Mechanism of Action of Penicillin: Triggering of the Pneumococcal Autolytic Enzyme by Inhibitors of Cell Wall Synthesis

Proc. Nat. Acad. Sci. USA Vol. 72, No. 10, pp. 4162-4166, October 1975 Microbiology

Mechanism of action of penicillin: Triggering of the pneumococcal autolytic enzyme by inhibitors of cell wall synthesis (release of lipoteichoic acid/autolysin) ALEXANDER TOMASZ AND SUSAN WAKS The Rockefeller University, New York, N.Y. Communicated by William Trager, July 9,1975

ABSTRACT During penicillin treatment of an autolysin in defective mutant." Most of the methods used in these in- defective mutant pneumococcus we have observed three vestigations have been described in detail in earlier publica- novel phenomena: (z) Growth of the mutant cultures is inhibited by the same concentrations of penicillin that induce tions. These include the composition of growth media and lysis in the wild type. (ii) Mutant bacteria treated with the monitoring growth by nephelometry (4), preparation of cell minimum growth inhibitory concentration of penicillin will walls (5), autolytic enzyme (6), and pneumococcal F-antigen lyse upon the addition of wild-type autolysin to the growth (7), and the assay of autolysin activity (8), "Loading" of the medium. Chloramphenicol and other inhibitors of protein mutant cells with wild-type autolysin was performed in the synthesis protect the cells against lysis by exogenous enzyme. following manner: Sensitivity of the cells to exogenous autolysin requires treatment with penicillin or other inhibitors of cell wall synthesis Fifty microliters of highly purified autolysin (containing (e.g., D-cycloserine or fosfonomycin) since exogenous autolys- 3.5 jg of protein) was added to 1 ml of autolysin defective in alone has no effect on bacterial growth. (ii) Treatment pneumococci (2 X 107 to 2 X 108 viable units/ml) and incu- with penicillin (or other inhibitors of cell wal synthesis) bated at 370 for 30 min. The cells were next filtered (Milli- causes the escape into the medium of a choline-containing pore, 0.45 Mm), washed-on the filter-three times with 2 macromolecule that has properties suggesting that it contains ml of warm (370) growth medium, and resuspended in pneumococcal lipoteichoic acid (Forssman antigen). Each one of these three phenomena (growth inhibition, sensitiza- growth medium. No significant amounts of enzyme seem to tion to exogenous autolysin, and leakage of lipoteichoic acid) escape to the medium during subsequent exponential shows the same dose response as that of the penicillin-in- growth of the enzyme-loaded bacteria. Detailed studies of duced lysis of wild-type pneumococci. this system will be described elsewhere*. On the basis of these findings we propose a new hypothe- All chemicals were of reagent grade. sis for the mechanism of penicillin-induced lysis of bacteria. It is suggested that inhibition of cell wall synthesis by any means triggers bacterial autolytic enzymes by destabilizing RESULTS the endogenous complex of an autolysin inhibitor (lipo Effects of penicillin on autolysin defective teichoic acid) and autolytic enzyme. Escape of lipoteichoic pneumococci acid-like material to the growth medium is a consequence of this labilization. Chloramphenicol protects bacteria against The use of a mutant pneumococcus defective in the major penicillin-induced lysis by interfering with the activity of the cellular autolytic enzyme (an N-acetyl-muramyl-L-alanine autolytic enzyme, rather than by depleting the concentration amidase) has allowed us to identify in the drug-treated bac- of the enzyme at the cell surface. teria three penicillin-induced effects that appear to be of general significance for the mode of action of bacteriolytic The rapid loss of viability and cellular lysis of pneumococci antibiotics. treated with penicillin and other cell wall inhibitory bacteri- Bacteriostatic Effect. Unlike the wild-type cells, which cidal drugs is known to require the