JOURNAL OF BACrERIOLOGY, Apr. 1970, p. 52-63Copyright 0 1970 American Society for Microbiology

Vol. 102, No. IPrinted In U.S.A.

Derepression of j-Lactamase (Penicillinase) inBacillus cereus by Peptidoglycans'

JOY HOCHSTADT OZER2, DOLORES L. LOWERY, AND ARTHUR K. SAZDepartment ofMicrobiology, Georgetown University Schools ofMedicine and Dentistry, Washington, D. C. 20007

Received for publication 7 January 1970

In Bacillus cereus 569 a cellular inducer of,-lactamase was isolated which has thesame constituents and basic structure as the soluble peptidoglycan found in sporula-tion, extracts from spores, and germination extracts, and which was previously called"spore-peptide." The material has been extensively purified and characterized. Twoacid-soluble, high-molecular-weight peptidoglycan fractions containing muramicacid, glucosamine, diaminopimelic acid, D-aspartate, and D- and L-alanine, -lysine,-glycine, and -glutamate, distinguishable on the basis of size and different amino acidto amino sugar ratios, have been found to be responsible for the observed induction.Both fractions are capable of inducing high levels of 3-lactamase in concentrationslower than those of benzyl penicillin required for optimal induction. Several experi-ments also suggest that it is the accumulation of such soluble peptidoglycan in peni-cillin-treated cells which leads to induction of f3-lactamase and not the penicillinitself. The "spore-peptide" inducer becomes available during sporulation, and en-dogenous derepression ofj-lactamase activity occurs simultaneously. Such derepres-sion also occurs in a strain of B. cereus very sensitive to penicillin and in which bothuninduced as well as "spore-peptide"-induced #-lactamase is a small fraction of thatproduced by the typical penicillinase producer. These results suggest that ,B-lactamasein B. cereus functions in cell wall metabolism during sporulation.

The hypothesis favored by many (see e.g., 34),that penicillinase evolved in bacteria as a resultof offering selective advantage to producingcells by its ability to hydrolyze penicillin, has beenquestioned by others almost since the time theenzyme was isolated nearly 30 years ago. Indeed,Abraham, who fiast reported the presence of theenzyme, expressed doubt both that penicillinasewas the cause of resistance to penicillins and thatits primary function was the hydrolysis of theantibiotic. In this regard, active penicillinaseproducers have been found to be susceptible toas little as 0.05 ,ug of penicillin/ml (13). Abraham(1) postulated that the widespread distribution ofpenicilhinase among various genera of bacteriaindicates that the enzyme might function in acellular reaction unique to the bacteria andinvolving the reaction which penicillin inhibitsin sensitive organisms. Enzymes related to cellwall metabolism fit such a description, and thepossible relationship between penicillinase andcross-linking enzymes was suggested soon after

I Portions of this work were used in partial fulfilment of therequirements for the Ph.D. degree awarded to Joy H. Ozer bythe Graduate School of Georgetown University.

' Present address: Laboratory of Biochemistry, NationalHeart and Lung Institute, National Institutes of Health, Be-thesda, Md. 20014.

52

the discovery of the transpeptidation reactions(43, 46).Previous studies in this laboratory (36, 37)

have shown that several cyclic and linear peptidesas well as penicillins are capable of inducinghigh levels of ,B-lactamase in Staphylococcusaureus and Bacillus cereus 569. Since numerouspeptides, both cyclic and linear, are produced bythe genus Bacillus and since penicillin is also acyclic peptide, we thought it feasible to look forinducing activity in cell-free preparations fromB. cereus. Such an inducer has been isolatedfrom sporulating cultures of B. cereus 569 andfound to be a soluble peptidoglycan with in-ducing activity superior to that of penicillin.Other strains of B. cereus also produced indcingpeptidoglycan. This report describes inductionwith the cellular inducer and presents its partialchemical characterization as well as evidencesuggesting involvement of the a-type f3-lactamaseof B. cereus in peptidoglycan metabolism duringsporulation.

MATERIALS AND METHODSOrganisms. B. cereus NRRL 569 was originally

obtained from N. Citri, Hebrew University-HadassahMedical School, Jerusalem. It is inducible for ,-

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DEREPRESSION OF j3-LACTAMASE IN B. CEREUS

lactamase and produces large amounts of extracellu-lar enzyme when exposed to a suitable inducer. Itwas maintained as a spore suspension (3 X 108 viablespores/ml in 0.15 M NaCi) at 4 C. Various (3-lacta-mase-producing strains of B. cereus (569/H, 5/Band 9592) as well as presumably non-fl-lactamase-producing strains (6464 9, 7064, 4342) were obtainedfrom the American Type Culture Collection, Rock-ville, Md. B. cereus 569 HPen- is a nonreverting,spontaneous, penicillinase-negative mutant obtainedfrom B. cereus 569H. All these strains, as well as alaboratory strain of B. cereus, were maintained onnutrient agar slants at room temperature in the dark.

Penicillins and other antibiotics. Benzyl penicillinused was Penicillin G (Squibb), and methicillin wasStaphcillin (Bristol).

Several other penicillins used were a gift of the lateA. Gourevitch, Bristol Laboratories, Syracuse, N.Y.Bacitracin A, 75% pure, was the gift of L. C. Craig,Rockefeller University, New York, N.Y., who hadpurified it by countercurrent distribution.

