JOURNAL OF BACTERIOLOGY, Nov. 1974, p. 748-758Copyright 0 1974 American Society for Microbiology

Vol. 120, No. 2Printed in U.SA.

Characterization of a Bacteriophage-Induced, Host-SpecificLytic Enzyme from Lysates of Bacillus stearothermophilus

Infected with Bacteriophage TP-8SYLVIA P. BREHM' AND N. E. WELKER

Department of Biological Sciences, Northwestern University, Evanston, Illinois 60201

Received for publication 12 July 1974

Phage TP-8 lysates of Bacillus stearothermophilus 4S or 4S(8) contain lyticactivity exhibiting two pH optima, one at pH 6.5 and the other at pH 7.5. Using avariety of fractionation procedures, the two lytic activities could not beseparated. At pH 7.5 the lytic enzyme is an endopeptidase which hydrolyzes theL-alanyl-D-glutamyl linkage in the peptide subunits of the cell wall peptidoglycanand at pH 6.5 it exhibits N-acetylmuramidase activity. Endopeptidase activity isinhibited by NaCl and neither lytic activity was significantly affected by divalentcations or ethylenediaminetetraacetic acid. Crude lysates contain 2.5 to 3.0 timesmore endopeptidase activity than N-acetylmuramidase activity. The ratio of thetwo lytic activities (endopeptidase/N-acetylmuramidase) changes to 1.3 to 1.7during the course of purification, to 1.0 after isoelectric focusing, and 3.9 and 6.00after exposure for 2 h at 60 and 65 C, respectively. We conclude that the two lyticactivities may be associated with a single protein or a lytic enzyme complexcomposed of two enzymes. Lytic activity at pH 7.5 is more effective insolubilizing cells or cell walls than the lytic activity at pH 6.5. LiCl extracts of4Sand 4S(8) cells contain lytic activity exhibiting endopeptidase activity at pH 7.5and N-acetylmuramidase activity at pH 6.5. Lytic activity in these LiCl extractsalso has a number of other properties in common with those in lysates of phageTP-8. We proposed that the lytic enzyme(s) are not coded for by the phagegenome but are part of the host autolytic system.

Proteins that catalyze the dissolution of bac-terial cell walls resulting in cell lysis are collec-tively referred to as lytic enzymes. Lytic en-zymes of bacterial origin may be categorized asphage lytic enzymes, which function in therelease of mature phage particles, or autolyticenzymes, which are thought to be associatedwith the processes of cell growth and divisionand sporulation.Phage-induced lytic enzymes have been de-

tected in lysates of various phage-host systems.With the exception of the N-acetylmuramidaseof phage T2 (13, 30) and T4 (28) and theendopeptidase of phage X (2, 3, 26); lytic en-zyme from phage P22 of Salmonellatyphimurium (19); N-acetyl-glucosaminidasefrom phage C-1 of group C streptococci (1; S. S.Barkulis et al., Abstr. Annu. Meet. Amer. Soc.Microbiol., p. 32, 1964); and the endopeptidasefrom phage TP-1 of Bacillus stearothermophilus(32, 33), most of the information has beendescriptive in nature. In each case only one lyticenzyme was detected in the crude lysate or

'Present address: Department of Microbiology, ScrippsClinic and Research Foundation, La Jolla, Calif. 92037.

purified enzyme preparation. More than onekind of lytic activity has been reported in crudelysates of phage 80 (6) or phage 53 (20) ofStaphylococcus aureus. It is generally assumed,however, that in most phage-host systems asingle phage-induced lytic enzyme is responsi-ble for the release of mature phage particles.Moo-Penn et al. (15, 16) reported that crude

lysates and purified lytic enzyme preparationsof coliphage N20F' exhibit lytic activity at twopH optima, one at pH 5.5 and the other at pH8.5. It was proposed that a monomer-dimertransition was responsible for lytic activity atpH 5.5 (monomer) and pH 8.5 (dimer).

In preliminary experiments we found thatmitomycin C-induced lysates of B.stearothermophilus 4S(8, 12) (lysogenized withtemperate phages TP-8 and TP-12) containedlytic activity exhibiting three pH optima (pH6.5, 7.5, and 9.0). Mitomycin C-induced lysatesof the lysogenic strain 4S(8) contained lyticactivity exhibiting pH optima at 6.5 and 7.5 andmitomycin C-induced lysates of lysogenic strain4S(12) contained lytic activity exhibiting a pHoptimum at 9.0. Lysates prepared by infection

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of the cured strain (4S) with phage TP-8 con-tained lytic activity exhibiting pH optima at 6.5and 7.5 and lysates prepared by infection ofstrain 4S with phage TP-12 contained lyticactivity exhibiting a pH optimum at 9.0.

In this paper we describe the isolation andsome general properties of the lytic activity inphage TP-8 lysates. The mode of action of thelytic activity on cell walls at each pH was alsodetermined. In addition, we present evidencethat indicates that the phage-induced lyticactivity is derived from the host autolytic sys-tem.

MATERLAL AND WETHODSThe organisms used in the study were B.

stearothermophiluw strains NCA 15034R and 4S(8).Strain 4S(8) is lysogenized with phage TP-8 (11) andwas isolated from a culture of 4S(8, 12) after aspontaneous loss of phage TP-12. A cured strain (4S)was isolated after treatment of strain 4S(8, 12) withN-methyl-N'-nitro-nitrosoguanidine. Phage TP-8 wasassayed on a nonrestricting mutant (4S r1,4) of strain4S (11) using the bacteriophage assay described byWelker and Campbell (31).

Cultures were grown in a 2% Trypticase (BBL),0.5% yeast extract, 0.5 glucose (TYG) or fructose(TYF) medium containing FeCl, .6H,O (7 mg/liter),MnCl, .4H,O (1 mg/liter), and Mg SO,4 7H,O (15mg/liter). Stock cultures were maintained on 2%Trypticase 2% agar (TA) slants or plates at 5 C. Thesolid medium contained the same salt mixture as theliquid medium. Growth was measured in a Bausch &Lomb Spectronic-20 colorimeter at 525 nm.

Preparation ofphage lysate. Each oftwo 2,800-mlFembach flasks (baffled-bottom) containing 1,100 mlof TYG medium were inoculated with 30 ml of cellinoculum prepared as follows. A 300-ml Erlenmeyerflask (baffled-bottom) containing 30 ml of TYGmedium was inoculated with ovemight 4S(8) or 4Scells from a TA plate and shaken in a New Brunswickgyratory water bath shaker (model G-76) for 2 h at55C.