activity of the pneumo- are lysed by penicillin, growing cultures of the mutant bac- coccal autolytic enzyme (an N-acetylmuramyl-L-alanine teria respond to the addition of penicillin by inhibition of amidase) (1, 2). However, the mechanism by which inhibi- growth (1). This bacteriostatic response has exactly the same tion of wall synthesis is coupled to the "suicidal" activity of drug concentration dependence as the lytic response of wild- this enzyme is not known. In this communication we report type cells (Fig. 1). observations that bear on this question and we propose a Sensitization to Externally Added Autolytic Enzyme. new hypothesis for the mechanism by which penicillin and Addition of wild-type autolysin preparation to the growth other lytic antibiotics cause destruction of the bacterial cell. medium of penicillin-inhibited mutant bacteria makes these cells lyse (Fig. 2). Several details of this observation are im- MATERIALS AND METHODS portant. (i) Penicillin treatment is essential for the sensitiza- An autolysin defective mutant derivative of the R36A strain tion of the cells to the enzyme, since autolysin without anti- of Diplococcus pneumoniae (3) was used as the source of biotic has no effect on the bacterial growth. (ii) Fig. 2 shows DNA with which the autolysin defect was introduced into that the doses of penicillin treatment needed for sensitiza- competent cells of the parent (wild-type) strain via genetic tion are the same as the doses required for inhibition of transformation. A culture of the transformant was kindly growth (in the mutant) or for lysis (in the wild type). (ii) given to us by Dr. S. Lacks (Brookhaven National Laborato- Autolysin and penicillin need not be present at the same ry), and cultures of these cells will be referred to as "autolys- * A. Tomasz and S. Waks, Biochem. Biophys. Res. Commun. Abbreviations: CAP, chloramphenicol; LTA, lipoteichoic acid. (1975), in press. 4162 Downloaded by guest on September 28, 2021 Microbiology: Tomasz and Waks Proc. Nat, Avad. Sci. USA 72 (1975) 4163

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10 1 2 3 4 5 HOURS FIG. 1. Bacteriostatic effect of penicillin in an autolysin defec- 400 tive pneumococcus. Exponentially growing cultures received peni- MINTES cillin G at the concentrations indicated; growth was monitored by 2. of nephelometry (Coleman FIG. Sensitlzution autolysin defective pneumococci to ex- Nephelometer). Numbers indicate the ogenous wild-type autolysin. Exponentially growing cultures re- concentrations of penicillin (in U/ml). ceived penicillin at the 60th min of growth and autolysin (109 cell equivalents per ml of medium) at the times lndicated by the ar- time. The bacteria could be sensitized to the enzyme by the rows. Growth was followed by nephelometry. Numbers indicate following sequence of treatments: the penicillin concentrationst 0.1 (1), 0.05 (2), 0.04 (3), 0.03 (4), exposure to penicillin (0.1 and 0.02 (5) U/ml. Tube 6 received no drug; tube 7 received only unit/ml for 60 min), treatment with penicillinase (Neutra- autolysin. pen, Rikers Labs; 10 units/ml for 5 min) followed by the addition of autolysin to the medium. Such bacteria lyse (Fig. parallel with the addition of penicillin prevents lysis. Lysis is 3). (iv) Sensitization is transient: after removal of penicillin also prevented by the addition of pneumococcal lipoteichoic (by penicillinase) bacteria recover their normal autolysin re- acid (Forssman antigen) to the medium (Fig. 4), sistance in parallel with the resumption of growth (see Fig. 3). (v) Chloramphenicol (CAP; 100,gg/ml) can block sensitization by penicillin if added in the following order: first CAP, 5 min later penicillin. (vi) Penicillin as a sensitizing 1000 7 agent can be replaced by other drugs, such as: vancomycin (50 jig/ml), cycloserine (50 jig/ml) (1), /3-chloro-D-alanine 2 (500 jig/ml), fosfonomycin (50 ug/ml) (1), cephalothin (1 tg/ml), and oxacyllin (1 ,ug/ml) (2, *). The common feature of these treatments is that they all interfere with cell wall biosynthesis. 