JouRNAL OF BACTERIOLOGY, Jan. 1976, p. 166-173Copyright0 1976 American Society for Microbiology

Vol. 125, No. 1Printed in U.S.A.

Analysis of Autolysins in Temperature-Sensitive MorphologicalMutants of Bacillus subtilis

W. C. BROWN,* C. R. WILSON, SHEILA LUKEHART,' F. E. YOUNG, AND M. A. SHIFLETTDepartment of Biology, University of California, San Diego, La Jolla, California 92093,* and Department of

Microbiology, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Received for publication 2 October 1975

The content and distribution of autolysin were measured in temperature-sensi-tive morphological mutants of Bacillus subtilis. Strains RUB1000 and RUB1012grew as rods at 30 C. At 45 C the mutants contained disproportionately lessteichoic acid than peptidoglycan and grew as irregular spheres. The amount ofenzyme that could be extracted from rods was at least 31 times the amountextracted from spheres. The rate of autolysis of cell walls was 7- to 28-fold greaterin rods than in spheres. The low activity found associated with the cell walls ofspheres was not compensated for by larger amounts of autolytic activity in thecytoplasm. 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 andrelatively resistant cell walls. Revertants of RUB1012 were isolated that had 13,23, and 55% of the normal proportions of teichoic acid when grown at thenonpermissive temperature. Cell walls from the revertants were as sensitive toadded amidase as the wild-type strain. None of the revertant strains regained thewild-type ability to produce more amidase at 45 C. However, the deficiency inautolysin observed with RUB1012 was partially restored in revertants containinghigher proportions of teichoic acid.

Any attempts to understand the modificationof prokaryotic cell surfaces must include athorough analysis of bacterial autolysins. Mostbaeteria contain one or more of these enzymes,and their biological roles have been the subjectof several investigations. Autolysins might par-ticipate in cell wall growth, cell separation,peptidoglycan turnover, and deoxyribonucleicacid-mediated transformation. The evidence forthese roles has been summarized in recentreviews (15, 18, 20). One approach being used toassess the roles of autolysins is the analysis ofmutants with defects in the enzymes (10, 12, 13,16). Often these deficiencies have been cor-related with morphological aberrations, such aschain formation (6, 10, 12, 24) or changes in cellshape (2). The isolation of these morphologicalvariants provides a convenient techniquewhereby potential autolysin-defective mutantsmight be selected. Conditional morphologicalmutants of Bacillus subtilis and B. licheniformiswere first reported by Rogers et al. (25). Thesemutants grew as irregular spheres on an inor-

IPresent address: Department of Medical Microbiology,Medical School, University of California, Los Angeles, Calif.90025.

ganic salts-glucose-tryptophan medium but asrods when extra sodium chloride or additionalnutrients were included (26). Studies on similarmorphological mutants were described byBoylan and Mendelson (1) and further char-acterized through the collaborative efforts ofBoylan et al. (2) and Cole et al. (8). Thesemutants grew as rods at 30 C but as irregularspheres at 45 C. Growth at the nonpermissivetemperature resulted in a marked decrease inboth the ratio of teichoic acid to peptidogly-can and N-acetylmuramyl-L-alanine amidaseassociated with the cell walls (2). It was not de-termined in the previous studies whether thedifferences in rates of autolysis of cell wallsformed at the different temperatures were at-tributable to alterations in enzyme distribution,activity, synthesis, or affinity of the enzymefor the substrate. The present investigation wasinitiated to evaluate these possibilities. Thispaper summarizes a study of the relationshipbetween the content and distribution of autoly-sin and morphological changes in temperature-sensitive rod- mutants of B. subtilis. The resultsindicated that rods had more autolysin andteichoic acid than did spheres.

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VOL. 125, 1976

MATERIALS AND METHODS

Strains of B. subtilis. All of the strains in thisstudy were derivatives of B. subtilis 168 (Table 1).Markers were introduced by congression (2, 9, 23).Media and growth conditions. The tryptose blood

agar base (Difco Laboratories, Detroit, Mich.) andSpizizen minimal medium with supplements (GMmedium) were prepared and utilized as describedpreviously (2). Cultures were grown with shaking in3-liter indented Fernbach flasks containing 1 liter ofGM medium. The flasks were inoculated with a 1%suspension of cells previously grown overnight in thesame medium. Growth was measured turbidimetri-cally with a Klett-Summerson photoelectric colorime-ter using a no. 66 filter.

