Isolation Characterization Mutant ofStaphylococcus Deficient in ... · AmutantofStaphylococcusaureus H(RUS3)wasisolatedaftermutagenesis withN-methyl-N'-nitro-N-nitrosoguanidine....

JOURNAL o0 BACTERIOLOGY, Mar. 1976, p. 961-967Copyright C 1976 American Society for Microbiology

Vol. 125, No. 3Printed in U.SA.

Isolation and Characterization of a Mutant of Staphylococcusaureus Deficient in Autolytic Activity

A. N. CHA¶TERJEE,' W. WONG, F. E. YOUNG,* AND R. W. GILPINDepartment ofMicrobiology, University ofRochester School ofMedicine and Dentistry, Rochester, New York

14642,* and Department of Microbiology, Medical College of Pennsylvania, Philadelphia,Pennsylvania 19129

Received for publication 4 September 1975

A mutant ofStaphylococcus aureus H (RUS3) was isolated after mutagenesiswith N-methyl-N'-nitro-N-nitrosoguanidine. The rate of autolysis of whole cellsand isolated cell walls of RUS3 was less than 10% of the parent strain. In addi-tion, the ability of the crude soluble enzyme isolated from RUS3 to degrade cellwalls was negligible compared with the parent strain. The cell wall compositionand the generation time of RUS3 were comparable to the parent strain. UnlikeS. aureus H, RUS3 grew in clumps and did not undergo cell wall turnover. Bothstrains exhibited identical kinetics of killing by penicillin G. This may indicatethat autolytic enzymes play a role in cell wall turnover and cell separation, butin S. aureus most of the autolytic activity is unrelated to the lethal effect of cellwall antibiotics.

Autolytic enzymes that degrade bacterial cellwalls have been the subject of intensive studyover the past decade and their mechanism ofaction has been well documented (9, 13). Inmarked contrast, the metabolic role of theseubiquitous enzymes is not clear. In one ap-proach to this problem, investigators havestudied mutant strains with altered autolyticactivity. Unfortunately in the majority of suchstudies the observed differences in the lyticbehavior were associated with changes in cellwall composition (7, 8, 16, 22), and not pri-marily due to a modification of the autolyticenzyme(s). Since Staphylococcus aureus H hasa well-characterized autolytic enzyme systemin which the N-acyl-muramyl-L-alanine ami-dase (amidase) activity predominates (20), wehave chosen this model system for our investi-gation. In the present article we describe theisolation and properties of a Lyt- mutant de-rived from S. aureus H. Evidence is presentedthat the observed phenotypic differences aredue to its defective autolytic enzyme and notrelated to any alteration of its substrate.

(This paper was presented in part at the 75thAnnual Meeting of the American Society forMicrobiology, New York, N.Y., 27 April to 2May 1975.)

MATERIALS AND METHODSStrains and growth conditions. General condi-

tions for maintenance, isolation, and characteriza-tion of the parental strain S. aureus H (Str) have

I Present address: Division of Biology, Birla Instituteof Technology and Science, Pilani, Rajsthan, India.

been described (1). All cultures were grown in phytone-yeast extract-K2HPO4 (PYK) broth containing0.2% glucose on a gyratory shaker at 37 C (12).

Selection procedure for Lyt- mutants. Nutrientagar plates containing either commercially availa-ble (Calbiochem) Micrococcus lysodeikticus or heat-killed S. aureus cells (1 mg/ml) (lyophilized) werespread with 100 to 200 colony-forming units (CFU)and incubated at 37 C for 24 h. They were thenplaced at 4 C and inspected every 24 h. With S.aureus H, a distinct zone of clearing appearedaround the colonies on M. lysodeikticus plateswithin 24 h at 4 C and increased with continuedincubation in the cold. The zone of clearing was lesspronounced on S. aureus plates but could be clearlyseen after 48 h of incubation at 4 C. Colonies thatfailed to exhibit any zone of clearing after 4 days ofincubation at 4 C (on both M. lysodeikticus and S.aureus plates) were selected for further study.