3 Mechanism of sensitization to autolysin It appeared possible that the mechanism of sensitization in- volves some nonspecific "damage" to the cell surface (caused by the inhibitor) which is needed to allow the at- tachment of extracellular enzyme molecules to the bacterial surface. However, this does not seem to be the case, since, by appropriate techniques, the autolysin molecules can be "ad- sorbed" into the surface of live, mutant bacteria prior to the treatment of the cells with the sensitizing agents. The autolysin, experimentally introduced into the cells, is retained in cell-bound form through several cell generations. 10 1 (The success of this "enzyme replacement therapy" is O 1 2 3 4 5 6 clearly indicated by the fact that these reconstituted mutant HOURS cells "revert" immediately to the wild phenotype in proper- FIG. 3. Sequential treatment of autolysin defective pneumo- ties that are associated with the in vivo activity of the auto- cocci with penicillin and autolysin. An exponentially growing cul- lytic amidase. ture was treated~~~~~~~~~~4with penicillin (0.1 U/ml, added at the time indi- Details of this system will be described elsewhere*.) Addition of penicillin (either at the minimum in- cated by the solid arrow) for 60 min. After this treatment, the cul- hibitory concentration, i.e., ture was divided into three portions: one was left to incubate with at 0.02 unit/ml, or at higher penicillin (3), the other two (2 and 4) received penicillinase (10 concentrations) to such "enzyme-loaded" bacteria (either units/ml for 5 min; time of addition indicated by solid arrow). Cul- immediately after enzyme adsorption or even after growth ture 4 received autolysin (109 cell equivalent units/ml of medium) for one generation) results in cellular lysis with a kinetics immediately after the penicillinase addition, while culture 2 was comparable to that of the wild type. Other bacteriolytic an- allowed to incubate for about 2.5 hr before addition of autolysin. tibiotics (oxacyllin, 1 gg/ml; cephalothin, 1 sg/ml; vancom- (Addition of autolysin is indicated by the winged arrows.) A con- ycin, 50 trol culture (1) received no additions at all. Growth (and lysis) gg/ml; D-cycloserine, 50 gg/ml) cause lysis, where- were monitored by following the turbidity of the cultures (OD660) as inhibition of protein and/or ribonucleic acid synthesis with a Zeiss spectrophotometer. Downloaded by guest on September 28, 2021 4164 Microbiology: Tomasz and Waks Proc. Nat. Acad. Sci. USA 72 (1975)

Table 1. Release of a choline-containing macromolecular material from penicillin-treated autolysin defective pneumococci Total radioactivity (cpm/ml) % Released Penicillin (U/ml) +CAP -CAP +CAP -CAP Exp. 1 None 24,320 9 0.03 23,320 21.6 0.05 22,170 18.6 0.07 21,300 17.1 0.1 22,490 - 18.1 Exp. 2 None 21,260 24,320 3.6 6.8 0.03 18,870 22,460 23.9 30.5 0.1 15,040 19,550 18.1 37.6 Bacteria were labeled by growth in [3H]choline-containing medium as described in the legend to Fig. 4. The cells were distributed into tubes containing various concentrations of penicillin with or without simultaneously added chloramphenicol (100 jg/ml). In Exp. 1, the prelabeled bacteria were allowed to grow for 60 min in isotope-free medium before the addition of drugs; in Exp. 2, this period was shorter, about 30 min. The cells were removed from the medium by centrifugation (10,000 x g, 10 min), and 100-pl portions of the supernatant were pipetted into 1 ml of cold, 10% trichloroacetic acid solution. The precipitates were collected onto glass fiber filter (GFA) and dried in an oven (1000, 10 min). Radio- activity was determined using a toluene base scintillation cocktail. Total, acid-precipitable radioactivity was determined by pipetting 100-ul portions of whole bacterial suspensions into trichloroacetic acid and measuring radioactivity in the precipitates, as described above.