Preparation of cell walls. Cell walls were pre-pared by one of the methods described previously (3,34). Substrate cell walls were prepared by treating thesuspensions with hot sodium dodecyl sulfate (SDS)(3).Assay of lytic activity. Lytic activity was mea-

sured turbidimetrically with a Beckman DBGT spec-trophotometer (3). Unless stated otherwise, the sub-strate was homologous cell walls prepared from cellsgrown at 30 C. One unit of autolysin activity repre-sents a decrease of 0.001 optical density unit at 600nm per min.

Analytical procedures. Glucose was measured bythe Glucostat assay (Worthington BiochemicalsCorp., Freehold, N.J.). Phosphorus was measured bythe method of Chen et al. (7). Teichoic acid wasestimated from the amount of glucose and phospho-rous present in the cell wall. A 1:1 ratio was found forthese compounds in strain 168 (32). Protein wasdetermined as described by Lowry et al. (22) or by themethod of Warburg and Christian (30).

Preparation of autolysin extracts. Crude autoly-sin was prepared by homogenizing cells in 5 M NaCl(3). The suspension was centrifuged at 12,000 x g for15 min at 4 C, and the supernatant fraction was usedas the crude autolysin.

RESULTSCharacteristics of autolysins from the par-

ent strain. It was shown previously that B.subtilis 168 and an asporogenic protease-defi-cient derivative (SR22) conta'-ied multiple au-

TABLE 1. Description of strains

Strain Origin strain Genotype

BR151 168 Iys-3 trpC2 metBRUB1000 Congressiona BR151 lys-3 trpC2 tag-lbRUB1012 Congression BR151 lys-3 trpC2 tag-2RUB2012c Congression BR151 Iys-3 trpC2 tag-2RUB2042c Congression BR151 Iys-3 trpC2 tag-2RUB2032c Congression BR151 Iys-3 trpC2 tag-2

a Markers introduced by transformation.b tag denotes teichoic acid glycerol.c Spontaneous revertant of RUB1012.

AUTOLYSINS AND MORPHOGENESIS 167

tolysins as defined by pH optima and by thechemical nature of the end groups solubilizedduring autolysis (5). This organism produces anN-acetylmuramic acid-L-alanine amidase at pH9.5, presumably an unspecified peptidase at pH8.0, and an unspecified hexosaminidase at pH5.5 to 6.0 (5). All organisms used in this inves-tigation were derivatives of strain 168 (Table1). One derivative, strain BR151, was used forthe development of all the mutant strains.Therefore, in this study strain BR151 is referredto as wild type although it carries three auxo-trophic requirements. The autolytic system instrain BR151 is similar to that of strains 168 andSR22.Cellular autolysis. The amounts of autolytic

activity were determined in the wild-type andmutant strains RUB1000 and RUB1012 previ-ously grown to log phase at 30 C or 45 C. Figure1 shows that the 30 C mutant cells autolyzed atrates similar to or greater than those of the 30 Cwild-type cells. The 45 C wild-type cells under-went a longer lag at pH 6.0 but then lysed at arate greater than the 30 C cells. At the alkalinepH optima, autolysis occurred at the same ratefor wild-type 30 C cells and 45 C cells. Con-versely, autolysis of the mutants was markedlyreduced at all three pH optima for cells grown atthe higher temperature. Although this latterobservation was made in all subsequent experi-ments, the most reproducible data was obtainedwith the autolysin active at pH 9.5. This en-zyme was characterized earlier as an N-acetyl-muramyl-L-alanine amidase (31) and was puri-fied recently by Herbold and Glaser (19). Theother autolysins in B. subtilis Marburg havebeen less thoroughly characterized. For thesereasons only the data obtained for the amidasewill be presented.Content and distribution of autolysin in the

wild-type and mutant strains. An experimentwas performed to determine whether the re-duced rates of autolysis were due to alterationsin the distribution of enzyme in the mutantstrains. The basic procedure is illustrated inFig. 2. No enzyme activity was detected in thedialyzed growth medium, suggesting that noneof the strains released autolysin during growth.There were significant differences in the

amounts of enzyme extracted from the variousstrains (Table 2). BR151 showed a modestincrease in total activity and specific activitywhen grown at 45 C. Both mutants showedreduced activity at the nonpermissive tempera-ture, although the reduction was more pro-nounced in strain RUB1012.Another portion of cells was disrupted to

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168 BROWN ET AL.