Preparation of labeled substrate. Peptidoglycanlabeled with _[3-H]lysine was prepared from cellsincubated in a defined medium in the presence ofpenicillin G (5 p.g/ml) as described previously (21).The peptidoglycan residue was repeatedly washedwith water and lyophilized. When needed, the mate-rial was suspended in 10 mM phosphate buffer (pH7.20), sonicated briefly to disperse clumps, and ad-justed to a concentration of about 8 mg/ml (about 3.3x 106 counts/min per ml).Preparation and assay of soluble autolytic en-

zyme. The soluble autolytic enzyme present in thecytoplasm was isolated by differential centrifuga-tion after mechanical disintegration of cells (21).The 100,000 x g supernatant was dialyzed overnightat 4 C against phosphate buffer and stored at -20 C.Enzyme preparations were thawed only once andused within a week of isolation.

Activity of the soluble autolytic enzyme wasmeasured by its ability to solubilize radioactivity

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962 CHATTERJEE ET AL.

from labeled peptidoglycan (21). Twenty microlitersof L_3H]lysine-labeled peptidoglycan substrate (8mg/ml) was mixed with crude enzyme and 10 mMphosphate buffer (pH 7.2) (final concentration) andincubated at 37 C. Final volume was usually 200 l.Reaction was stopped by adding 1 ml of distilledwater followed by a transfer to a boiling-water bathfor 2 min. The mixture was filtered (0.45-,Am HAWPmembrane filter, Millipore) and 0.5 ml ofthe filtratewas assayed for radioactivity. A control containingheat-inactivated enzyme was run with every experi-ment.

Assay of cell wall turnover. Cell wall turnover, asindicated by the decrease in the amount of radioac-tivity associated with the cell wall peptidoglycanand teichoic acid was determined as previously de-scribed (24). For turnover studies, cells were labeledwithN-[P4C]acetylglucosamine (GlcNAc). Specificityof GlcNAc in labeling the two cell wall polymers ofS. aureus has been demonstrated previously (24).

Other methods. Methods for the preparation andanalysis of cell walls, isolation of native cell walls,and the methods (both turbidimetric and radioac-tive) for following the rate of lysis of whole cells andcell walls have been all described previously (3, 10,11). All determinations of CFU were done on platesof nutrient agar. The mutant cell suspension wasroutinely sonicated for 1 min (Biosonik-111; powersetting, 20) before dilution and plating. Effective-ness of this level of sonication in the selective disag-gregation of clumped cells of S. aureus has beendemonstrated (2). Phosphate was determined by themethod of Chen et al. (4), and protein was measuredby the method of Lowry et al. (14). All radioactivitywas measured in a Beckman LS 230 scintillationcounter using a toluene-based scintillation fluidcontaining Triton X-100 (15). Counting efficiencywas 78% for 14C and 28% for 3H.

RESULTSIsolation and autolytic characteristics of

Lyt- mutant. S. aureus H (Str) cells suspendedin buffer were treated with N-methyl-N'-nitro-N-nitrosoguanidine (1). About 80%o of the cellswere killed by the mutagen. Survivors werediluted into fresh PYK broth, and after 2 h at37 C aliquots were diluted and spread on M.lysodeikticus plates. Colonies that failed toshow any zone of clearing (about 1 in 6 x 103)were further tested on S. aureus plates. Of the16 colonies selected on the M. lysodeikticusplates, 7 failed to exhibit any clearing on the S.aureus plates. These 7 colonies were cloned andgrown in PYK broth, and their autolytic prop-erties were compared with the wild type. Ofthese seven isolates, one strain designatedRUS3 appeared to be the most promising andwas selected for further study.We first compared the rate of whole-cell lysis

of S. aureus H with RUS3. In a typical experi-ment, the turbidity of S. aureus H cells (at 585nm) suspended at 37 C in 10 mM phosphate