After the addition of the cell inoculum, the Fem-bach flasks were shaken in a New Brunswick gyratoryincubator shaker (model G-25) at 55 C. At an opticaldensity (OD) at 525 nm of 0.17 to 0.2 (7 x 107 to 10'cells/ml) mitomycin C (0.1 zg/ml) or phage TP-8(multiplicity of infection [MOI] of 1.0) was added tocultures of 4S(8) and 4S, respectively. After lysis wascomplete (usually 2 to 2.5 h after the addition ofmitomycin C or infection with phage TP-8) the lysateswere cooled to 40 C.Lyic enzyme assay. The substrate (acetone-

treated cells of B. stearothermophilus NCA 1503-4R)for the lytic enzyme assay was prepared by theprocedure described by Welker (32). Lytic activitywas measured in a Gilford model 2400 automaticspectrophotometer equipped with dual thermospac-ers. The assay temperature was maintained at 55 Cwith a Haake model F constant temperature circula-tor. The reaction mixture contained 2.8 ml of bufferand 0.1 ml of enzyme. After equilibration at 55 C, 0.1

ml of substrate (0.38 mg) was added. The initial ODat 410 nm was between 0.6 and 0.75. A unit of lyticactivity is defined as a decrease in OD of 0.1 per minbetween 2 and 5 min after the addition of thesubstrate. Protein was determined by the method ofLowry et al. (12) using bovine serum albumin as astandard.Column chromatography. Carboxymethyl (CM)-

cellulose (standard capacity, 0.7 fi= 0.1 meq/g) wasobtained from Schwarz/Mann and a suspension wasprepared by the procedure described by Welker (32).A chromatographic column (2.5 by 15 cm) was packedby gravity flow at 5 C to a height of 10 to 12 cm andequilibrated with 0.05 M sodium acetate buffer, pH5.6 (SA buffer).

Diethylaminoethyl (DEAE)-cellulose (standard ca-pacity, 0,9 X 0.1 meq/g) was obtained from Schwarz/Mann and phosphocellulose (exchange capacity, 0.87meq/g) from Bio-Rad Laboratories. Sephadex G-75and G-100 were obtained from Pharmacia Inc.

Characterization of lytic activity on cell wails.Cell walls of B. stearothermophilus NCA 1503-4Rwere prepared as described by Sutow and Welker (24).Cell walls (2.5 mg) were incubated with a purifiedlytic enzyme preparation (63 5Lg) in 11 ml of theappropriate buffer. Duplicate samples (1.0 ml) wereremoved at intervals and placed in tubes (11 by 75mm) containing 0.25 ml of 1% Na,B47.- 10 H2O or 1.0ml of 0.53% Na,CO,-0.065% KCN solution for thedetermination of N-terminal amino groups (8) andreducing power (17), respectively. The released aminogroups and reducing power were determined by theprocedure described by Welker (32, 33) and expressedrelative to D-alanine and N-acetylglucosamine, re-spectively.

Fractionation of cells into cell wail and nonwallfractions. Cells were ruptured and separated into twofractions (cell walls and nonwall material) by amodification of the procedure described by Sutow andWelker (24). Cells (2.5 to 3.5 g) were transferred to a40-ml Duran flask containing an amount of acid-washed glass beads (0.2 mm) that equaled three timesthe wet weight of the cells. Distilled water was addedto produce a 50% suspension of bacteria (wt/vol) andthe mixture was shaken for three 1-min intervals at2,000 rpm in a Braun model MSK mechanical homog-enizer (Braun Co., Melsunger, Germany). The sus-pension was cooled with intermitent jets of liquidCO,. The slurry was diluted with 30 ml of distilledwater, and the glass beads, intact bacteria, and celldebris were removed by centrifugation at 1,000 x g for10 min. Disruption of the remaining bacteria in theresidue was achieved by a second treatment in theBraun cell homogenizer. After centrifugation at 1,000x g for 10 min, the cell wall suspensions werecombined, diluted to 100 ml with distilled water, andtreated with deoxyribonuclease (10 gg/ml) for 30 minat 37 C. The cell walls were collected by centrifuga-tion at 27,000 x g for 15 min. The cell wall pellet wassuspended in 20 ml of distilled water and centrifugedat 1,000 x g for 10 min. The cell walls were againcollected by centrifugation at 27,000 x g for 15 min,suspended in 10 ml of distilled water, and lyophilized.The low-speed centrifugation pellets (cell debris) andthe high-speed centrifugation supernatant fluids (cy-

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toplasmic fractions) were combined and lyophilized.Extraction of lytic activity from intact cells and

cell fractions. Lyophilized cell walls and nonwallmaterial or intact cells were suspended in 20 to 40 mlof 5 M LiCl and stirred for 1 h at 5 C. The insolublematerial in each sample was removed by centrifuga-tion at 16,000 x g for 20 min and the supernatantfluids containing lytic activity were dialyzed exhaus-tively against 0.04 M tris(hydroxymethyl)amino-methane (Tris)-hydrochloride buffer (pH 7.0).

RESULTSEffect ofpH on lytic activity. In preliminary

experiments lytic activity in crude lysates of B.stearothermophilus 4S(8) was assayed in abuffer (pH 5.7 to 9.0) composed of equal vol-umes of 0.1 M sodium phosphate and 0.1 MTris. Lytic activity was detected over a pHrange of 5.7 to 9.0 with two broad pH optima,one near pH 6.5 and the other near pH 7.5 (Fig.1, dashed line). Similar results were obtainedwhen the lytic activity was assayed in 0.05 MNaH2PO4 (pH 5.7 to 8.0) or 0.05 M Tris-hydro-chloride buffer (pH 7.2 to 9.0) or when H,PO,or maleic acid was used in place of HCI in the

400 -

320 _

:1240 -

( 10I-

pHFIG. 1. Effect of pH on lytic activity. Reaction

mixtures contained 2.8 ml of 0.033M Tris-hydrochlo-ride buffer (pH 7.2 to 9.0) plus 10-3 M EDTA (0),0.017M sodium phosphate buffer (pH 5.7 to 8.0) plus10-3M NaCl (0), or a buffer (pH5.7 to 9.0) composedof equal volumes of 0.1 M Tris and 0.1 M sodiumphosphate (0), and 0.1 ml of crude lysate (21 to 51jsg/ml). After preincubation at 55 C the reaction wasinitiated by the addition of 0.1 ml of substrate (0.375mg acetone-treated cells). One unit of activity isdefined as a decrease in optical density at 410 nm of0.1 in 1 min at 55 C.