30C

100

50

0

100

50

0

100

50

0

0

45C

20 40 0 20 40

TIME (minutes)FIG. 1. Cellular autolysis of wild-type and mutant strains of B. subtilis. Cells were grown in GM medium to

midlog phase, washed, and resuspended at a concentration of 1 mg/ml in 50 mM acetate (pH 6.0),tris(hydroxymethyl)aminomethane (pH 8.0) or glycine (pH 9.5). The mixture was incubated at 37 C. Thenumbers at the top of the figure represent the temperature used during growth of the cells. Symbols: 0, BR151;A, RUB1000; 0, RUB1012.

determine the amounts of activity associatedwith the cell wall and cytoplasmic fractions.The rate of cell wall autolysis of strain BR151cells grown at 45 C was about twice that of the30 C cells (Table 3). The rates of autolysis of30 C cell walls from RUB1012 and RUB1000were 7 and 28 times more rapid than the ratesfor the 45 C cells, respectively. Autolysis of 45 Ccell walls from these mutants was not completeeven after 24 h. Although the cytoplasm con-

tained very little enzyme as compared with thecell walls, it is clear that the levels of autolysinwere higher in the cytoplasmic fraction fromstrain BR151 grown at 45 C (Table 4). Strain

RUB1000 had more enzyme than the parentwhen grown at 30 C but not at 45 C. Bothmutants had significantly lower amounts ofamidase in the cytoplasm when grown at thenonpermissive temperature.When these data are compared with the

results presented in Table 2, it is concluded thatonly small amounts of the active amidase wereobtained in cells grown at either temperature. Itis significant that a reduction in extractableactivity in the 45 C cells was not compensatedfor by larger amounts of activity in the cyto-plasm or growth medium. When aliquots con-taining equal mixtures of 30 C and 45 C ex-

0

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1-CD C

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AUTOLYSINS AND MORPHOGENESIS 169

LOG CULTURE

centrifuge

LOG CELLS [CULTURE MEDIUM|

FRENCH PRESS

centrifuge

r .~~~~~~~~~~~~~~~~~CELL WALLS

wash wi

ICELL WALLS

CYTOPLASM/H20

Procedure for determining the distribution of autolysins in wild-type and mutant strains of B.

TABLE 2. Total activity and specific activity ofamidase extracted from cells with 5 M sodium

chloride

Total SpactStrain Temp activityb Ratio (U/ig of(C)o (U/gof

poencells) poen

BR151 30 1,200 1.0 7545 1,800 1.5 118

RUB1000 30 2,380 1.0 3145 420 0.18 15

RUB1012 30 1,200 1.0 4145 _C NAd NAd

a Temperature used during growth of cells.b Activity at pH 9.5.c Below the limit of the assay.dNA, Not applicable.

TABLE 3. Rate of autolysis of cell walls from wild-typeand mutant strains of B. subtilisd

Strain Temp (C)& Ratec Ratio

BR151 30 1.32 1.045 2.4 1.8

RUB1000 30 2.24 1.045 0.08 0.035

RUB1012 30 2.8 1.045 0.4 0.14

aCell walls were isolated from midlog-phase cellsgrown in GM medium at 30 C or 45 C. The walls weresuspended in 50 mM glycine buffer (pH 9.5) at aconcentration of 1.0 mglml and incubated in a waterbath at 37 C.

b Temperature used during growth of cells.c Percent decrease in turbidity per minute.