buffer (pH 7.20) decreased about 50%o during a6-h period. The decrease in turbidity of RUS3was less than 5% under comparable conditions.When isolated cell walls of the two strains wereallowed to lyse by endogenous autolytic activ-ity, similar results were obtained (Fig. 1). Theoptical data were confirmed by incubating na-tive cell walls labeled with GlcNAc at 37 C.Over a period of24 h at 37 C, less than 6% oftheradioactivity present in cell walls of RUS3 wassolubilized, whereas with S. aureus H cell wallsprepared and incubated under identical condi-tions the value was about 80%.The autolytic rate of RUS3 (both whole cells

and isolated cell walls) could not be stimulatedby changes in pH or ionic strength of the incu-bation medium. Also, growth in the presence of1.0 M NaCl, which has been shown in certaincases to stimulate the rate of autolysis of S.aureus (10), had no discernible effect on RUS3.The generation time of RUS3 as judged by

increase in optical density (OD) was compara-ble to S. aureus H: about 30 min at 37 C in PYKbroth. Observation by phase contrast micros-copy showed that RUS3 grew in large clumpsin a fashion analogous to another autolyticmutant (10), whereas S. aureus H appeared aswell-separated cocci with little or no clumping.We tried to disaggregate the clumps by addi-tion of exogenous lytic enzymes (lysostaphin orthe soluble autolytic enzyme from S. aureus H).Although the soluble enzyme had no observ-able effect on the cells of RUS3 (it should benoted that this enzyme also does not lyse wholecells ofS. aureus H), addition of lysostaphin ledto a gross lysis of cells. All attempts to disag-gregate RUS3 with lysostaphin (e.g., low con-centration, incubation at 20 C) were unsuccess-ful. The RUS3 cells could be disaggregated bymild sonication, and the number of CFU ob-tained from a suspension of RUS3 cells after

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TIME (hours)

FIG. 1. Autolysis ofisolated cell walls ofS. aureusH and RUS3. Native cell walls of S. aureus H andRUS3 suspended in 10 mM phosphate buffer (pH720) were incubated at 37 C. At intervals, sampleswere withdrawn for determination of OD. S. aureusH walls (a), RUS3 walls (A).

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AUTOLYTIC MUTANT OF S. AUREUS

such a treatment was comparable to that of S.aureus H. The corresponding values for S.aureus H and RUS3 were 4 x 107 to 8 x 107 perml and 3 x 107 to 6 x 107 per ml, respectively(per 0.1 OD unit at 585 nm).One interesting property of RUS3 that might

be related to its negligible autolytic activity isits marked ability to survive under nongrowingconditions. Thus, although the titer of S. au-reus H cells suspended in distilled water at 37 Cdecreased from 7 x 1010 to 6 x 108 in 7 days, thecorresponding decrease in the titer of RUS3cells was only from 6 x 109 to 2 x 108. Similarresults were obtained when the cells were sus-pended in phosphate buffer. Therefore, it mightbe possible to use this phenotype as an enrich-ment technique in the future isolation of Lyt-mutants.

Autolytic properties of the cytoplasmic en-zyme. We next compared the activity of thesoluble autolytic enzyme of the Lyt- mutant tothat of S. aureus H. This was important sincethe bulk ofthe autolytic amidase ofS. aureus His known to be located in the cytoplasm (20). Toincrease the sensitivity of assay, the labeledsubstrate was isolated from cells incubated inthe presence of penicillin (21). It may be seenthat the ability of the crude enzyme from RUS3to degrade cell wall peptidoglycan is negligiblecompared to the wild type (Fig. 2). Further-more, in marked contrast to the parental en-zyme, increasing additions of the mutant en-zyme had very little effect on the release ofradioactivity (Fig. 3). Identical results were ob-tained when the substrates were switched, e.g.,when the labeled peptidoglycan was isolatedfrom RUS3 instead ofS. aureus H. The possibil-ity of the presence of an inhibitor of lytic activ-ity in RUS3 was ruled out by suitable mixingexperiments.