Tris buffer (pH 7.2 to 9.0). The trough in thepH-lytic activity curve (Fig. 1, dashed line) be-tween pH 6.5 and 7.5 may indicate a differen-tial effect of phosphate and Tris buffers onlytic enzyme activity rather than the presenceof two lytic enzymes or one lytic enzyme withtwo pH optima. A similar pH-lytic activitycurve was obtained when 0.05 M histidinebuffer (pH 6.0 to 6.5), 0.05 M maleate buffer(pH 6.3 to pH 6.7), 0.05 M imidazole buffer (pH6.6 to 7.5), or 0.05 M Veronal buffer (pH 7.5 to8.5) were used in the assay. These results indi-cate that crude lysates contained lytic activityexhibiting two pH optima. Lytic activity at pH7.5 was assayed in Tris-hydrochloride bufferand lytic activity at pH 6.5 was assayed in so-dium phosphate buffer. In the various crudelysate preparations the relative amount of lyticactivity at pH 7.5 to the lytic activity at pH 6.5(pH 7.5/pH 6.5) was between 2.5 and 3.0.

Effect of buffer concentration and sodiumchloride on lytic activity. Lytic activity at pH7.5 was inhibited by 10-i to 10-2 M NaCl (Table1). A slight stimulation of lytic activity at pH7.5 and pH 6.5 was observed with 10-3 M and10' M EDTA, respectively. Lytic activity in acrude or dialyzed crude lysate was not signifi-cantly affected by Co2+, Mg2+, Ca2+, or Mn2+(10-5 M to 10-3 M). The slight stimulation oflytic activity by ethylenediaminetetraaceticacid (EDTA) must be by some function otherthan by chelating divalent cations.The effect of buffer concentration on lytic

activity was determined over a range of 0.01 to0.1 M (Table 1). Maximum lytic activity at pH6.5 and 7.5 occurred around 0.017 M sodiumphosphate (P buffer) and 0.033 M Tris-hydro-chloride (T buffer), respectively.When the lytic activity in a crude or dialyzed

crude lysate was assayed in P buffer (pH 5.7 to8.0) plus 10-i M NaCl (PN buffer) or T buffer(pH 5.7 to 8.0) plus 10-3M EDTA (TE buffer) adiscontinuous pH-lytic activity curve was ob-tained between pH 6.5 and 7.5 (Fig. 1).The selective use of NaCl or EDTA allows a

clear differentiation and quantitative estima-tion of the two lytic activities in crude lysates.

Isolation and purification of lytic activity.The ,appearance of lytic activity in the superna-tant fluids of mitomycin C-induced cultures ofB. stearothermophilus 4S(8) is shown in Table2. Mitomycin C was added to cultures (0.1jsg/ml) in the early exponential phase of growth(OD at 525 nm of 0.2; 7 x 107 to 108 cells/ml).Lytic activity and phage TP-8 were not detectedin the supematant fluids until the culture beganto lyse (90 min after the addition of mitomycinC). Purified preparations of phage TP-8 (7 x

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TABLE 1. Effect of NaCI, EDTA, and bufferconcentration on lytic activitya

Relative rate of lySisbAddition Lytic act at Lytic act at

pH 7.5 pH 6.5

NaCl and EDTACNone 1.00 1.00NaCl

10-2M 0.19 1.0010-3 M 0.38 1.0410-4M 0.38 1.0010-6M 1.07 0.95

EDTA10-'M 1.20 1.0510-4M 1.07 1.20

Buffer concnd0.01 M 0.06 0.440.017M 0.65 1.000.033M 1.00 0.600.05 M 0.85 0.600.10 M 0.65 0.20

aReaction mixtures contained 2.8 ml of buffer, 0.1ml of crude lysate (21 to 51 ;g/ml), and 0.1 ml ofsubstrate.

D The results are presented as a comparison to therate of lysis in Tris-hydrochloride buffer (lytic activityat pH 7.5) or sodium phosphate buffer (lytic activityat pH 6.5) that were arbitrarily assigned at a rate of1.0.

Additions were made to 0.05M Tris-hydrochloridebuffer (pH 7.5) or 0.05 sodium phosphate (pH 6.5buffer).

d The Tris-hydrochloride buffers (pH 7.5) con-tained 10-3 M EDTA and the sodium phosphatebuffers contained 10-3 M NaCl.

TAmLE 2. Appearance of lytic activity and phageTP-8 in the supernatant fluids of mitomycin Cinduced cultures of B. stearothermophilus 4S(8)

Time after Lytic act"addition of Optical Plaque- (units/ml)mitomycin density formingC (min) (525 nm) units per mla pH 7.5 pH 6.5

30 0.35 0 0 060 0.60 0 0 090 0.75 6.0 x 104 1 21120 0.36 1.1 x 10' 80 60150 0.09 2.8 x 109 225 110180 0.07 4.0 x 10' 470 170

a Phage TP-8 were assayed on restriction-negativestrain 4Sr,4, m- (11).

b Assay conditions as described in Fig. 1.

1010 plaque forming units [PFUJml) did notexhibit lytic activity and lytic activity was notdetected in the supematant fluids of a culturegrown to the stationary phase of growth. Ly-sates of B. stearothermophilus 4S infected with

phage TP-8 also contained lytic activity exhib-iting maximal activity near pH 6.5 and pH 7.5.These data indicate that lysates prepared bymitomycin C induction of strain 4S(8) or phageTP-8 infection of strain 4S contain lytic activityexhibiting two pH optima.

Lytic activity in crude lysates was concen-trated in good yield by the following procedure.The crude lysate (2 liters) was treated with 0.5ALg/ml each of deoxyribonuclease and ribonu-clease for 30 min at 37 C. All subsequentprocedures were carried out at 5 C. The pH ofthe crude lysate was adjusted to 6.0 with 1 NHCl and 472 g per liter of solid ammoniumsulfate was slowly added with stirring (finalsaturation of 70%). After addition of the ammo-nium sulfate, stirring was continued for 12 to 16h. The precipitate was collected by centrifuga-tion at 16,300 x g for 20 min, suspended in 30 to40 ml of SA buffer, and dialyzed exhaustivelyagainst SA buffer. The insoluble material wasremoved by centrifugation at 12,000 x g for 20min.The dialyzed ammonium sulfate fraction was

applied to a column of CM-cellulose. The col-umn was first washed with 1 liter of SA bufferfollowed by a linear gradient of NaCl (0 to 2 M)in SA buffer (total volume of 500 ml). Theelution profile is shown in Fig. 2. Lytic activitiesat pH 6.5 and pH 7.5 were eluted in the samefractions with 0.025 to 0.03 M NaCl. Fractions

I-z

lo

IL00.5 -.