penicillin-induced escape of a radioactive choline-labeled macromolecular component to the growth medium. Low concentrations of penicillin (near the minimal inhibitory concentration) were sufficient to induce release. It should be emphasized that this release occurs without added autolysin, i.e., in the complete absence of cellular disintegration. Fig. 5 shows the sedimentation of this component during centrifugation in sucrose density gradients. The shift in the apparent molecular size (disaggregation) of the material in the deter- gent (sodium dodecyl sulfate)-containing gradient is typical of the behavior of the pneumococcal lipoteichoic acid (LTA) (7). The autolysin inhibitory activity of the penicillin-treated FIG. 4. Protection of "autolysin-loaded" mutant pneumococci against penicillin induced lysis by chloramphenicol and lipo- culture filtrate is shown in Table 2. It can be seen that the teichoic acid. Autolysin defective pneumococci were "loaded" with inhibitory factor is nondialyzable, it is resistant to heating at wild-type autolysin (see Materials and Methods). After resuspen- 1000 for 30 min, and is sensitive to periodate. Deoxycholate sion in growth medium the bacteria were distributed into three added together with the filtrate has reversed the inhibition test tubes, one of which received chloramphenicol (100 ug/ml; tube of autolysin activity. A); pneumococcal lipoteichoic acid (200 pg/ml) was added to the Another observation suggesting the escape of LTA into second tube (B), the third tube received no additions (control, not shown C). After 30 min of incubation at 370, penicillin G (0.1 the growth medium is illustrated in Table 3, which shows U/ml) was added to each of the three cultures and incubation con- that after exposure of the cells to a relatively small dose of tinued for 3 hr. At this time samples were examined by phase con- penicillin (0.1 U/ml for 1.5 hr) the bacteria seem to have lost trast microscopy (Zeiss R.A. microscope equipped with 100/1.25 the powerful autolysin inhibitory activity that can be nor- planachromat oil immersion lens). Photographs were made on mally found in the lipoteichoic acid fraction of pneumococci Panatomic X film. Upper photo, tube A; lower photo, tube B. Con- (9). In fact, some stimulatory activity may be present. The trol showed only amorphous debris (not documented). nature of this activity is presently being investigated. Inhibition of DNA synthesis under similar conditions (by Two other inhibitors of cell wall synthesis, D-cycloserine mitomycin C, 5-fluorodeoxyuridine, or 5-hydroxyphenyla- (50 gsg/ml) and fosfonomycin (50 jug/ml), also caused re- zauracil) had no effect (inhibitory or stimulatory) on the lease of choline-labeled material. On the other hand, inhibi- penicillin-induced lysis*. tion of protein synthesis (by chloramphenicol) has substan- Penicillin-Induced "Leakage" of a Lipoteichoic Acid- tially reduced the penicillin induced release of LTA-like Like Substance from the Cells. Table 1 demonstrates the material (Table 1). Downloaded by guest on September 28, 2021 Microbiology: Tomasz and Waks Proc. Nat. Acad. Sci. USA 72 (1975) 4165 13,000

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FRACTION NUMBER FIG. 5. Sedimentation behavior of the choline-labeled macromolecular material leaked into the growth medium during penicillin treatment. A culture of the autolysin defective pneumococci was labeled with radioactive choline ([methyl-3H]choline; 0.1 ,Ci and 5 Mg/ml) by three generations of growth in 10 ml of isotope-containing medium. The cells were washed free of extracellular radioactivity (Millipore filtra- tion), resuspended in isotope-free growth medium and (after 30 min of growth at 370) penicillin was added to the culture (0.1 U/ml). After further incubation for 1.5 hr the cells were removed by centrifugation (10,000 X g, 15 min) and the supernatant medium was collected, dialyzed (overnight) against water, and concentrated to a small volume (0.5 ml) by embedding the dialysis bag in Aquacide II. Portions (100 Ml) of the solution were layered on sucrose gradients [5-25% sucrose, in 0.15 M NaCl solution with (--- -) and without (-) 2% sodium dodecyl sulfate], and centrifuged at 35,000 rpm at 180 for 16 hr in a Spinco model L3-50 ultracentrifuge. A swinging bucket (SW 50.1) rotor and po- lyallomer tubes were used. The gradients were analyzed for radioactivity; 8-drop fractions were collected through a pinhole pierced through the bottom of the tubes, and the radioactivity was measured using the "Readysolv" scintillation liquid (Beckman) and a Mark II scintillation spectrometer (Nuclear Chicago Corp.). Table 2. Appearance of autolysin inhibitory activity DISCUSSION in the culture filtrate of penicillin-treated pneumococci Suppression of the endogenous