Enzymes. Purified B. cereus 569 (3-lactamase per-pared by the method of Kogut et al. (22) was a giftfrom Jacqueline Formica of this department. Bovinespleen deoxyribonuclease I, bovine pancreatic ribo-nuclease A, and crystalline egg white lysozyme werepurchased from Sigma Chemical Co., St. Louis, Mo.Twice-crystallized trypsin was purchased from Worth-ington Biochemical Corp., Freehold, N.J. ChalaropsisN, 0 diacetyl muramidase was the gift of J. T. Park,Tufts University, Boston, Mass. Rabbit muscle L(+)-lactic dehydrogenase, glycerol kinase, and a-glycero-phosphate dehydrogenase were purchased from SigmaChemical Co. Hog kidney D-amino acid oxidase waspurchased from Worthington Biochemical Corp.Butyribacterium rettgeri D(-)-lactate dehydrogenasewas the gift of C. L. Wittenberger, National Institutesof Health, Bethesda, Md.

Specialized chemicals. Pyridine 2,6 dicarboxylicacid [dipicolinic acid (DPA)] was purchased fromAldrich Chemical Co., Milwaukee, Wis. A sample ofchromatographically homogeneous diaminopimelicacid was a gift of the Sigma Chemical Co. Muramicacid was first obtained from J. T. Park of TuftsUniversity, and subsequently purchased from CycloChemicals, Los Angeles, Calif. Both preparationswere homogeneous and identical on paper chroma-tography in several solvents. A purified sample of theN-acetylpyridine derivative of nicotinamide adeninedinucleotide was the gift of C. L. Wittenberger.

Preparation of spores. One liter of casein hydrolysate(CH) medium (22) was inoculated with 3 X 108 viablespores of B. cereus 569 and incubated at 35 C for 48hr with shaking. The culture was then heated to 80 Cfor 5 min and centrifuged at 2 C; the pellet was washedthree times with 0.15 M NaCI, resuspended in 200 mlof 0.15 M NaCl, and dispensed in 5.0-ml portions instoppered test tubes. Spores of B. cereus 569/H, 9592,and 5/B were prepared similarly.

Growth from spore suspensions for induction of (.-lactamase. A 50-ml amount of CH medium wasinoculated with 1.5 X 107 viable spores and incu-bated without shaking for 16 hr at 30 C, after whichincubation was continued for 1.5 to 2 hr at 35 C

with shaking. The cells were collected by centrifuga-tion and resuspended in fresh CH medium to anoptical density of 0.13 at 660 nm. Induction of theenzyme was followed as previously described (36, 37).

Preparation of endogenous inducer (see Fig. 1).A 100-ml amount of an exponential culture in CHmedium was used to inoculate each of four 20-literNalgene bottles containing 15 liters of CH medium;5 ml of n-tributyl citrate was added to each bottleas antifoam, and the sporulating cultures were aeratedfor 24 hr at 35 C. When desired, induced cultures wereprepared by the initial addition of methicillin at afinal concentration of 1 Ag/ml or of benzyl penicillinat a concentration of 3,000 ,xg/ml added initiallyand then twice more at conveniently spaced intervals.In both instances, when cultures were harvested bycentrifugation in a Sorvall continuous-flow apparatusat 2 C, they contained a total of 106 to 107 interna-tional units (IU) of penicillinase in the filtrate, aninternational unit being defined as that amount ofenzyme which will hydrolyze 1 ,m of benzyl penicillin/min at 30 C, pH 7.0. (To convert the IU to the morecommonly used Perret unit, multiplication by 60may be performed.) The collected cells were stored asa paste at -20 C. For inducer preparation, the or-ganisms were thawed at room temperature; a 15%suspension (v/v) was made in distilled water andsonically disrupted in a Branson 20-kc Sonifier. Thesupernatant fluid after sonic disruption was extractedwith 10% (final) trichloroacetic acid at 1 C for 10min. The soluble fraction was collected, extracted threetimes with three volumes of ethyl ether to remove thetrichloroacetic acid evaporated to remove the ether,and then stored at -20 C. Such treatment yieldedmaterial which was stable for at least 3 to 4 monthsat -20 C.

Enzymatic treatment of extract. Crude and acid-soluble extracted materials were treated as follows.A 1-ml amount of the supernatant fluid obtained bycentrifugation of the crude extract at 12,000 X gfor 10 min, or about 20 ,ug of acid-soluble inducingmaterial, was treated with 1 mg each of deoxyribo-nuclease, ribonuclease, or trypsin, or a combinationof all three, in a volume of 1 ml. For reasons to bediscussed later, 20 ,ug/ml bovine pancreatic ribonucle-ase A and 20 Mg/ml of bovine spleen deoxyribonucle-ase I were employed in the preparation of highlypurified inducing material. Incubations were at 35 Cfor 90 min in the following buffered solutions. Deoxy-ribonuclease I treatment was at pH 5.4 in 0.01 Msodium acetate, 0.02 M MgSO4; ribonuclease treat-ment was at pH 7.8 in 0.02 M tris(hydroxymethyl)-aminomethane (Tris)-hydrochloride; trypsin treat-ment was at pH 7.0 in 0.05 M Tris-hydrochloride.Enzyme was removed by trichloroacetic acid precipi-tation (10% final). The trichloroacetic acid, salts, andany hydrolytic products were then removed by dialy-sis. In experiments where treatment was with morethan one enzyme, the order was deoxyribonuclease,ribonuclease, trypsin. Prior to treatment with eachsucceeding enzyme, dialysis was often carried outagainst the buffer used with the subsequent enzyme.