I.-

0.4 I0CDcoi

0.2 00

10. 20 30 40 50 60FRACTION NUMBER

FIG. 2. Purification of lytic activity by CM-cel-lulose column chromatography. Experimental detailsare as described in the text. Elution was with acontinuous gradient of NaCI (O to 2 M) in 0.05 sodiumacetate buffer (pH 5.6). Fractions (6.7 ml) werecollected at the start of the NaCI gradient. Absorb-ancy at 280 nm, A; lytic activity at pH 7.5, 0; lyticactivity at pH 6.5, *.

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containing lytic activity were pooled and storedat 5 C. The lytic activity in the pooled materialwas stable for several weeks at 5 C if theconcentration of protein was relatively high (1to 2 mg/ml). It was necessary with some prepa-rations, however, to reduce the volume of theeluate (increase the protein concentration) bylyophilization. Lytic activity was not adsorbedto DEAE-cellulose and was eluted from phos-phocellulose with 0.3 M NaCl. The two' lyticactivities were not separated by chromatogra-phy on columns of Sephadex G-75 or G-100.

Considerable inactivation of lytic activity wasobserved when the purification procedure wascarried past the CM-cellulose fractionationstep. Table 3 summarizes the results of thepurification through the CM-cellulose fraction-ation. The pH 6.5 and pH 7.5 lytic activitieswere purified 539- and 340-fold, respectively.Recovery of the pH 6.5 and 7.5 lytic activity was37 and 23%, respectively. Lytic activity was notdetected in the fractions that were discardedand the purified lytic preparation did not con-tain TP-8 phage.Although the two lytic activities were not

separated, lytic activity at pH 7.5 was inacti-vated to a greater extent than the lytic activityat pH 6.5. In this preparation (Table 3) the ratioof lytic activity at pH 7.5 to the lytic activity atpH 6.5 was reduced from 2.7 to 1.6.The lytic activity assay was valid for amounts

of protein (purified lytic preparation) whichresulted in changes in OD of not less than 0.01and not more than 0.1 during the 1-min assayperiod. A linear dependence of lytic activity onprotein concentration was observed over a pro-tein concentration of 52 to 343 Wg/ml and 68 to515 ,g/ml for lytic activity at pH 7.5 and 6.5,respectively.The lytic preparations used in subsequent

experiments were obtained from the CM-cel-lulose fractionation step (Table 3).

Effect of temperature on lytic activity.Lytic activity was determined over a tempera-ture range of 20 to 70 C. Maximum lytic activityat pH 6.5 and 7.5 was observed between 60 and65 C. The stability of the lytic activity withrespect to temperature was determined by ex-posing samples of a purified lytic preparation(175 pg) in either P buffer, T buffer, PN buffer,or TE buffer at 60 or 65 C. Samples wereremoved at 1- and 2-h intervals, diluted 10-foldin either PN buffer or TE buffer and assayed forlytic activity at 55 C. The two lytic activitieswere inactivated 96 to 100% after a 1-h exposureat each temperature. The presence ofEDTA orNaCl in the buffer did not affect the extent ofthe inactivation. After 2 h of exposure, theextent of inactivation was 89 to 90% in TE

12

200 -10

6

500

4

2

06df250 20 30 40 50 60

FRACTION NUMBR

FIG. 3. Isoelectric focusing of a purified lytic en-zyme preparation. Experimental details as describedin the text. The pH was determined at 4 C and thelytic activity at pH 7.5 and pH 6.5 was measured ineach fraction (2 ml). Lytic activity atpH 7.5, 0; lyticactivity at pH 6.5, 0; pH, A.

TAsz 3. Purification of Lytic activity

Lytic act at pH 7.5 Lytic act at pH 6.5

ProteinStep" (mg/mlI)Unitu/ Total Pur YieldmP

units Sp act cationM1 nt patcation(10'1) (-fold) (% P (10') (-fold)

Crude lysate (2.7) 10.3 450 9.3 44 1 100 170 3.5 17 1 100Ammonium sulfate 11.2 6,000 5.3 536 12 57 3,850 3.4 344 20 97

fractionation (1.6)CM-cellulose adsorption 0.3 4,500 2.1 15,000 340 23 2,750 1.3 9,167 539 37and elution (1.6)

The number in parenthesis is the ratio of the lytic activity at pH 7.5 to the lytic activity at pH 6.5.h Assay conditions as described in Fig. 1. One unit of activity is defined as a decrease in optical density at 410

nm of 0.1 in 1 min at 55 C.

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buffer, 30 to 58% in PN buffer, and 30 to 50% inP buffer. Inactivation was greatest at 65 C andno differential inactivation of the two lyticactivities was noted.

After 2 h of exposure in T buffer pH 7.5 lyticactivity was stimulated (144 to 150%), whereaspH 6.5 lytic activity was inactivated by 42 to60%. The ratio of lytic activity at pH 7.5 to thelytic activity at pH 6.5 was increased from 1.6 to3.9 and 6.0 after exposure for 2 h at 60 and 65 C,respectively.

Isoelectric focusing. A purified lytic prepa-

ration (specific activity of 16,667 and 10,185units per mg of protein for lytic activity at pH7.5 and 6.5, respectively) was dialyzed againstglycine (1% wt/vol) for 12 to 15 h at 5 C. Thedialyzed preparation was subjected to isoelec-tric focusing in a 110 ml column (LKB Produk-ter AB, Bromma, Sweden) on an Ampholine(pH 7 to 10) and sucrose (0 to 50%) gradientcontaining arginine (1.25 mg/ml) to extend thepH gradient in the alkaline range. The cathodewas at the bottom of the column. After the lyticpreparation was focused for 72 h (4 C) at 700 V,fractions (2 ml) were collected from the bottomof the column and assayed for lytic activity. Thetwo lytic activities were not separated (Fig. 3)and the recovery of the lytic activity at pH 7.5and 6.5 was 23 and 39%, respectively. The ratioof lytic activities at pH 7.5 and 6.5 was identicalin each fraction. These results indicate thatlytic activity at pH 7.5 was inactivated to a

greater extent than the lytic activity at pH 6.5.Ninety percent of the lytic activity was detectedin fractions 14 to 20 (isoelectric point [pI] of 9.5