tracts were added to the reaction mixture, therate of lysis of the substrate was not reducedbelow the amount caused by dilution (unpub-lished data). This suggested that the 45 Cextracts did not contain an inhibitor of autoly-sin.To determine whether a modification of the

substrate was also responsible for the inhibitionof autolysis, SDS cell walls from the differentstrains were tested for their sensitivity to exoge-nous amidase. Strain BR151 formed rod-shapedcell walls at both temperatures. These wallswere equally sensitive to added amidase (Table

TABLE 4. Total activity and specific activity ofamidase in the cytoplasm of wild-type and mutant

cellsa

Total SpactStrain Temp activityc Ratio (U/mg of(C)b (U/g of protein)

cells)

BR151 30 133 1.0 0.6645 277 2.1 1.36

RUB1000 30 306 1.0 3.2745 10 0.03 0.22

RUB1012 30 81 1.0 1445 _d NAe NAe

a Growth conditions were the same as described inTable 3. Amidase activity was measured in thedialyzed cytoplasmic fraction after disruption of 1 g(wet weight) of cells.

bTemperature used during incubation of cells.c Activity at pH 9.5.d Below the limit of the assay.eNA, Not applicable.

5 MNaCI

centrifuge

FIG. 2.subtilis.

SALT EXTRACT

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170 BROWN ET AL.

5). Rod walls from strain RUB1000 were 22times more sensitive to amidase than were

sphere walls (Table 5). Rod walls from strainRUB1012 were eight times more sensitive toamidase than were sphere walls. By contrast,sphere walls from both mutants and walls fromwild-type cells grown at 45 C (rods) were more

sensitive to lysozyme than were the respectiverod walls formed at 30 C (Table 6). In addition,sphere walls from the mutants were more sensi-tive to lysozyme than were the 45 C walls fromthe wild-type strain. The results indicate thatthe mutation expressed at 45 C caused theformation of cell walls that were more resistantto amidase but more sensitive to lysozyme.Examination of revertants isolated from

strain RUB 1012. Strains RUB1000 andRUB1012 do not form colonies when plated on

tryptose blood agar base medium at 45 C. Thisproperty was utilized in isolating revertants.RUB1012 was the parent organism for thesestudies (M. Shiflett, Ph.D. dissertation, Univ.of Rochester, Rochester, N.Y., 1975). Washedcells of RUB1012 were plated on tryptose bloodagar base medium and incubated at 45 C.Revertants were recognized by their ability toform small colonies under these conditions.Three such revertants, RUB2032, RUB2012,and RUB2042, had 13, 23, and 55% of thenormal porportion of teichoic acid to peptido-glycan in their cell walls, respectively (M.Shiflett and F. E. Young, Abstr. Annu. Meet.Am. Soc. Microbiol. 1973, P 75, p. 153). Thesestrains were examined for cell wall autolysis,substrate sensitivity, and amounts of extracta-ble amidase. The rates of autolysis for cell wallsobtained from revertants grown at 30 C were

similar to the rates of the wild-type and parentstrains (Table 7). When compared with the datafor RUB1012 in Table 3, the rates of autolysis of45 C revertant cell walls were lower than ex-

TABLE 5. Sensitivity of rod and sphere walls to addedhomologous amidasea

urce of S o urce of SDS walls Activityautolysin (U)

BR151 (30 C) BR151 (30 C) 5.1BR151 (30 C) BR151 (45 C) 5.1RUB1000 rods RUB1000 rods 19.4RUB1000 rods RUB1000 spheres 0.87RUB1012 rods RUB1012 rods 5.3RUB1012 rods RUB1012 spheres 0.65

a SDS cell walls were prepared from log-phase cellsas described previously (5). The walls were suspended(1 mg/ml) in 50 mM glycine buffer, pH 9.5. Amidasewas added, and the mixture was incubated at 37 C.

TABLE 6. Sensitivity of rod and sphere wallsto lysozymea

Source of SDS walls Activity (U) Ratio

BR151 rods(30 C) 7.4 1.0BR151 rods (45 C) 11.2 1.5RUB1000 rods 6.7 1.0RUB1000 spheres 13.2 2.0RUB1012 rods 5.8 1.0RUB1O12 spheres 15.9 2.7

a Log-phase cell walls were suspended (1 mg/ml) in0.1 M sodium phosphate buffer, pH 7.0. The finalconcentration of lysozyme was 5.0 ,g/ml.