Gel filtration. For further purification by gel filtra-tion, the acid-soluble enzyme-treated fraction (Fig. 1)

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Supernatant fraction after sonic disruption

I 10% TCA 1 CCentrifuge

Ether extract 3X,Neutralize

RNAse, DNAse

10% TCACentrifuge

Supernatant fraction Sediment

Dialysis, 10- M EDTA

Dialysis against water 3X

Fractionation on Biogel P150

Resolution of three major components

FIG. 1. Preparation of cellular inducer of penicil-linase. Abbreviations: TCA, trichloroacetic acid;RNAse, ribonuclease; DNAse, deoxyribonuclease;EDTA, ethylenediaminetetraacetic acid.

was first dialyzed against 10-4 M ethylenediamine-tetraacetate (EDTA), pH 8.0, to remove residualDPA, followed by dialysis three times against dis-tilled water. The dialysate was concentrated by evapo-ration under reduced pressure at 40 C to containapproximately 5 mg/ml (dry weight) and then cen-trifuged for 75 min at 48,000 rev/min in the no. 50rotor of a Spinco L-2 centrifuge. The rather rigorouscentrifugation for removing particulate matter wasnecessary because of the apparent viscosity of theconcentrated preparation. Filtration was throughBiogel P-150; 100 to 200 mesh, in 0.01 M Tris-hydro-chloride buffer (pH 7.6) in a column with bed di-mensions (1.5 by 113 cm). A flow rate of 10.8 ml/hrwas regulated by pump. The total included volumewas marked by applying the sample to the column inbuffer containing tritiated water. Effluent was moni-tored continuously at either 224 or 226 nm with aBeckman DB spectrophotometer, and fractions of1.8 ml were collected. Tubes with absorption peakswere analyzed for inducing capacity and chemicalconstituents.

Sedimentation. Samples (0.2 ml) of inducing ma-terial suspended in 5% sucrose were layered on thetop of a 5 to 20% (v/v) sucrose gradient formed by a

pump over 0.4 ml of CsSO4 in 25% sucrose (a = 1.78)in cellulose nitrate tubes. All solutions used in pre-paring the gradients were made up to 0.15 M NaCl in0.01 M Tris-hydrochloride buffer, pH 7.4. A replicatetube containing three sedimentation markers wasincluded in the run. The markers were chosen todiffer not only in their sedimentation characteristicsbut also in their absorption spectra, thus allowing forsimple determination of their positions. The markersused were thyroglobulin (1 1.6S, detected by absorp-tion at 280 nm) hemoglobin (4.3S, detected by ab-sorption at 430 nm) and cytochrome c (1.2S, de-tected by absorption at 550 nm). Centrifugation wasfor 28 hr at 35,000 rev/min at 4 C in a Spinco L-2/50ultracentrifuge with an SW39 rotor. Fractions (0.2ml) were collected by puncturing the bottom of thecentrifuge tube, and equivalent fractions from twocentrifuge tubes were pooled.

Preparation of hydrolysates of inducing extractsand cell fractions. Chemical analyses and paperchromatography of amino acids and hexosamines wereperformed after hydrolysis of appropriate sanples in6 N HCI in Teflon-capped tubes for 4, 7, or 24 hr at121 C. The HCI was removed by evaporation to dry-ness at 40 C under reduced pressure. Distilled waterwas added and the evaporation procedure was re-peated three times to remove residual HCI.

Hexosamines were quantitatively determined chem-ically after hydrolysis in 2 N HCI for 2 hr at 100 C.Hydrolysates of materials to be run through an auto-mated amino acid analyzer were prepared fromlyophilized samples suspended in a large tube in con-stant boiling HCI. The tube was evacuated andsealed. Hydrolysis was carried out at 110 C for 14 to40 hr. HCI was removed by lyophilization. For analy-sis of nucleotides in certain preparations, the samplewas dried by evaporation under reduced pressure andthen over P205 in a vacuum desiccator for 2 hr. A0.2-ml amount of 72% perchloric acid was added at0 C; the sample was then treated at 100 C for 1 hrand brought to 0 C, and 0.5 ml of water at 0 C wasadded. The pH was then adjusted to between 4 and 5with KOH at 0 C. The sample was centrifuged and thesupernatant fluid was analyzed for free bases bypaper chromatography. Two measured amounts ofpurified deoxyribonucleic acid and, ribonucleic acidwere treated in the same way as the experimentalsamples.

Analytical procedures. Paper chromatography ofhydrolysates was performed on Whatman no. 1 paperin descending one- an two-dimensional chroma-tography by using the following solvent systems:(I) phenol-water, 1: 1 (lower phase); (II) n-butylalcohol-acetic acid-water, 4:1:5 (upper phase); (III)n-butyl alcohol-pyridine-acetic acid-water, 30:20:6:20; (IV) pyridine-water, 80:20 (15); (V) isobutyricacid-i N NH4OH, 5:3 (25); (VI) n-butyl alcohol-acetic acid-water, 3:1:1 (44); (VII) tertiary butanol-methyl ethyl ketone-formic acid-water, 40:30:15:15.

Ascending chromatography was used with thefollowing solvent: (VIII) 11.5 N HCI-isopropanol-water, 16.7:65:18.3.The solvent systems listed were employed to sepa-

rate chemical compounds as follows.

