either the LiCl or the detergent method. The data suggest that these techniques have sufficient sensitivity to detect small differences in autolytic activity among mutants and various organisms and are also suitable for large-scale isolation of autolysin for purification and characterization studies.     

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.     

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.     

Site of Initiation of Cellular Autolysis in Streptococcus faecalis as Seen by Electron Microscopy

Low concentrations of glutaraldehyde (0.1% or higher) blocked cellular and wall autolysis. The site of autolytic activity was studied by allowing cell autolysis to proceed for very short periods (0 to 15 min) before addition of glutaraldehyde. Electron microscopy of ultrathin sections showed that the primary site of autolytic activity was the leading edge of the nascent cross wall. The base of the cross wall seemed more resistant than the tip. Evidence supporting the involvement of autolysin activity in continued wall extension and in cell separation as well as in the initiation of new sites of wall extension was obtained. In cells exposed for 10 min to chloramphenicol, wall dissolution was very much slower but occurred at the same cross wall site.     

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.     

Analysis of autolysins in temperature-sensitive morphological mutants of Bacillus subtilis.

The content and distribution of autolysin were measured in temperature-sensitive morphological mutants of Bacillus subtilis. Strains RUB1000 and RUB1012 grew as rods at 30 C. At 45 C the mutants contained disproportionately less teichoic acid than peptidoglycan and grew as irregular spheres. The amount of enzyme that could be extracted from rods was at least 31 times the amount extracted from spheres. The rate of autolysis of cell walls was 7- to 28-fold greater in rods than in spheres. The low activity found associated with the cell walls of spheres was not compensated for by larger amounts of autolytic activity in the cytoplasm. No activity was found in the growth medium at either temperature. The failure of the mutant cells to autolyze was due to low amidase activity and relatively resistant cell walls. Revertants of RUB1012 were isolated that had 13, 23, and 55% of the normal proportions of teichoic acid when grown at the nonpermissive temperature. Cell walls from the revertants were as sensitive to added amidase as the wild-type strain. None of the revertant strains regained the wild-type ability to produce more amidase at 45 C. However, the deficiency in autolysin observed with RUB1012 was partially restored in revertants containing higher proportions of teichoic acid.     

Specific recognition of choline residues in the cell wall teichoic acid by the N-acetylmuramyl-L-alanine amidase of Pneumococcus.

Pneumococci growing on choline-containing medium are known to incorporate this amino alcohol into the wall teichoic acid and produce autolysin-sensitive cell walls. In contrast, bacteria grown on the choline analogue, ethanolamine, incorporate ethanolamine into the teichoic acid and synthesize cell walls that are resistant to the homologous autolysin. In this communication, we report experiments aimed at understanding the biochemical mechanism of this phenomenon. Ethanolamine-containing (autolysin-resistant) cell walls were methylated in vitro with methyl iodide. Under appropriate conditions, virtually all of the ethanolamine residues could be converted to choline. After methylation, the formerly autolysin-resistant walls could be quantitatively hydrolyzed by the pneumococcal autolysin. Methylated walls also recovered another property typical of cell walls isolated from choline-grown bacteria: they could induce the in vitro "conversion" of an inactive form of autolysin to the catalytically active form (Tomasz, A., and Westphal, M. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 2627-2630). The results suggest that the autolysin-catalyzed hydrolysis of amide bonds in the peptidoglycan requires an additional interaction between the enzyme protein and choline residues in the teichoric acid portion of the cell wall.     

A mutant of Streptococcus pneumoniae that exhibits thermosensitive penicillin tolerance and the paradoxical effect

Mutants of Streptococcus pneumoniae that contain active autolysin and yet cannot be induced to lyse during treatment with penicillin (Lyt+Tol+ mutants) have been described. We have now shown that these mutants are temperature dependent (32 degrees C); at 37 degrees C these bacteria underwent penicillin-induced lysis. In addition, mutants at the lysis-permissive temperature showed the so-called 'paradoxical response' to penicillin. Temperature shift experiments indicated that the change from tolerant to lytic response or vice versa is a fast process. No differences were detected in autolysin specific activity or in the kinetics of inhibition of protein, peptidoglycan and teichoic acid syntheses in cells treated with penicillin at 32 and 37 degrees C. The results of genetic crosses indicated that the thermosensitivity of penicillin-induced autolysis in the Lyt+Tol+ mutants is not a property of the autolytic enzyme itself. The observations suggest that the thermosensitive process in the mutants represents either a step(s) in autolysin regulation or involves some difference in the structure of the cell walls produced at 32 degrees C versus 37 degrees C.