Cell wall composition. The fact that both thepeptidoglycan substrates (from S. aureus H andRUS3) could be degraded at the same rate bythe parental enzyme suggested that there wereno significant variations in these polymers.This was confirmed by the quantitative analy-sis of the cell walls (Table 1). It is evident thatthe cell wall composition ofRUS3 (both peptido-glycan and teichoic acid) is very similar if notidentical to the wild type. In addition, the abil-ity of isolated cell walls of RUS3 to bind phage52A was similar to that reported for S. aureusH (1). Since the phage receptor site in S. aureusinvolves both peptidoglycan and teichoic acid(1, 19), this further supports the contention thatthere are no significant variations in the cellwall composition of RUS3.

Cell wall turnover. We have previously dem-

24

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4 12

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8

41do

0 10 20 30 40 50 60TIME (min.)

FIG. 2. Kinetics of solubilization of labeled pepti-doglycan by soluble autolytic enzyme from S. aureusH and RUS3. Peptidoglycan substrate labeled withL-[3H]lysine (from S. aureus H) was incubated withthe 105,000 x g supernatant isolated from S. aureusH or RUS3 at 37 C. At intervals, reaction was termi-nated by boiling and the released radioactivity wasdetermined. See text for details. S. aureus H enzyme(a), RUS3 enzyme (A).

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400 800

jug PROTEIN1200

FIG. 3. Solubilization of labeled peptidoglycan byincreasing additions of soluble autolytic enzyme.Varying amounts of the crude autolytic enzyme fromS. aureus H or RUS3 were incubated with peptido-glycan labeled with L-[3H]lysine (from S. aureus H)at 37 C for 30 min and the released radioactivity wasassayed as described in the text. Symbols as in Fig. 2.

onstrated the role of amidase in the turnover ofcell walls of S. aureus (24). It was of interest toexamine if the cell walls of the Lyt- mutantcould undergo turnover. The experimental de-

, I I

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sign for detection of cell wall turnover has beenpreviously described (24). Briefly, the cells werelabeled with [14C]GlcNAc for four generationsand then diluted into fresh prewarmed growthmedium containing an excess of cold GlcNAc.Loss of radioactivity from cell wall polymersduring growth in the nonlabeled medium is adirect measure of cell wall turnover. Figure 4shows that the radioactivity associated with thecell wall peptidoglycan and teichoic acid ofRUS3 did not change while the cells were grow-ing, establishing the absence of cell wall turn-over. This is in striking contrast to the behaviorof S. aureus H where both peptidoglycan andteichoic acid were undergoing active turnover(Fig. 4).

Effect of cell wall antibiotics. It has beenpostulated that autolytic enzymes play a majorrole in the bactericidal effect of cell wall anti-biotics (17, 18). In a Lyt- mutant ofDiplococcus

pneumoniae, marked resistance to cellularlysis and killing was observed with several an-tibiotics that inhibit the biosynthesis of cellwall (23). In the present study we compared theeffects of penicillin G on the growth and viabil-ity of S. aureus H and RUS3. The minimuminhibitory concentration ofpenicillin G (by tubedilution assay) for both S. aureus H and RUS3was determined to be 0.05 pzg/ml. Figure 5shows the effect of adding penicillin G to expo-nentially growing cultures of S. aureus H andRUS3. The growth rate of both cultures de-creases in the presence of penicillin (especiallyat 5.0 ,ug/ml), but there is no evidence of cellu-lar lysis even after 180 min of incubation withthe antibiotic. The absence of gross cellulardisintegration was further confirmed by exami-nation of the cells from both cultures by phasecontrast microscopy. Furthermore, penicillin-treated cells and control cells did not display

TABLE 1. Cell wall composition of S. aureus H and RUS3Wall componenta

strain used Muramic Glucosa- Glutamicacid mine acid Glycme Lysine Alanine Phosphorus

H 0.37 0.73 0.42 1.84 0.47 1.12 0.82RUS3 0.41 0.78 0.42 1.98 0.48 1.15 0.84

a Expressed as micromoles per milligram of cell wall.