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Two-dimensional chromatography in solvent VIIfollowed by I was used for detection and separation ofpossible imino acids.

Two-dimensional chromatography in solvent VIfollowed by IV was used for separation of peptido-glycan components.

One-dimensional chromatography in solvent IIwas used to separate glucosamine from alanine andmuramic acid.

One-dimensional chromatography in solvent IIIwas used to separate muramic acid from alanine andglucosamine.

One-dimensional chromatography in solvent Vwas used to compare the mobility of the soluble B.cereus peptidoglycan, uridine diphosphate precursorsand their hydrolysates with soluble intermediatesreported to participate in peptidoglycan synthesis (25).

One-dimensional ascending chromatography for 48hr in solvent VIII was used for the detection of purinesand pyrimidines. Samples were examined under ultra-violet illumination for comparison with known purinesand pyrimidines.Amino acid and amino sugar spots were developed

with 0.2% ninhydrin in acetone, or 0.2% ninhydrinin a solvent containing: cadmium acetate, 0.5 g;water, 50 ml; acetic acid, 10 ml; and acetone, 500 ml.Chromatograms were developed at room temperature.Reducing sugars were detected by the alkaline AgNO3method of Trevelyn et al. (44). Commercial sprays(Sigma Chemical Co.) containing bromocresol green,tetrazolium salts, and isatin were also used for thedetermination of certain ionized substances, reducingcompounds, and certain amino acids, respectively.Identifications of amino acids were made by com-parison with known standards in four or more solventsystems.

Phosphate determinations. Inorganic phosphate wasdetermined by the method of Fiske and SubbaRow(14) and total phosphorus was determined by themodification of King (21).

Carbohydrate determinations. The presence ofsimple sugars was evaluated as glucose equivalentsby the phenol-sulfuric acid technique of Dubois et al.(11) as described by Ashwell (2). Differentiation ofsimple hexoses was determined by the method ofDische and Shettles (10) and later by the recent modi-fication of Dische and Danilchenko (9). Glucose andrhamnose were used as standards. Reducing sugar wasdetermined as glucosamine equivalents by the methodof Park and Johnson (30). Free hexosamine wasdetermined by the Elson-Morgan reaction with themodification of Immers and Vasseur (18), which isnecessary when determinations are being carried outin the presence of amino acids, particularly lysine.Muramic acid was determined by measurement of itscarboxyethyl moiety as lactate by a modification ofthe Barker anrd Summerson procedure (3, 4) specificfor muramic acid, suggested by Strange and Dark(40, 41).

Diaminopimelic acid determination. Diaminopimelicacid was determined by the method of Work (47).DPA determination. DPA was detected qualita-

tively only, by the method of Janssen et al. (19).Enzymatic determinations. Glycerol and fl-glycero-

phosphate were determined on hydrolysates made bytreatment in 2 N H2SO4 for 2 hr at 100 C. The hy-drolysates were neutralized by addition of Ba(OH)2,and the resultant precipitate was removed by centri-fugation. Enzymatic analyses, with glycerolkinaseand a-glycerophosphate dehydrogenase, were thenperformed (20).The stereospecificity of lactate derived from

muramic acid was determined on hydrolysates byusing rabbit muscle L(+)-lactic dehydrogenase(Sigma) and B. rettgeri D(-)-lactate dehydrogenase.The stereospecificity of amino acids in hydrolysates

of peptidoglycan fractions was carried out with D-amino acid oxidase by using the conditions describedby Burton (8). Assay was by comparison of quantita-tive amino acid oxidase-treated and untreated hy-drolysates (26). L-Arginine (0.2 Am) and D-leucine(0.2 ,um) were included in the assay mixtures as speci-ficity and reaction completion controls, respectively.

Amino acid analysis. Amino acids were automati-cally analyzed by the method of Miller and Piez (26)after hydrolysis. Chromatography was performed atseveral temperatures to ascertain that all peaks ofelution behaved as would be expected of homogeneoussubstances. Diaminopimelic acid was found to elutewithout overlap between valine and methionine.Muramic acid was not completely separated fromserine, but corrections could be made because ofdifferential rates of destruction upon prolonged hy-drolysis. The same was true of the unsubstitutedhexosamine which eluted near valine and overlappedit when large quantities of hexosamines were found.Amino sugars were measured colorimetrically by othermethods already described.

Standardization of dry weight of inducer with nin-hydrin. Duplicate samples of between 200 and 250 mgof lyophilized extract from each of two batches of cellswere further purified as described; they were thendried, and the final products were weighed in pre-weighed vessels. Weight was standardized againstninhydrin values of an amino acid standard. Theninhydrin test used throughout the study was that ofMoore and Stein (28). All weight values for inducerfractions therefore are based on their reactions withninhydrin.

Preparation of other cell fractions. Germinationfiltrates were prepared by the method of Powell andStrange (32). Insoluble peptidoglycan was preparedby the method of Park and Hancock (31) from thecold trichloroacetic acid-precipitable material de-rived from both vegetative and sporulating cultures.Spore peptidoglycan was prepared by partial disrup-tion of a 4% (v/v) suspension of spores by sonicdisintegration for 20 min at a temperature below10 C. The resulting suspension was treated with tri-chloroacetic acid (10% final) in the cold for 10 minand centrifuged; the supernatant fluid was dialyzedagainst many changes of distilled water.