autolytic activity greatly re- Amount of cell wall duced the rate with which pneumococci lose their viability material solubilized during penicillin treatment and virtually abolished cellular in 10 min lysis by this antibiotic (1, 2). These findings indicate that the (% in control) pneumococcal autolysin-an N-acetylmuramyl-L-alanine amidase-plays an essential role in the penicillin-induced Autolysin (control) 100 disintegration of these bacteria. Autolysin + culture filtrate 43 However, it is not clear how the inhibition of bacterial cell Autolysin + culture filtrate wall synthesis by penicillin brings about the deranged activi- + deoxycholate (0.1%) 95 ty of the autolytic enzyme. The observations described in Autolysin + periodate-treated this paper suggest a mechanism by which the primary (in- culture filtrate 90 hibitory) activity of penicillin may lead to the autolytic de- A culture of exponentially growing wild-type pneumococci (100 ml; 2.5 x 107 cells per ml) received penicillin (0.1 unit/ml). After Table 3. Loss of autolysin inhibitory activity from the 1.5 hr of incubation, both cultures were harvested (centrifugation Forssman antigen (lipoteichoic acid) fraction of at 10,000 x g, 20 min). The supernatant fluid was dialyzed against several changes of distilled water and lyophilized to dryness; the pneumococci treated with penicillin residue was dissolved in water (500 d). Before testing for autolysin inhibitory activity the solution was heated at 1000 for 30 min to in- Amount of cell wall material activate possible residual autolysin. A 100-Al portion of the solution solubilized in 10 min was treated with periodate (0.025 M paraperiodic acid in 0.05 M (cpm/ml and % of control) sodium acetate buffer, pH 4.8) at 00 in the dark for 24 hr. Excess reagent was destroyed by the addition of 10 Ml of glycerol (0.1 M) Autolysin (control) 1039 (100) and incubation at 370 for 60 min. Autolysin + cell extract .362 (35) The inhibitory activity was tested in the following manner: 5-Ml Autolysin + penicillin- portions of the filtrate were added to the following reaction mix- treated cell extract 1912 (184) ture: 200 Al of buffer (Tris-maleate, 0.05 M, pH 6.9), 10 ,l of [3H]- choline-labeled cell wall suspension (containing 200 Mg dry weight Two cultures of wild-type pneumococci (100 ml each) were used and 108 cpm/ml), and 10 Al of autolysin (109 cell equivalents per for the preparation of F antigen (7). One of the cultures was treated ml). The enzyme reaction was allowed to run at 370 for 10 min. with penicillin (0.1 unit/ml) for 1.5 hr before the preparation. The Each reaction tube received 25 Ml of formaldehyde (38% solution) F antigen fraction of each preparation was dissolved in 0.5 ml of and 25 Ml of carrier bovine serum albumin (4% solution), and was water. This fraction contained 1.3 x 1010 cell equivalents of mate- centrifuged at 10,000 x g for 15 min in an Eppendorff centrifuge. rial in the case of the penicillin-treated culture and 6.9 X 109 cell Portions (150 Ml) of the supernatant solutions were assayed for the equivalents in the control. Portions (100 Ml) of the extracts were determination of solubilized radioactivity as described in the tested for their effect on the activity of the autolysin by the method legend to Table 1. described in the legend to Table 2. Downloaded by guest on September 28, 2021 4166 Microbiology: Tomasz and Waks Proc. Nat. Acad. Sci. USA 72 (1975) struction of the cell. We propose that the key event in this surface, is incorrect.,The experiments described here and in mechanism is the labilization or inactivation of an endoge- an accompanying communication* clearly show that inhibi- nous autolysin inhibitor that (under normal conditions) tion of protein synthesis interferes with the in situ activity keeps the activity of this enzyme suppressed. The existence of the autolysin. Inhibition of the activity of the streptococ- of a powerful and specific autolysin inhibitor in pneumococcal muramidase by chloramphenicol has been suggested ear- ci has recently been demonstrated and identified as the lier by several lines of evidence (15). Our results fully con- complex, choline-containing LTA (Forssman antigen) of this firm this suggestion. The mechanism by which CAP inter- bacterium (9). The results summarized in Table 2 indicate feres with autolysin activity is not known at present. How- that this inhibitory activity is not detectable in penicillin- ever, the substantial reduction by CAP of the penicillin-in- treated cells. The escape of choline-containing macromole- duced LTA-1 release hints that inhibitors of protein synthe- cules (resembling the Forssman antigen) from penicillin- sis may inhibit in situ autolytic activity via modulation of treated pneumococci may represent the release of some the metabolism