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TIME (min.)FIG. 4. Turnover ofcell wall polymers in S. aureus H and RUS3. S. aureus H and RUS3 ceUs growing in

PYK broth were uniformly labeled with ['4C]GlcNAc (specific activity, 56 2i4Lmmol; 02 ulCi/ml). The cellswere then harvested and diluted into fresh prewarmed PYK broth containing 100 mM cold GlcNAc. Sampleswere withdrawn at intervals and the radioactivity associated with the cell wall peptidoglycan and teichoic acidwas determined after chemical fractionation as described in text. Growth of the cultures was monitored tur-bidimetrically. Symbols: 0, OD; A, teichoic acid; 0, peptidoglycan. Open symbols, S. aureus H; closedsymbols, S. aureus RUS3.

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AUTOLYTIC MUTANT OF S. AUREUS 965

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TIME (mir)FIG. 5. Effect ofpenicillin G on the growth ofS. aureus H and RUS3. Penicillin G (at 0.05 and 5.0 pg/mi)

was added to exponentially growing cultures ofS. aureus H and RUS3 at zero time and the turbidity of thecultures was monitored at 585 nm. Symbols: 0, control; A and V, penicillin at 0.05 and 5.0 pg/mi, respec-tively. Open symbols, S. aureus H; closed symbols, S. aureus RUS3.

differences in the extent of release ofmacromol-ecules that absorb at 260 nm or in the sucepti-bility to lysis in the presence of 1% sodiumdodecyl sulfate. In marked contrast, penicillinwas found to have a dramatic effect on viabil-ity. Figure 6 shows the exponential decrease inthe number of CFU of S. aureus H growing inthe presence of penicillin G. The rate of killingis significantly higher with the higher concen-tration of penicillin. To our surprise, the kinet-ics of killing of RUS3 cells by penicillin G wasidentical to that of S. aureus H (Fig. 6). Theabove data were confirmed in three separateexperiments. Similar results were obtainedwith n-cycloserine (data not shown). These find-ings strongly suggest that at least in S. aureusthe primary lethal effect of penicillin may notbe directly coupled to the autolytic activity.

DISCUSSIONData presented in this article support the

hypothesis that the primary defect of RUS3 isin the amidase. This could be caused either be adecreased level of autolytic enzyme or by analteration in the structure of the protein. Thepossibility that the Lyt- phenotype is related to

a modification of the cell wall substrate is un-likely because cell walls of the parental andmutant strains have identical composition andcan bind phage 52A at similar rates. Further-more, the isolated cell walls of both strains aredigested at the same rate by the amidase iso-lated from the parental strain. In a previousstudy with a Lyt- mutant in Streptococcus fae-calis, Pooley et al. showed that the mutantcell wall peptidoglycan was less cross-linkedthan the wild type and as a result was degradedmore rapidly by the wild-type autolytic enzyme(16). Though we have not directly analyzed thedegree of cross-linking of RUS3 walls, the sub-strate characteristics of RUS3 wall suggest nosignificant variation in its cross-linking. Itshould be further noted that, in contrast to thebehavior of the S. faecalis mutants, the autoly-tic rate of RUS3 could not be stimulated bychanges in ionic strength. It is remarkable thatthe mass doubling time of RUS3 is comparableto that of S. aureus H. This implies that thebulk of the autolytic activity (over 90%) in S.aureus is not essential for growth.A relationship between alteration of autoly-

tic behavior and clumping of cells in liquidculture has been noted before in other systems,

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10 r

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TIME (mirn)FIG. 6. Effect ofpenicillin G on the viability of S.

aureus H and RUS3. Penicillin G (at 0.05 and 5.0pg/ml) was added to exponentially growing culturesof S. aureus H and RUS3 at zero time. At suitableintervals, samples were withdrawn, suitably diluted,andplated on nutrient agar. The RUS3 samples weresonicated before plating as described in text. Sym-bols: 0, control; A and 0, penicillin at 0.05 and 5.0pg/ml, respectively. Open symbols, S. aureus H;closed symbols, S. aureus RUS3.