Similar fractions from purified cell walls of S.aureus and Escherichia coli were prepared by thesame method, except that 15% (v/v) suspensions ofthe highly purified cell walls of S. aureus and partiallypurified walls of E. coli were used. The sonic disrup-tion time was 5 min.

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Soluble nondialyzable peptidoglycan from Micro-coccus lysodeikticus (strain from this laboratory) wasprepared by the method of Mirelman and Sharon(27). Nucleotide-amino sugar-peptide precursors ofpeptidoglycan were prepared from B. cereus 569 bythe method of Park and Chatterjee (29) with thesingle following modification. Nucleotides wereadsorbed on charcoal, the nonnucleotide-containingmaterials were washed away with water, and thenucleotides were then eluted with 0.02 M (NH4)2CO3.The eluent was made to pH 5.0 with 0.1 N HCI, andnucleotide-amino sugar peptides were separated fromother nucleotides on Dowex 50 at 4 C by water elu-tion of most nucleotides and 1 N NH4OH elution ofpeptide-containing nucleotides. NH4OH was removedby immediate lyophilization. Both charcoal-elutedand Dowex 50-eluted fractions were neutralized andtested for inducing activity and were analyzed chem-ically.

Cell wall preparations. Highly purified cell walls ofS. aureus (Oeding strain 8507) were generouslydonated by E. Huff, National Institutes of Health,Bethesda, Md. Fractionated cell wall material fromE. coli 0113 was a gift from A. L. Jackson, MelparCorp., Falls Church, Va.

Treatment of soluble peptidoglycans with chalarop-sis N,O-diacetylmuramidase, egg white lysozyme,and B. cereus 569-jS-lactamase. Equal weights ofpeptidoglycan and Chalaropsis enzyme, prepared bythe method of Hash and Rothlauf (16), were mixedtogether. For treatment with enzyme, purified B.cereus-soluble peptidoglycan was exposed to 10 timesas much lysozyme (by weight). Finally, to determinethe effect of ,3-lactamase on the inducing material, 20,ug of soluble peptidoglycan was exposed to 100 unitsof the enzyme in 0.02 M phosphate buffer (pH 6.8)for 30 min. The O-lactamase was then removed bylowering the pH to 5.0 with 0.5 M sodium acetatebuffer, and was adsorbed on celite 545 at 4 C for 3hr. The celite was removed by centrifugation, and thesupernatant fluid was readjusted to pH 6.8 by dilutionin the buffer. Most of the ,B-lactamase activity wasremoved, and the small residual activity was sub-tracted from subsequent induction assays.

Treatment of insoluble peptidoglycans and- wholecells. Treatment of insoluble peptidoglycan and wholecells was the same as for soluble peptidoglycan exceptthat cell walls or cells were diluted to an optical densityof 0.5 at 660 nm and were reacted overnight withshaking. Chalaropsis enzyme was at a concentrationof 100 ,g/ml, egg white lysozyme was used at 50 to1,000 ,ug/ml, and B. cereus ,3-lactamase was at 102 to104 units/ml.

RESYULTSInduction of /3-lactamase with crude and par-

tially purified cell extracts. Experiments per-formed with 10,000 X g supernatant fluids ofcell homogenates prepared by treatment of 15%(v/v) cell suspension in a Branson 20-kc Sonifierrevealed that, though all cultures were preparedidentically, some supernatant fluids induced/3-lactamase in the homologous strain whereas

others did not. The lack of reproducibility wasfound to be due to a factor present in such crudeextracts which was capable of either breakingdown or inhibiting ,B-lactamase. Such activityfrequently could be lessened by dialysis againstdistilled water or simply by dilution of the ex-tract. No attempt was made to determine thenature of the factor. The inducing material inthe crude extract was labile but could be sta-bilized by treatment with cold 10% (final) tri-chloroacetic acid. The precipitate was discardedand the trichloroacetic acid was removed fromthe supernatant fluid by dialysis or ether extrac-tion. Such acid-soluble extracts retained in-ducing activity for several months at -20 Cwithout diminution and were heat-stable, re-taining more than 75% of the original inducingactivity after treatment at 100 C for 10 min. Thematerial responsible for induction was notinactivated by treatment with ribonuclease,deoxyribonuclease, or trypsin, alone or in com-bination.When the 10% acid-soluble preparation was

scanned in a spectrophotometer between 230and 800 nm, the only peak observed was at 262nm. This absorbancy was only partially removedby ribonuclease, and treatment in addition withdeoxyribonuclease did not lower it. The residualabsorbancy was found not to be due to nucleicacids, since chromatographic analysis of 70%perchloric acid hydrolysates of the fractiondesigned to detect nucleic acid components,even if they contributed only one third of theabsorbancy observed at 262 nm, did not revealpurines or pyrimidines. Ether extraction of thepartially purified preparation from 10% tri-chloroacetic acid did not reduce activity nor wasthere any noticeable quantity of lipid present inthe ether layer.