0.2). A small fraction (7%) of the lytic activitywas detected in a white fluffy precipitate (pI of3.0 a 0.2). The lytic activity was separated fromthe precipitate by washing with 5 M LiClfollowed by centrifugation and when subjectedto a second isoelectric focusing exhibited a pI of9.5. Lytic activity could not be separated fromthe precipitate by washing with distilled water,NaCl, or buffer (PN or TE buffer). The natureof the precipitate was not determined in thisinvestigation. If arginine was not added to theAmpholine gradients (pH 3 to 10 or pH 7 to 10),lytic activity was detected in the cathode com-

partment.Polyacrylamide gel electrophoresis. The

purified lytic enzyme preparation (34 Mg) was

subjected to polyacrylamide gel electrophoresisusing the acid gel system (number 8) describedby Mauer (14) for basic proteins. Duplicate gelswere subjected to 3 mA per gel for 135 min (12C). One gel was stained with 0.01% Analineblue-black in 7.5% acetic acid and the other was

cut into 2-mm segments with a razor blade.Each segment was placed into a tube containing0.3 ml of 0.05 M sodium phosphate buffer (pH7.3) and held at 5 C for 5 days. Lytic activitywas measured in each fraction and a densitome-ter trace of the stained gel was obtained at 560nm on a recording Gilford spectrophotometerwith a linear transport for gel scanning. Thelytic activity in each fraction along with thecorresponding densitometer tracing is shown inFig. 4. Lytic activity (85% recovery) was associ-ated with the major protein band (segments 6through 9). The two minor protein stainingbands did not have lytic activity. After poly-acrylamide gel electrophoresis of a purifiedpreparation (34 to 50 ug) in gels recommendedfor the separation of neutral and acidic proteins(14), five minor protein staining bands weredetected; however, none of them contained lyticactivity. Although three of the eight proteinbands were too faint to appear in the densitome-ter trace, we estimate that nonlytic enzymeprotein contaminants account for approxi-mately 10 to 15% of the total protein. It ispossible that one or more of the protein stainingbands represents inactive enzyme.

Ultracentrifugation. Purified lytic prepara-tions were subjected to ultracentrifugation in

!0.4-

I-0.34

0.2 20

jO.1~~ ~~~~~~~-10 -n

.I IU

52

0 02 4 6 8 10 12 14 16

SEGMENT NUMBERFIG. 4. Polyacrylamide-gel electrophoresis of a pu-

rified lytic enzyme preparation. Experimental detailsas described in the text. Gel was stained with analineblue-black and a densitometer trace was obtained at560 nm on a recording Gilford spectrophotometer witha linear transport for gel scanning. A duplicate gel wascut into 2-mm segments. The lytic activity at pH 7.5and 6.5 was measured in each segment. Lytic activityat pH 7.5, open; lytic activity at pH 6.5, cross lines.

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linear sucrose gradients (5 to 20%) at 208,000 xg for 18 h or in CsCl (1.3527 g/ml of lytic en-zyme) at 176,592 x g for 69 h. The two lyticactivities were not separated. Lytic activity wasinhibited by urea (4 M), sodium dodecyl sulfate(1%), and guanidine-hydrochloride (4 M).These results precluded the use of these protein-denaturing agents in ultracentrifugation stud-ies.Action of lytic enzyme on cell wails. Cell

walls were rapidly solubilized by the purifiedlytic enzyme at pH 7.5 with a 95% reduction inOD after 20 min of incubation. The solubiliza-tion of N-terminal amino groups parallels thereduction in OD and reaches a level of 0.73 gmolof cell wall/mg after 20 min of incubation (Fig.5B).

After 30 min of incubation at pH 6.5, the ODof a cell wall suspension was reduced by 40 to50%. Continued incubation (180 min) did notfurther reduce the OD of the cell wall suspen-sion. Lytic activity at pH 6.5 results in thesolubilization of reducing groups, reaching alevel of 0.04 pmol/mg of cell wall after 30 minof incubation (Fig. 5A).No release of reducing groups or NH2-termi-

nal amino acids occurred in cell wall controls atpH 6.5 or 7.5 after incubation at 55 C for 3 h.

Lysis of cell walls at pH 7.5 was accompaniedby the release 0.42 Mmol of COOH-terminalalanine and 0.39 umol of NH2-terminal glu-tamic acid (Table 4). The release of reducinggroups (1.6-fold increase over untreated cellwalls) probably reflects the presence of residualautolytic enzymes in the cell wall (32). Lysis ofcell walls at pH 6.5 results in an eight-foldincrease in reducing groups and the release of0.04 gmol of COOH-terminal alanine and 0.05Mmol of NH2-terminal glutamic acid. Theamounts of COOH-terminal (0.14 umol of cellwall per mg) and NH2-terminal (0.25 ,mol/mgof cell wall) diaminopimelic acid in the cell wall(33) were unchanged after lysis at pH 7.5 and6.5. No other COOH-terminal or NH2-terminalamino acids were detected. The solubilizationof cell walls with lysozyme unmasked 0.01 ,umolof NH2-terminal amino acid per mg of cell wall.

It could be argued that the release of COOH-terminal alanine and NH,-terminal glutamicacid in cell walls incubated with lytic enzyme atpH 6.5 is a result of endopeptidase activity or tothe presence of autolytic activity in the cellwall. These arguments however, do not ade-quately explain the release of reducing groups atpH 6.5 since the complete solubilization of cellwalls with the lytic endopeptidase of thermo-philic phage TP-1 (32) does not result in the

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FIG. 5. Solubilization of N-terminal amino groupsand reducing power from cell walls treated with apurified lytic enzyme at pH 7.5 and 6.5. (A) Reducingpower at pH 6.5 (0) and pH 7.5 (0). (B) N-terminalamino groups at pH 7.5 (0) and pH 6.5 (0).

release or unmasking of additional reducinggroups.From the data presented we conclude that the

lytic enzyme exhibits some glycosidase activityat pH 6.5.The liberation of COOH-terminal alanine

accompanied by an increase in NH2-terminalglutamic acid is evidence that the lytic activity

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VOL. 120,1974 PHAGE TP-8-INFECTED LYSATES OF B. STEAROTHERMOPHILUS

at pH 7.5 catalyzes the hydrolysis of the L-ala-nyl-D-glutamyl linkage in the peptide subunitsof the cell wall peptidoglycan. The mode ofaction on cell walls is identical to that of thelytic endopeptidase of phage TP-1 (33).The release of reducing power at pH 6.5

indicates the presence of glycosidase activity.The nature of the glycosidic bond was deter-mined after reduction of the cell wall digest (8).Samples of digested and undigested cell wallswere treated with 0.1 M sodium borohydride for3 h at room temperature. The samples weredialyzed against distilled water, lyophilized,and hydrolyzed in sealed tubes with 4 N HCl for4 to 6 h at 105 C. The HCl was removed in vacuoover NaOH and the hydrolysates were exam-ined by two-dimensional chromatography on