TABLE 7. Autolysis of cell walls from revertantsderived from RUB1012a

Strains Temp Rater Ratio(C)b

RUB2032 (13) 30 1.10 1.045 d NAe

RUB2012 (23) 30 2.39 1.045 0.096 0.04

RUB2042 (55) 30 2.21 1.045 0.26 0.12

a Procedure is the same as that described in Table3. The number in parentheses represent the percent-age of wild-type teichoic acid estimated from thephosphorus content of the walls.

"Temperature used during growth of cells.c Percent decrease in turbidity per minute.d Below the limit of the assay.e NA, Not applicable.

pected. Nevertheless, it was observed that theautolysis of these cell walls increased concomi-tantly with the increase in teichoic acid content(Table 7). Likewise, extracts from RUB2012and RUB2042 grown at 45 C (Table 8) con-tained more autolysin than extracts fromRUB1012 (Table 2). None of the mutants pro-duced more amidase at 45 C than at 30 C, ascharacteristically shown by the wild type. WhenSDS walls from the revertants were incubatedwith added amidase, it was observed that rodand sphere walls from all of the revertantstrains were about equally sensitive to theamidase. Table 9 shows that the revertantsdiffered in their sensitivity to lysozyme. StrainRUB2032 regained the normal pattern of sensi-tivity associated with the wild-type strain,whereas strain RUB2012 was similar to theparent (RUB1012). There was only a slightdifference in the rates of lysis of rod and spherewalls of strain RUB2042. These results did notdemonstrate a good correlation between lyso-zyme sensitivity and the ratio of teichoic acid topeptidoglycan in the walls.

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AUTOLYSINS AND MORPHOGENESIS 171

TABLE 8. Total activity and specific activity ofamidase extracted from revertants with

5Msodium chloridea

Total Sp actStrain Temp activ- Ratio (U/mg(C)b ityc of pro-

(U/g) tein)

RUB2032 (13) 30 296 1.0 13345 28 0.09 32

RUB2012 (23) 30 560 1.0 43545 354 0.63 138

RUB2042 (55) 30 368 1.0 24745 164 0.45 94

a Cells were grown in GM medium at 30 C or 45 C.Cells were harvested in midlog phase, washed, andextracted with 5 M NaCl as described previously (3).The numbers in parentheses represent the percentageof wild-type teichoic acid.

b Temperature used during growth of cells.c Activity at pH 9.5.

TABLE 9. Sensitivity of revertant cell wallsto lysozymea

Source of SDS walls Activity (U) Ratio

RUB2032 rods 4.6 1.0RUB2032 spheres 7.0 1.5RUB2012 rods 4.7 1.0RUB2012 spheres 11.8 2.5RUB2042 rods 7.0 1.0RUB2042 spheres 8.2 1.2

a Log-phase cell walls were suspended (1 mg/ml) in0.1 M sodium phosphate buffer, pH 7.0. The finallysozyme concentration was 5.0 ug/ml.

DISCUSSION

Previous studies with the mutant RUB1000demonstrated a transition from rods to sphereswhen grown under nonpermissive conditions(1). The spheres contained thickened cell wallsand a significant reduction in the amount ofteichoic acid relative to peptidoglycan. It wassuggested that the relative reduction in teichoicacid content either directly or indirectly con-tributed to the morphological alterations. Onepossibility suggested for indirect effects wasthat teichoic acid could regulate the synthesis ofpeptidoglycan and the formation or activity ofN-acetylmuramyl-L-alanine amidase. This au-tolysin could subsequently modulate the struc-ture of the peptidoglycan. Preliminary experi-ments showed that the sphere walls autolyzedmore slowly than the rod walls (2). In thisinvestigation we found a simultaneous reduc-tion in all of the autolytic activities. Thefollowing results indicate that the reduction in