Figure 1 shows the method of purification ofthe inducing material. The material present inthe fraction just prior to EDTA and Biogeltreatment, and which by analytical ultracen-trifugation had three components, representsless than 1% by weight of the material presentin the supematant fluid after centrifugation ofthe crude extract arising from treatment of thecell suspension in a Branson 20-kc Sonifier.Its activity was undiminished by treatmentwith lysozyme, Chaloropsis N,O-diacetylmur-amidase or ,B-lactamase. Three experimentsshowing typical results obtained with inducingmaterial purified as far as the above stage areshown in Table 1. A certain amount of varia-bility in levels of induction observed was foundfor all inducers, but the particular variabilityof the crude extract was not so much in thelevel of induction observed on different days but

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was largely determined by factors related to thebatch of cell extract (compare crude superna-tants fluids in experiments 2 and 3). In experi-ment I, soluble peptidoglycan from S. aureus

induced f-lactamase in B. cereus 569; however,no induction was observed when intact cell wallswere used. The result shown here was obtainedonly after highly purified cell walls of S. aureus

known to contain an autolytic enzyme (17) were

ultrasonically disrupted and then acid-extractedand dialyzed. The mechanism of action of thisenzyme is not yet known.

It did not seem to matter whether or not theculture giving rise to the inducing material wasinduced with penicillin prior to disruption. It wassubsequently learned, however, that culturesforming active inducer usually contained largeamounts of extracellular f,-lactamase. As cellswere washed before being disrupted, the jl-lactamase did not contribute to values measuredin the assay of the crude extract. Table 2 showsthat soluble peptidoglycans prepared from otherB. cereus stains induce ,B-lactamase formation instrain 569. (When these experiments were per-

formed, it was already known that the inducingmaterial was a peptidoglycan.) Conversely, pep-

tidoglycan from strain 569 also stimulated ,B-lac-tamase production in strain 9592, the other in-ducible strain tested. These results indicate thatthe observations made are not specific solely tostrain 569.

Extracts from partially purified cell wallsprepared in similar manner from E. coli did notinduce ,3-lactamase in B. cereus, nor did acid-soluble lysozyme digest of M. lysodeikricus.Uridine nucleotide cell wall precursors of B.cereus 569 cell walls were prepared by a modifi-cation of the method of Park and Chatterjee(29) and did not induce; however, no deter-mination was made as to whether these phos-phorylated compounds could be taken into the569 cells. Insoluble mucopolymer preparedaccording to Park and Hancock (31) also didnot induce, but a trichloroacetic acid-soluble,nondialyzable preparation obtainable from theinsoluble mucopeptide after the latter was storedfor many months at 4 C did induce. The identityof each of these materials as cell wall precursor orfragment was confirmed by paper chromatog-raphy after acid hydrolysis. Bacitracin andvancomycin, known to interfere with cell wallmetabolism, did not cause induction of ,8-lac-tamase. Vancomycin caused lysis but bacitracindid not.When it was observed that sporulating cells

were a particularly good source of inducingpeptidoglycan, the association of DPA with theinducing fractions was investigated. DPA was

TABLE 1. Induction in Bacillus cereus 569

Inducera O-Lactamaseobservedb

Expt I

None... 0.2Benzyl penicillin, 1 ,g/ml. 3.8Methicillin, 1,ug/m. 4.3Trichloroacetic acid-soluble fraction,

1.8jAg/mg 1.9Trichloroacetic acid-soluble fraction,

enzyme treated, 0.75 jAg/mI. 2.5Soluble peptidoglycan from S. aureus. 1.7Whole cell walls from S. aureus. 0.2

Expt 2None. 0.2Benzyl penicillin, 1 ug/ml. 6.0Methicillin, 1 Ag/ml. 5.7Crude supernatant fraction, 500 jAg/ml.... 3.8Trichloroacetic acid-soluble fraction

1.8 jg/ml. 1.81.2,ug/ml. 3.80.75 g/mi.m 3.1

Expt 3None. 0.5Benzyl penicillin, 1 j,g/m. 4.5Methicillin, 1 ,ug/ml.. 3.2Crude supernatant fraction, 500 jig/ml.... 0.7Trichloroacetic acid-soluble fraction,

1.8jAg/ml. 3.6Trichloroacetic acid-soluble fraction,

enzyme treated1.2,/g/ml. 2.70.75 5Ag/mlg 1.9

a The trichloroacetic acid-soluble fraction rep-resents the supernatant fluid obtained after treat-ment of the cell suspension in the Branson 20-kcSonifier followed by centrifugation (crude super-natant) and then ether extraction to remove theacid (see Fig. 1). "Enzyme treated" means thisfraction treated with ribonuclease and deoxyribo-nuclease. Soluble peptidoglycan from S. aureuswas prepared as described in the Methods section.Crude supernatant fractions were different prep-arations: trichloroacetic acid-soluble fractionswere the same preparations but were used in dif-ferent experiments.

b Expressed as international units per milliliterof inducing system.

found associated with all peptidoglycan materialfrom B. cereus 569 except when the peptido-glycan was derived from vegetative cells. DPAcould be removed from the soluble material bydialysis against 104 M EDTA (pH 8) withoutloss of inducing activity. DPA alone did notinduce when tested at 10 to 50 ,ug/ml, nor didany of the peptidoglycan constituents alone orin combinations induce, unless all were added

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in 100 times the concentrations represented bytheir proportion in the amounts of peptidoglycanactive in induction. The results of such experi-ments are shown in Table 3.