Whatman no. 4 paper with pyridine:water (4:1vol/vol) and 2,6-lutidine:water (4:1, vol/vol).Glucosaminitol and muramitol were preparedby borohydride reduction of samples of stan-dard glucosamine (Schwarz/Mann) and mura-mic acid (Cyclo Chemical Corp., Los AngelesCalif.), respectively. Amino sugars and hex-osaminitols were detected with ninhydrin. Two-dimensional paper chromatography of a cellwall hydrolysate before incubation at pH 6.5revealed that muramic acid and glucosaminewere present in approximately equal amounts.After incubation at pH 6.5 followed by reduc-tion, a new component moving with the same Rfvalue as muramitol was detected. These resultsare consistent with but do not prove that thelytic activity at pH 6.5 catalyzes the hydrolysisof the glucosidic bond linking Cl of muramicacid to C4 of glucosamine.Characterization of lytic activity in

autolysates. The effect of pH on autolysis ofcells of strains 4S and 4S(8) is shown in Fig. 6.Autolysis of each strain occurs over a pH rangeof 5.0 to 10.0 with two broad pH optima, onenear pH 6.5 and the other near pH 7.5. Theextent of autolysis of strain 4S(8) was greaterthan that of strain 4S. No TP-8 phage weredetected in the autolysates of strain 4S(8). Cellsof strain 4S(8) exposed to mitomycin C for 90min (10 to 20 min before culture lysis) undergoautolysis to the same extent as the nontreatedculture. The autolysates of induced cells how-ever contained TP-8 phage (106 to 5 x 10. PFUper ml). The data indicate that the presence ofTP-8 prophage and not the events associatedwith phage replication enhance cell autolysis.

Lytic activity can be released from whole cellsor cell walls after autolysis at pH 7.5 or 6.5 or bytreatment with LiCl (7; W. C. Brown, Abstr.Annu. Meet. Amer. Soc. Microbiol., p. 48,

TABLE 4. Reducing power and COOH-terminal andNH,-terminal amino acids in cell walls before and

after treatment with purified lytic enzyme atpH 6.5 and pH 7.5a

pH Reducing NH,- Ala- NH,- Dap-power Glu COOH Dap COOH

6.5Initial 0.005 0 0.03 0.24 0.14Final 0.040 0.05 0.07 0 0Change 0.035 0.05 0.04 0 0

7.5Initial 0.005 0 0.03 0.24 0.14Final 0.008 0.39 0.45 0.24 0.14Change 0.003 0.39 0.42 0 0

a Cell walls (4 to 6 mg) were suspended in 5 ml ofTE buffer or PN buffer and incubated with a purifiedlytic enzyme preparation (200 to 350 gg) at 55 C for 4h. NH,-terminal and COOH-terminal amino acidsand reducing groups were quantitated by the proce-dure described by Welker (33). Values are expressedas micromoles per milligram of cell wall.

1972). Extraction with LiCl was used because(i) strain 4S does not undergo complete autol-ysis even after several hours of incubation at55 C and (ii) autolysates contain cell compo-nents that are difficult to remove and wereinhibitory to lytic activity.Lithium chloride extracts of cells of strains 4S

and 4S(8) were assayed for lytic activity in PNbuffer (pH 5.7 to 8.0) or TE buffer (pH 7.2 to9.0). Two broad pH optima were observedwhich coincided with the pH optima for cellautolysis (Fig. 6) and the lytic activity in phagelysates. The lytic activity in LiCl extracts ofstrains 4S and 4S(8) had a number of otherproperties in common with the lytic activitydetected in phage lysates (data not shown).Lytic activity at pH 6.5 was inhibited (80%) byNaCl (10-3 M). At pH 6.5 and pH 7.5 the lyticenzyme exhibited N-acetylmuramidase and en-dopeptidase (hydrolysis of the L-alanyl-D-glu-tamyl bond of the cell wall peptide subunit)activity, respectively. The two lytic activitiescould not be separated by ion exchange chroma-tography (adsorbs and elutes from carboxy-methyl cellulose with 0.03 M NaCl), isoelectricfocusing (pl 9.5), or polyacrylamide gel electro-phoresis. No attempt was made to free the lyticenzyme of contaminating cell components.

Since cells of the lysogen contained more lyticactivity than the cured strain we were inter-ested in determining whether the lytic enzymewas associated with the cell wall or located inthe cell cytoplasm. Cells of strains 4S and 4S(8)and strain 4S(8) treated with mitomycin C wereruptured in distilled water and separated into

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5.0 6.0 7.0 8.0 9.0 10.0

FIG. 6. Effect of pH on autolysis of B. stearother-mophilus 4S and 45(8). Cultures (1.1 liters of TYGmedium) of strains 4S and 4S(8) were grown at 55 Cto the early stationary phage of growth (opticaldensity at 525 nm of 0.65 to 0.70; 6 x 106 to 7 x 10cells/ml), divided into thirteen 30-mI samples, andthe cells were collected by centrifugation at 4,000 x g

for 15 min. Cells were suspended in 5 ml of 0.017 Msodium phosphate buffer (pH 5.0 to 7.5) plus 10-'MNaCI or 0.033M Tris-hydrochloride buffer (pH 7.0 to10.0) plus 10-3 EDTA. The initial optical density at525 nm (Bausch & Lomb Spectronic -20 colorimeter)was between 0.50 and 0.65. The cells were incubatedat 55 C for 5 min and the optical density of eachsample was again measured. The extent of autolysiswas taken as the percent reduction in optical density(turbidity) after 5 min at 55 C. Strain 4S(8) (circles)and strain 4S (squares). Autolysis in Tris-hydrochlo-ride buffer plus 10-' M EDTA (open symbols) and0.017 M sodium phosphate buffer plus 10-8 M NaCI(closed symbols).

two fractions, cell walls and nonwall material.Lytic activity was extracted from each fractionwith 5 M LiCl and the total lytic activity (at pH6.5 and 7.5) in each fraction was compared inthe two strains (Table 5). The lysogen containedmore lytic activity than the cured strain and inboth strains 82 to 86% of the lytic activity wasassociated with the cell wall. Lytic activity inthe cells of strain 4S(8) treated with mitomycinC for 90 min was detected predominantly (80 to81%) in the nonwall fraction. Most (95%) of thelytic activity in the nonwall fraction was de-tected in the cytoplasmic fluids fraction (high-

speed centrifugation supernatant fluids). Thetotal lytic activity extracted from disruptedcells was usually 10 to 15% higher than thatextracted from intact cells.