cellular autolysis was not due to an alteration inthe distribution of autolysin: (i) no autolysinwas found in the growth medium when cellswere grown under permissive or nonpermissiveconditions, although the presence of protease inthe medium cannot be ruled out; (ii) cell wallsfrom 45 C mutant cells underwent autolysis at aslower rate than did walls from 30 C cells, and(iii) a reduction in extractable autolysin at 45 Cwas not compensated for by larger amounts ofactivity in the cytoplasm of unextracted cells.The reduction in substrate sensitivity to ami-dase demonstrated by 45 C walls partially ex-plains the observed inhibition of cellular autol-ysis, but the salt extraction data indicate thatautolysin was not being produced. The possibil-ity that an autolysin inhibitor was being pro-duced at 45 C was eliminated by appropriatemixing experiments. Another possible explana-tion was that autolysin synthesized at 45 C inthe mutant was more sensitive than 30 C au-tolysin to the salt used during extraction. Sensi-tivity to concentrated salt solutions was re-ported for the N-acetyl-muramyl-L-alanineamidase of B. licheniformis (15).A strong correlation was made in our study

between the level of autolysins and the relativeproportion of teichoic acid in the walls. Onesignificant observation was that all autolyticactivities were simultaneously depressed whenthe organisms had lower relative levels of tei-choic acid. This was further indicated by therevertant studies that showed that the autolysindeficiency was partially restored concomitantlywith the increase in teichoic acid relative topeptidoglycan. Earlier, we provided evidencefor a good association between autolysin andteichoic acid and suggested that this moleculemight regulate the synthesis and activity ofautolysin and other surface polymers (4). Thishypothesis is substantiated by studies reportedby other investigators. Tomasz (28, 29) showedthat substitution of ethanolamine for choline onteichoic acid in pneumococci reduced cellularautolysis and inhibited cell separation. Re-moval of D-alanine residues on teichoic acid inStreptococcus zymogenes resulted in increasedsensitivity of the walls to endogenous autolysinin Staphylococcus aureus 52A5. Finally, Her-bold and Glaser showed that teichoic acid-freewalls autolyzed at a slower rate than normalwalls and that the binding of enzyme to teichoicacid-free walls was 20- to 30-fold less than tointact walls (19).Although a positive correlation was observed

between autolysin production and rod forma-tion in this investigation, these data did not

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demonstrate that autolysin deficiency was acausal factor in the observed morphologicalchanges. The simplest explanation for our find-ings is that the pleiotrophic effects were due to a

mutation in a regulatory gene that controlsseveral functions related to the cell surface.

Several investigations have focused on therole of autolysins in cellular morphogenesis(reviewed in reference 18). Evidence was pre-

sented previously to suggest that changes in cellshape were directly related to levels of autolysinin Arthrobacter crystallopoietes (21). Duringstudies on sphere-to-rod transition in this orga-

nism, it was observed that higher levels ofmuramidase and shorter polysaccharide chainswere associated with spheres. Other studieshave suggested that autolysins function primar-ily in cell separation (6, 11, 14, 25). It should beemphasized that only a few systems have beenstudied so that no firm conclusions can bemade with respect to the roles of autolysins inmorphogenesis. The continued analysis of a

variety of mutants with abnormal autolysismight be beneficial in such studies. Two majorclasses may be generated, depending on

whether the mutation occurs in the structuralgene or in a regulatory gene. The examples citedhere cover only defects in amidase. Obviously,defects in other autolysins are possible. Prelimi-nary designation of the structural gene classmight be amiE, whereas the regulatory classmight be designated as amiR. The cellularautolysis of amiE mutants will be reduced dueto a defect in enzyme synthesis. There havebeen no conclusive reports describing a muta-tion in a structural gene for autolysins. Possiblecandidates have been isolated from B. subtilis(12) and S. aureus (W. Wong, R. W. Gilpin, andA. N. Chatterjee, Abstr. Annu. Meet. Am. Soc.Microbiol. 1975, K109, p. 165). Regulatory ef-fects might be caused by changes in moleculesthat stimulate or inhibit autolysin activity di-rectly or indirectly through modifications of thesubstrate. For example, increased levels of auto-lysin have been found associated with a proteasedeficient mutant of B. subtilis (5). Changes inan allosteric activator (19) might lead to an

increase or decrease in the activity of autolysin.Several reports have appeared in which a lowerrate of autolysis was correlated with alterationsin the substrate (6, 33), as well as mutants withdefects both in autolysin and in the substrate(15, 16, 24). When suspected autolytic defectivemutants are isolated, it is important to distin-guish clearly whether the defects are caused bychanges in the primary structure of the enzymeor by changes in substrate.