f3-Lactamase production in uninduced acltesand distribution of the inducer. Under the con-ditions of the induction assay, the high levels ofj3-lactamase occurring in the absence of the addedinducer occasionally made it necessary to discardthe results of entire experiments. Such highlevels indicated that control of synthesis of P-lactamase did not depend solely on the additioffof exogenous inducers. Since all experiments wereperformed with precisely identical techniquesand procedures, it seemed likely that unknownand subtle factors were responsible for thesevariations. It was considered possible that thephysiological state of the cell could be responsiblefor the discrepancies. Accordingly, experimentswere performed to correlate j3-lactamase pro-duction with various stages of the growth cycle.An experiment with B. cereus 569 observed hourlyfor 19 hr after inoculation of a spore suspensioninto nutrient broth is shown in Fig. 2 (data col-lected by C. F. Garon of this laboratory). ,B-Lactamase appears endogenously without addedinducers at that point where sporulation beginsand increases as sporulation proceeds. Figure 3shows this same experiment with the ,-lactamaseactivity calculated per viable cell. Here highlevels of 13-lactamase are present in germinatingspores as well as during spore formation. Whencultures of different ages were assayed for ,B-lactamase activity, the activity per milliliter ofculture was low until the end of logarithmicgrowth and high during sporulation (Table 4).This was equally true of the availability of thepeptidoglycan inducer (Table 5). Sporulationand sporulating cells are defined as the appear-ance of refractile spores within sporangia. Table5 also shows that the soluble, nondialyzablepeptidoglycan is available from sporulating cells,partially disrupted spores, and germinationfiltrates, making it similar in all observed respectsto the material called spore peptide as describedby Powell and Strange (32, 42). The derepressionof sporulating cultures of several ,-lIctamase-producing strains of B. cereus (Table 4) indicatesthat both inducible strains (defined by theirenhanced production when treated with penicil-lins) and constitutive strains (defined by theirunresponsiveness to penicillin) produce oraccumulate increased amounts of ,B-lactamase atsporulation. The only strain which produced no,-lactamase was the ,-lactamase-negative HPen7which is a nonreverting mutant of 569/H.

,B-Lactamase production In a penicillin-senitivestrain. When strains of B. cereus were tested for

ability to produce ,-lactamase spontaneously, alaboratory strain of B. cereis (sensitive to <1.0,ug/ml) previously considered not to produce,-1ctamase was found to form s i tlevels of the enzyme, though lower than any ofthe other strains. Not only does the sensitivestrain produce ,-lactamase when indoced bypenicillin or spore peptide but, most intesinly,it is endogenously fully induced at sportion(Table 6).

Effect of j3-actanase on cells and cell faI_Exposure of insoluble peptidoglycan to 3-lac-tamase at 37 C for 18 hr did not reduceturbidity.8-Lactamase had no obvious effect on the in-ducing peptidoglycan, growth or morpholodgo oflog-phase cells, morphology of spore suspen- s,or subsequent ability of spore suspensions togerminate. Assay for morphology and gr6wthwas after 3 hr of incubation of the enzyme withspores or cells.

Charcterization and activity of furthr-pwMredinducr. Preliminary characterization of unpurefractions indicated that the inducing activityresided in a peptidoglycan structure in materialsfrom both B. cereus and S. aureus.

Greater purification of the B. cereus. 569peptidoglycan-rich material wasby further resolution of the material on BgelP150 (see Fig. 1). All the material was taken A-tothis gel. A previous experiment using BiogelPE6showed two peaks to be included and one'.ex-cluded. Figure 4 shows the fractionadon 6,b-tained by use of Biogel P150. Only frctois Iand II exhibited inducing activity. Fractiw Itaand III did not induce. (Peak IA was assumed tobe contaminated with peak I and was not tested.)Absorption spectra were carried out on all thefive fractions. Only fractions H and Ha shoWedany absorption peaks, both absorbing at 260nm, where fraction II had the same opticalactivity as did Ha. Fraction IHa did not haveinducing activity nor did it react with ninhydrin.It was not characterized further. The,relitfreareas proscribed under the curves by absorbanceat 226 nm did not give an accurate reprnokof the relative amount of total material in achipeak. By adding the concentrations of consit-uents found upon hydrolysis, peak I and tarepresent 40% of the total; peak II represnts45%, and peak HI, 15%. Peak Im, the scomponent, which elutes just before the triti-tedwater marker, was found to be entirely j -peptide and contained small amounts of di-aminopimelic acid. Table 7 shows that iqthinducing components are peptidoglycans. Ratiosof the components of the inducing fractions -(and H) were compared with glutamate whisarbitrarily given a value of 1.0. Fraction H',

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I Ia

z

>

0

4a>- %

70

r,60 30

so

40 cz

30

20;

I 0

0

SPORES I VEGETATIVE GATM IAAULATICH IREFCTILE SPOSa A a a a a

1 3 5 7 9 I1 1 13 15 17 1TIME IN HOURS

FIG. 2. Growth and ,j-lactamase activity in Bacilluscereus 569; spontaneous appearance in the absence ofexogenous inducer. Viable spores (9.1 X 106) of B.cereus 569 were inoculated into 100 ml of nutrientbroth and incubated at 37 C. At zero time and at hourlyintervals thereafter, viable cell counts were made,j3-lactamase activity in the culture was determined,and spore counts were made under bright-phase mi-croscopy. j3-Lactamase units plotted here are Perretunits; to convert to international units, divide by 60.