Coyette and Shockman (5) reported that 3.2to 4.5 times more autolytic enzyme activity wasassociated with the cell wall when cells ofLactobacillus acidophilus were disrupted in thepresence of 0.01 M sodium phosphate, pH 7.8,than when disrupted in distilled water.The distribution of the lytic activity between

the cell wall and the nonwall material in strain4S(8) and strain 4S(8) treated with mitomycinC did not significantly change (3 to 7% morelytic activity associated with the cell wall) whencells were ruptured in either 0.1 sodium phos-phate (pH 6.5), 0.1 M Tris-hydrochloride (pH7.5), or 0.1 M NaCl.

Since very little activity was detected in thesupernatant fluids of cultures treated with mi-tomycin C for 90 min and identical amounts oflytic activity were obtained from induced andnoninduced cells of strain 4S(8), we concludethat lytic enzyme is released from the cell wallinto the cytoplasm during the pbage lytic cycle.

TABLE 5. Lytic activity associated with the cell wallsof B. stearothermophilus 4S, 4S(8), and 4S(8) treated

with mitomycin C"

Lytic act in strain" (units/lo' cellsc)

4S 4S(8) ~~4S(8) plusFraction tS4S(8) mitomycin C'

pH pH pH pH pH pH6.5 7.5 6.5 7.5 6.5 7.5

Cytoplasm 0.3 0.5 23 65 95 335and celldebris

Cell wall 1.9 2.6 101 325 24 80

a Cultures (1.1 liters ofTYG medium) of strains 4Sor 4S(8) were grown at 55 C to the early stationaryphage of growth (optical density at 525 nm of 0.65 to0.70; 6 x 101 to 7 x 10" cells/ml).

b Assay conditions for lytic activity as described inFig. 1.

c Bacterial counts were determined using a Coultercounter model ZB. The counter was operated with a 30&m diameter orifice and maximum amplification.Coulter counter readings were maintained in the 2 x10' to 20 x 10' range and were performed afterdilution in normal saline which had been filteredthrough a type HA Millipore filter. No correctionswere made in the data for background or coincidencecounts.dA culture of strain 4S(8) was grown for 90 min in

the presence of mitomycin C (0.1 ;&g/ml). A duplicateculture began to lyse 100 to 110 min after the additionof mitomycin C.

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It is possible however that these results do notreflect the actual in vivo distribution of lyticactivity in the lysogen and cured strains.

DISCUSSIONThe present studies were concerned with the

characterization of the lytic activity in phageTP-8 lysates. Crude lysates and purified lyticenzyme preparations exhibit lytic activity withtwo pH optima, one at pH 6.5 and the other atpH 7.5. At pH 7.5 the lytic enzyme is anendopeptidase which hydrolyzes the L-alanyl-D-glutamyl linkage in peptide subunits of the cellwall peptidoglycan and at pH 6.5 it exhibitsN-acetylmuramidase activity. A variety of en-zyme fractionation procedures including ammo-nium sulfate fractionation, ion exchange chro-matography, gel filtration, isoelectric focusing,and polyacrylamide gel electrophoresis wereemployed but the two lytic activities (pH op-tima at 6.5 and 7.5) could not be separated. Thelytic enzyme is a basic protein with a pI of 9.5.Endopeptidase activity is inhibited by NaCland neither lytic activity was significantly af-fected by divalent cations or EDTA. Crudelysates contain 2.5 to 3.0 times more endopepti-dase activity than N-acetylmuramidase activ-ity. The ratio of the two lytic activities (en-dopeptidase/N-acetylmuramidase) decreases to1.3 to 1.7 during the course of purification andincreases from 1.6 to 3.9 and 6.0 after exposurefor 2 h at 60 and 65 C, respectively. Thecombined results indicate that two differentlytic activities are associated with the lyticenzyme. The two lytic activities can be differen-tiated with respect to pH optimum and sensitiv-ity to NaCl, EDTA, and thermostability. Therelatively small number of reducing groupsreleased at pH 6.5 as opposed to the largenumber of NH,-terminal amino acid groupsreleased at pH 7.5, however, makes it difficultto prove the existence of two specific lyticactivities.The combined data indicate that phage ly-

sates contain a lytic enzyme complex composedof two enzymes, each with a different mode ofaction. It is unlikely that the two lytic activitiesare associated with a single protein or that amonomer-dimer transition may account for theendopeptidase activity and N-acetylmurami-dase activity. An example of such a lytic en-zyme was reported by Moo-Penn et al. (15, 16);however, the mode of action of the monomer(pH 5.5) and dimer (pH 8.5) on Escherichia colicell walls was not determined. A final decisionas to the nature of the lytic enzyme(s) in phageTP-8 lysates cannot be made until lytic prepa-

rations are obtained free of other contaminatingproteins.

It is generally accepted that a single, phage-specific lytic enzyme is responsible for therelease of mature phage particles from infectedcells. In most of the phage-host systems re-ported to date only one lytic activity wasdetected in phage lysates. There have beenreports, however, wh.ere crude lysates con-tained more than one kind of lytic activity (6,20). It is possible that some investigators ne-glected to examine the lysates for the presenceof additional lytic enzymes or that additionallytic enzymes were not detected because theywere present in low concentrations. The pres-ence of more than one lytic enzyme in a lysatecan best be detected by a determination of theCOOH- and NH,-terminal amino acids (endo-peptidase of N-acetylmuramyl-L-alanine ami-dase) or the nature of the reducing group (N-acetylglucosaminidase or N-acetylmuramidase)released as a result of lytic activity on cellwalls. Since lytic enzymes present in low con-centrations may be lost during the purificationof the predominant lytic enzyme, these studiesmust be made with crude lysates.

Stewart and Marmur (22) suggested that thelytic enzyme responsible for lysis of B. subtilisafter infection with phage SP82 was not codedfor by the phage genome but was a host auto-lytic enzyme. This hypothesis was based pri-marily on the observation that cells of a mutantof B. subtilis lysed after infection with phageSP82 in the absence of detectable phage-specific DNA synthesis (21). Several other ob-servations suggested that the phage SP82 ge-nome does not code for its own lytic enzyme: (i)lytic activity extracted from phage SP82-infected cells is similar to that from uninfectedcells with respect to the effect of ionic inhibitorsand of temperature on lytic activity and withrespect to substrate specificity (C. R. Stewartand J. Marmur, unpublished data cited in ref.22). (ii) No lysin-deficient phage SP82 mutantshave been isolated (D. M. Green, personalcommunication cited in ref. 22). (iii) No lyticenzyme activity was detected in an in vitroprotein-synthesizing system in which phage T4lysozyme and phage SP82 deoxycytidylate de-aminase were synthesized (L. Gold and M.Schweiger, personal communication cited inref. 22). All of these observations suggest thatphage SP82 lacks a specific lytic enzyme.More than one lytic enzyme (autolysin) has

been identified in autolysates of E. coli (9, 18,29), Staphylococcus aureus (25, 27), B. $ubtilis(4), and B. thuringiensis (10). No one kind of

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BREHM AND WELKER

lytic enzyme can be classified as only phagespecific or host specific (autolytic). This is notsurprising since both classes of lytic enzymeshave as their substrate the bacterial cell wall.