suggesting a role of autolytic enzyme in cellseparation (2, 7, 16, 22). To function in this role,the activity of these endogenous enzymes mustbe under exquisite control resulting in the nick-ing of selected bonds. Therefore, we were notsurprised at the inability of exogenously addedenzymes to disaggregate the clumps of RUS3. Itis significant that viable cells can be recoveredfrom the clumps (with an increase in CFU)after mild sonication. This suggests that therole of amidase in cell separation occurs at alate stage of the division cycle- after partition-ing of the genome and growth of a functionalcross wall.The inability of the cell walls of RUS3 to

undergo turnover is a nice illustration of therole of autolytic amidase in this process. To ourknowledge, this is the first time such a correla-tion has been demonstrated in a Lyt- mutantstrain. This also reinforces the concept that theprocess of cell wall turnover is not essential forcell growth and division.

The observed effect of cell wall antibiotics onRUS3 is in striking contrast with the pneumo-coccal system where a Lyt- strain was shown tohave a marked resistance to similar antibiotics(23). Though we cannot explain the reason forthis discrepancy, one major difference betweenthe two systems is in the lytic effect of penicil-lin. Thus, unlike the pneumococcal system, ad-dition of penicillin G to a growing culture of S.aureus does not result in any visible lysis orcellular disintegration-at least not within thefirst 2 to 3 h, during which time the viable titerdeclines exponentially (Fig. 5 and 6). This istrue for both the wild-type strain, with its nor-mal complement of autolytic enzyme, and theLyt- strain. This suggests that the role of auto-lytic enzyme in the killing of sensitive cells byantibiotics could vary widely and in S. aureusit probably plays a relatively minor role. Thisis reinforced by our observation that both withthe wild-type and the mutant strains the rateof killing is proportional to the penicillin con-centration (Fig. 6). It might be recalled thatin Escherichia coli, which is lysed rapidly bypenicillin, lower concentrations of penicillinlead to a more rapid loss of viability, a phenom-enon referred to as the "zonal effect" (5, 6). Thissuggests that, unlike S. aureus, the lytic en-zymes do play a significant role in E. coli; atlow pencillin levels the biosynthetic machinerycould stumble along making more uncross-linked wall material that in turn would bedigested more rapidly by the autolytic enzymes.The three observations reported in this arti-

cle: (i) absence of cell lysis, (ii) identical killingrates for both strains, and (iii) absence of zonaleffect, suggest that autolytic enzymes are notprimary killing agents when S. aureus istreated with penicillin. It might be that killingis related to the irreversible nature of damagecaused by penicillin, i.e., once penicillin is fixedthe cell loses its viability. Obviously, morework is needed to define the nature of the pri-mary defect(s) responsible for penicillin-in-duced killing in S. aureus.

ACKNOWLEDGMENTSThis investigation was supported by Public Health Ser-

vice grants AI-10141 and AI-07211 from the National Insti-tute of Allergy and Infectious Diseases. William Wong is apredoctoral trainee supported by Public Health ServiceTraining grant no. 1-T32-GM07102 from the National Insti-tute of General Medical Sciences.

LITERATURE CITED1. Chatterje, A. N. 1969. Use of bacteriophage-resistant

mutants to study the nature of the bacteriophagereceptor site of Staphylococcus aureus. J. Bacteriol.98:519-527.

2. Chatterjee, A. N., D. Mirelman, H. J. Singer, and J. T.Park. 1969. Properties ofthe novel pleiotrophic bacte-

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3. Chatterjee, A. N., and F. E. Young. 1972. Regulation ofthe bacterial cell wall: isolation and characterizationofpeptidoglycan mutants ofStaphylococcus aureus. J.Bacteriol. 111:220-230.