ACKNOWLEDGMENTS

This study was supported by National Science Foundationgrant GB 40035 to W. C. B. and Public Health Service grant5-RO1-AI-10141 from the National Institute of Allergy andInfectious Diseases to F. E. Y. M. A. S. is a predoctoral fellowwith Public Health Service grant 5-TO1-GM-00592 from theNational Institute of General Medical Sciences.

LITERATURE CITED

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2. Boylan, R. J., N. H. Mendelson, D. Brooks, and F. E.Young. 1972. Regulation of the bacterial cell wall:analysis of a mutant of Bacillus subtilis defective inbiosynthesis of teichoic acid. J. Bacteriol. 110:281-290.

3. Brown, W. C. 1973. Rapid methods for extracting autoly-sins from Bacillus subtilis. Appl. Microbiol.25:295-300.

4. Brown, W. C., D. K. Fraser, and F. E. Young. 1970.Problems in purification of Bacillus subtilis autolyticenzyme caused by association with teichoic acid. Bio-chim. Biophys. Acta 198:308-315.

5. Brown, W. C., and F. E. Young. 1970. Dynamic interac-tion between cell wall polymers extracellular proteasesand autolytic enzymes. Biochem. Biophys. Res. Com-mun. 38:546-568.

6. Chatterje, A. N., D. Mirelman, H. J. Singer, and J. T.Park. 1969. Properties of a novel pleiotrophic bacterio-phage-resistant mutant of Staphylococcus aureus. J.Bacteriol. 100:846-853.

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

8. Cole, R. M., T. J. Popkin, R. Boylan, and N. H.Mendelson. 1970. Ultrastructure of a temperature-sen-sitive rod mutant of Bacillus subtilis. J. Bacteriol.103:793-810.

9. Davie, J. M., and T. D. Brock. 1966. Effect of teichoicacid on resistance to the membrane lytic agent ofStreptococcus zymogenes. J. Bacteriol. 92:1623-1631.

10. Dubnau, D., C. Goldthwaiter, I. Smith, and J. Marmur.1967. Genetic mapping in Bacillus subtilis. J. Mol.Biol. 27:163-185.

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12. Fan, D. P., and M. M. Beckman. 1973. Mutant ofBacillus subtilis with a temperature-sensitive autolyticamidase. J. Bacteriol. 114:798-803.

13. Fan, D. P., M. M. Beckman, and W. P. Cunningham.1972. Ultrastructural studies on a mutant of Bacillussubtilis whose growth is inhibited due to insufficientautolysin production. J. Bacteriol. 100:1247-1257.

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

15. Forsberg, C. W., and H. J. Rogers. 1974. Characterizationof Bacillus licheniformis 6346 mutants which havealtered lytic enzyme activities. J. Bacteriol.118:358-368.

16. Forsberg, C. W., P. B. Wyrick, J. R. Ward, and H. J.Rogers. 1973. Effect of phosphate limitation on themorphology and wall composition of Bacilluslicheniformis and its phosphoglucomutase-deficientmutants. J. Bacteriol. 113:969-984.

17. Ghuysen, J.-M., and G. D. Shockman. 1973. Biosynthesisof peptidoglycan, p. 37-130. In Loretta Levie (ed.),Bacterial membranes and walls. Marcel Dekker, Inc.,New York.

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18. Glaser, L. 1973. Bacterial cell surface polysaccharides.Annu. Rev. Biochem. 42:91-112.

19. Herbold, J. R., and L. Glaser. 1975. Bacillus subtilisN-acetyl-muramic acid L-alanine amidase. J. Biol.Chem. 250:1676-1682.

20. Higgins, M. L., and G. D. Shockman. 1971. Procaryoticcell division with respect to wall and membranes. CRCCrit. Rev. Microbiol. 1:29-71.

21. Krulwich, T. A., and J. C. Ensign. 1968. Activity of an

autolytic N-acetylmuramidase during sphere-rod mor-

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