of LTA. form of the LTA into the medium. Similar leakage can be The three penicillin-induced phenomena described in this observed in both autolysin defective (mutant) and the wild- paper (growth inhibition, sensitization to exogenous autolys- type cells (undocumented). We suggest, therefore, that the in, and leakage of LTA-like material) occur even at the low- sensitization of autolysin defective pneumococci to exog- est dose of drug treatment that causes lysis in the wild-type enous enzyme and the triggering of autolysin activity in the cells. Furthermore, these phenomena are all transient, since wild-type cells are analogous phenomena: they are both the upon addition of penicillinase the penicillin-treated mutant consequences of the removal of an amidase inhibitor. cultures can resume growth (Fig. 3). These facts indicate It should be emphasized that the exact chemical nature of that penicillin inhibits the same enzymatic target reaction in the choline-containing material released to the medium is both the wild type and in the autolysin defective cells and not known at present, and we shall refer to it in this discus- that this inhibition itself is not sufficient to cause cellular sion as "lipoteichoic acid-like material" (LTA-1 for brevity's disintegration. The irreversible effects of penicillin (lysis- sake). and, to some extent, loss of viability) seem to be the conse- How penicillin induces the release of LTA-1 material is quences of the triggering of the activity of autolytic ami- not clear at the moment. It is important to note, however, dase. that all cell wall inhibitors tested-irrespective of their exact It will be important to test the validity of these suggestions site of action-cause release of LTA-1. Since inhibition of in other species of bacteria. it is possible that a better under- even the earliest step in cell wall synthesis (by fosfonomycin) standing of the in vio controls of autolytic enzymes may causes LTA-1 release, it seems unlikely that biosynthetic in- yield ways to improve the chemotherapeutic efficacy of termediates of the Park-peptide type are mediators in this bacteriolytic antibiotics. effect. Possibly, the interruption of the flow of biosynthetic This investigation has been supported by a grant from the Na- precursors might lead to the accumulation of Glycosyl Car- tional Institutes of Health. rier Lipid (10) and/or the lipoteichoic acid-like carrier described by Glaser and his associates (11). Such carrier, "un- 1. Tomasz, A., Albino, A. & Zanati, E. (1970) Nature 227, 138- charged" with the biosynthetic building blocks of cell wall, 171. may play a regulatory role in the labilization of the inhibi- 2. Tomasz, A. (1974) Ann. N.Y. Acad. Sd. 235,439-447. tor-autolysin complex. This possibility is presently being 3. Lacks, S. (1970) J. Bacteriol. 101, 373483. tested. Alternatively, autolysin triggering by cell wall inhibi- 4. Tomasz, A. (1970) J. Bacteriol. 101, 860-871. tors may have a more complex mechanism. Whatever the 5. Mosser, J. L. & Tomasz, A. (1970) J. Biol. Chem. 245, 287- mechanism of this effect is, it seems clear that the site at 298. 6. Tomasz, A. & Westphal, M. (1971) Proc. Nat. Acad. Sci. USA which a particular inhibitor interrupts cell wall synthesis is 68,2627-2630. relatively unimportant from the point of view of the eventu- 7. Briles, E. B. & Tomasz, A. (1973) J. Btol. Chem. 248, 6394- al lysis of the cells. 6397. The experiments described in this paper also suggest a 8. Holtje, J. V. & Tomasz, A. (1974) J. Biol. Chem. 249, 7032- new interpretation for the role of protein synthesis in peni- 7034. cillin-induced lysis (12-14). The penicillin-induced autolysin 9. Holtie, J. V. & Tomasz, A. (1975) Proc. Nat. Acad. Sd. USA sensitivity of enzyme-defective pneumococci is blocked by 72,1690-1694. chloramphenicol in spite of the ample supply of exogenous 10. Anderson, J. S., Matsuhashi, M., Haskin, M. & Strominger. J. autolytic enzyme in the medium. Furthermore, enzyme- L. (1965) Proc. Nat. Acad. Sci. USA 54,587-593. 11. Fiedler, F. & Glaser, L. (1974) J. Biol. Chem. 249,2684-2689. loaded mutant pneumococci (i.e., mutant cells into which 12. Jarvetz, E., Gunnison, J. B., Speck, R. C. & Coleman, V. R. the missing autolysin was reintroduced experimentally) are (1951) Arch. Int. Med. 87,349-359. protected against penicillin lysis by inhibition of protein syn- 13. Prestidge, L. S. & Pardee, A. (1957) J. Bacterlol. 74,48-59. thesis*. These results indicate that the currently widespread 14. Rogers, H. J. (1967) Nature (London) 213,31-3. belief, namely, that chloramphenicol protects bacteria from 15. Shockman, G. D., Daneo-Moore, L. & Higgins, M. L. (1974) lysis by lowering the concentration of autolysin at the cell Ann. N.Y. Acad. Sd. 235,161-195. Downloaded by guest on September 28, 2021