Since the lytic activity in lysates of phageTP-8 and LiCl extracts of 4S and 4S(8) cells or

cell walls have a number of properties in com-

mon and we have not been successful in thesearch of lysin-deficient mutants of phage TP-8,it is tempting to speculate that the lytic en-

zyme(s) is not coded for by the phage genome

but is part of the host autolytic system.Definite proof as to the relationship between

the lytic enzyme in phage lysates and the lyticenzyme extracted from whole cells will beprovided when the enzymes from these twosources are freed of all contaminating protein.

ACKNOWLEDGMENTSThis investigation was supported by Public Health Service

research grant AI-06382 from the National Institute of Allergyand Infectious Diseases. S.P.B. was a predoctoral traineesupported by Public Health Service grant GM-43617 from theNational Institute of General Medical Sciences.

LITERATURE CITED

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2. Black, L. W., and D. S. Hogness. 1969. The lysozyme ofbacteriophage A. I. Purification and molecular weight.J. Biol. Chem. 244:1968-1975.

3. Black, L. W., and D. S. Hogness. 1969. The lysozyme ofbacteriophage A. II. Amino acid and end group analy-sis. J. Biol. Chem. 244:1976-1981.

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

5. Coyette, J., and G. D. Shockman. 1973. Some propertiesof the autolytic N-acetylmuramidase of Lactobacillusacidophilus. J. Bacteriol. 114:34-41.

6. Doughty, C. C., and J. A. Mann. 1967. Purification andproperties of a bacteriophage-induced cell wall pepti-dase from Staphylococcus aureus. J. Bacteriol.93:1089-1095.

7. Fan, D. P. 1970. Cell wall binding properties of theBacillus subtilis autolysin(s). J. Bacteriol.103:488-493.

8. Ghuysen, J. M., D. J. Tipper, and J. L. Strominger. 1966.Enzymes that degrade bacterial cell walls, p. 685-699.In S. P. Colowick and N. 0. Kaplan (ed.), Methods inenzymology, vol. 8. Academic Press Inc., New York.

9. Hartmann, R., J. V. Holte, and U. Schwarz. 1972.Targets of penicillin action in Escherichia coli. Nature(London) 235:426-429.

10. Kingan, S. L., and J. C. Ensign. 1968. Isolation andcharacterization of three autolytic enzymes associatedwith sporulation of Bacillus thuringiensis var.

thuringiensis. J. Bacteriol. 96:629-38.11. Lees, N. D., and N. E. Welker. 1973. Restriction and

modification of bacteriophage in Bacillusstearothermophilus. J. Virol. 11:606-609.

12. 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.

13. Maass, D., and W. Weidel. 1963. Final proof for theidentity of enzymic specificites of egg-white lysozymeand phage T2 enzyme. Biochim. Biophys. Acta78:369-370.

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15. Moo-Penn, W., and H. Wiesmeyer. 1969. The isolationand characterization of coliphage NIOF', and propertiesof the induced lytic enzyme. Biochim. Biophys. Acta178:318-329.

16. Moo-Penn, W., C. Mowry, and H. Wiesmeyer. 1969. Thepurification and physiochemical properties of a lyticenzyme induced by coliphage N,,F'. Biochim. Biophys.Acta 178:330-346.

17. Park, J. T., and M. J. Johnson. 1949. A submicrodetermi-nation of glucose. J. Biol. Chem. 181:149-151.

18. Pelzer, H. 1963. Mucopeptidhydrolasen in Escherichiacoli B. I. Nachweis und Wirkungsspezifitat. Z. Natur-forsch. 18b:950-956.

19. Rao, G. R. K., and D. P. Burma. 1971. Purification andproperties of phage P22-induced lysozyme. J. Biol.Chem. 246:6474-479.

20. Sonstein, S. A., J. M. Hammel, and A. Bondi. 1971.Staphylococcal bacteriophage-associated lysin: a lyticagent active against Staphylococcus aureus. J. Bacte-riol. 107:499-504.

21. Stewart, C. R., M. Cater, and B. Click. 1971. Lysis ofBacillus subtilis by bacteriophage SP82 in the absenceof DNA synthesis. Virology 46:327-336.

22. Stewart, C. R., and J. Marmur. 1970. Increase in lyticactivity in competent cells of Bacillus subtilis afteruptake of deoxyribonucleic acid. J. Bacteriol.101:449-455.

23. Strange, R. E., and F. A. Dark. 1957. Cell-wall lyticenzymes at sporulation and spore germination ofBacillus species. J. Gen. Microbiol. 17:525-537.

24. Sutow, A. B., and N. E. Welker. 1967. Chemical composi-tion of the cell walls of Bacillus stearothermophilus. J.Bacteriol. 93:1452-1457.

25. Takebe, I., Y. J. Singer, E. M. Wise, Jr., and J. T. Park.1970. Staphylococcus aureus H autolytic activity: gen-eral properties. J. Bacteriol. 102:14-19.

26. Taylor, A. 1971. Endopeptidase activity of phage A-

endolysin. Nature N. Biol. 234:144-145.27. Tipper, D. J. 1969. Mechanism of autolysis of isolated cell

walls of Staphylococcus aureus. J. Bacteriol.97:837-847.

28. Tsugita, A., M. Inouye, E. Terzaghi, and G. Streisinger.1968. Purification of bacteriophage T4 lysozyme. J.Biol. Chem. 243:391-397.

29. Van Heijenoort, Y., and J. Van Heijenoort. 1971. Study ofthe N-acetylmuramyl-L-alanine amidase in Esche-richia coli. FEBS Lett. 15:137-141.

30. Weidel, W., and W. Katz. 1961. Reindarstellung undCharakterisierung des fur die Lyse T2-infizierter Zellenverantwortlichen Enzyms. Z. Naturforsch.16b:156-162.

31. Welker, N. E., and L. L. Campbell. 1965. Induction andproperties of a temperate bacteriophage from Bacillusstearothermophilus. J. Bacteriol. 89:175-184.

32. Welker, N. E. 1967. Purification and properties of a

thermophilic bacteriophage lytic enzyme. J. Virol.1:617-625.

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