4. Chen, P. S., T. Y. Toribara, and H. Warner. 1956.Microdetermination of phosphorus. Anal. Chem.28:1756-1758.

5. Eagle, H., and H. D. Musselman. 1948. The rate ofbactericidal action of penicillin in vitro as a functionof its concentration and its paradoxically reducedactivity at high concentrations against certain orga-nisms. J. Exp. Med. 88:99-131.

6. Eagle, H. 1951. Further observations on the zone phe-nomenon in the bactericidal action of penicillin. J.Bacteriol. 62:663-668.

7. Forsberg, C. W., and H. J. Rogers. 1971. Autolyticenzymes in growth of bacteria. Nature (London)229:272-273.

8. Forsberg, C. W., P. W. Wyrick, J. B. Ward, and H. J.Rogers. 1973. Effect of phosphate limitation on themorphology and wall composition ofBacillus licheni-formis and its phosphoglucomutase-deficient mu-tants. J. Bacteriol. 113:969-984.

9. Ghuysen, J. M. 1968. Use of bacteriolytic enzymes indetermination of wall structure and their role in cellmetabolism. Bacteriol. Rev. 32:425-464.

10. Gilpin, R. W., A. N. Chatterjee, and F. E. Young. 1972.Autolysis of microbial cells: salt activation of autoly-tic enzymes in a mutant of Staphylococcus aureus. J.Bacteriol. 111:272-283.

11. Gilpin, R. W., S. Narrod, W. Wong, F. E. Young, andA. N. Chatterjee. 1974. Autolysis in Staphylococcusaureus: preferential release of old cell wall duringautolysis. J. Bacteriol. 119:672-676.

12. Hebeler, B. H., A. N. Chatterjee and F. E. Young.1973. Regulation of the bacterial cell wall: effect ofantibiotics on lipid biosynthesis. Antimicrob. AgentsChemother. 4:346-353.

13. Higgins, M. L., and G. D. Shockman. 1971. Procaryotic

cell division with respect to walls and membranes.CRC Crit. Rev. Microbiol. 1:29-72.

14. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

15. Patterson, M. S., and R. C. Greene. 1965. Measurementof low beta-emitters in aqueous solution by liquidscintillation counting of emulsions. Anal. Chem.37:854-857.

16. Pooley, H. M., G. D. Shockman, M. L. Higgins, and J.Porres-Juan. 1972. Some properties of two autolytic-defective mutants of Streptococcus faecalis ATCC9790. J. Bacteriol. 109:423-431.

17. Rogers, H. J. 1967. Killing of staphylococci by penicil-lins. Nature (London) 213:31-33.

18. Rogers, H. J. 1970. Bacterial growth and the cell enve-lope. Bacteriol. Rev. 34:194-214.

19. Shaw, D. R. D., and A. N. Chatterjee. 1971. O-acetylgroups as a component of the bacteriophage receptoron Staphylococcus aureus cell walls. J. Bacteriol.108:584-585.

20. Singer, H. J., E. M. Wise, Jr., and J. T. Park. 1972.Properties and purification ofN-acetylmuramyl-L-al-anine amidase from Staphylococcus aureus H. J. Bac-teriol. 112:932-939.

21. Takebe, I., H. J. Singer, E. M. Wise, Jr., and J. T.Park. 1970. Staphylococcus aureus H autolytic activ-ity: general properties. J. Bacteriol. 102:14-19.

22. Tomasz, A. 1968. Biological consequences of the re-placement of choline by ethanolamine in the cell wallof Pneumococcus: chain formation, loss of transfor-mation and loss of autolysis. Proc. Natl. Acad. Sci.U.S.A. 59:86-93.

23. Tomasz, A., A. Albino, and E. Zanati. 1970. Multipleantibiotic resistance in a bacterium with suppressedautolytic system. Nature (London) 227:138-140.

24. Wong, W., F. E. Young, and A. N. Chatteijee. 1974.Regulation of bacterial cell walls: turnover of cellwall in Staphylococcus aureus. J. Bacteriol. 120:837-843.

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