TABLE 2. ,3-Lactamase induction in various strainsof Bacillus cereus by peptidoglycans from

various strains of B. cereus

P-LactamaseStrain induced Inducer (0.5 pg/ml) activitya

B. cereus 569 0.1B. cereus 569 Benzyl penicillin 2.7B. cereus 569 569 Peptidoglycanb 1.9B. cereus 569 569H Peptidoglycan 1.6B. cereus 569 5/B Peptidoglycan 2.0B. cereus 569 9592 Peptidoglycan 1.5B. cereus 569 569H Pen- Peptido- 1.5

glycanc

B. cereus 9592 <0.1B. cereus 9592 Benzyl penicillin 6.3B. cereus 9592 569 Peptidoglycan 0.9

B. cereus 569Hd 0.9B. cereus 569H 569 Peptidoglycan 1.0

a Expressed as international units per milliliterof induction system.bWeight of this material determined after batch

preparation; weight of inducers from other strainsestimated by adjusting to equal ninhydrin values.Peptidoglycans used here are those fractions priorto EDTA and Biogel treatment.

- Prepared after growth in potato extract me-dium; others grown in casein hydrolysate me-dium.

d B. cereus 569H is a constitutive producer of3-lactamase. B. cereus 9592 is an inducible pro-ducer.

TABLE 3. Induction of,-lactamase inBacillus cereus 569

Limit ofInducer Concn inductionInducer ~~(#g/mi) ratio"

observed

Benzyl penicillin...1 >25.0Peptidoglycan inducer. 1 >10.0Muramic acid...... 5-20 <1.5D-Glucosamine.. 10-30 <2.0N-Acetyl glucosamine. 20 <1 .5Diaminopimelic acid... 10 <1 .5Lysine..... 10 <1.5Glycine...... 10 <1.5L-Alanine...... 10 <1.5D-Alanine. 5-20 <2.0L-Glutamate. 10 <1.5D-Glutamate. 10 <1.5Dipicolinic acid.. 10 <1.5Amino acid-amino sugarb 1-20 <2.0Amino acid-amino sugarb.. 100 4.0-7.0

a Since the results are compiled from a numberof separate experiments, an induction ratio rep-resenting units per milliliter induced/units permilliliter uninduced has been calculated for eachexperiment. Twofold differences are occasionallyseen between duplicates in the assay system; five-fold differences are considered significant.

b A solution containing N-acetylglucosamine,D-alanine, L-alanine, D-glutamate, glycine, dia-minopimelic acid, and lysine in molar ratios of2.0:1.0:1.0:1.0:0.2:1.0:0.2, respectively. Peptido-glycan inducer is fraction treated with EDTA, butprior to Biogel chromatography.

I

<< 10\u~~~~~

4~~~~~~

-a10.

O ~~~~g0

z0 . I ID 0 1 3 5 7 9 11 13 15 17 19

TIME IN HOURS

FIG. 3. ,8-Lactamase activity during growth. Samedata as Fig. 2, but j8-lactamase activity calculated perviable cell. Units plotted are Perret units; to convertto international units, divide by 60.

which is less substituted than fraction I as indi-cated by higher hexosamine-to-amino acid ratiosthan fraction I and more glycine than alanine,thus resembles spore peptidoglycan more than

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TABLE 4. Appearance of fl-lactamasein sporulation"

Stai -Lactamase activit P-atmsStrain at end of log phase activity whenespores are visible'

Bacillus cereus569.. 0.2 2.3569He... 1.0 4.0H Pen-.. <0.2 <0.25/Bc..... 4.7 >199592......... <0.2 6.2

a Assay performed on whole culture.b Expressed as international units per milliliter.e Constitutive strain.

TABLE 5. Soluble peptidoglycaninducer availability

Total SolubleBaclluceeus569 soluble peptido; Aint used #_Lac-Baclluceeus569

peptido- glycan, o t-sstarting materiala P j mlof iin-b amaSeglca packed ductionb activityC

mg mg pgVegetative cells.. . 0.088 0.009 4.4 0.7Spores .. ; 0.061d 0.350 1.2 0.7Germination fil-

trate ......... . 0.720 3.6 3.6 2.2Sporulating cells. . 40 0.67 1.5 4.0Uninduced. <0.1Penicillin G in-duced. 1.0 3.8

a Vegetative cells were cells grown in CH me-dium to late-log phase (no spores visible). Cellswere then centrifuged, and a 10-ml volume ofpacked cells was used for peptidoglycan extrac-tion. Spores represent 0.2 ml of packed spores sus-pended in appropriate amount of buffer and pepti-doglycan extracted. Germination filtrate is filtratederived from inoculation of 0.2 ml of packedspores into 5 ml of CH medium. Sporulating cellsare derived from sporulating cultures which werecentrifuged, and a 60-ml volume of sporulatingcells was used for extraction of peptidoglycan.

b Amounts based on ninhydrin assay with aminoacid-amino sugar mixture simulating B. cereuspeptidoglycan composition used as standard. Dryweights of peptidoglycan hydrolysates corre-sponded to equal weights of standard hydrolysate.Soluble peptidoglycan represents material priorto placing on Biogel column (see Fig. 1).

c Expressed as international units per milliliterof induction system.

d Even after prolonged sonic treatment (25min), spores did not disintegrate.

vegetative cell peptidoglycan (35). Table 7 alsoindicates that traces of ornithine or diamino-butyric acid (or both), characteristic of anti-biotics synthesized by Bacillus and produced at

TABLE 6. ,B-Lactamase activity in a penicillin-sensitive strain of Bacillus cereus

Cell state Inducer P-Itamreactivity

Vegetative None 0.1Benzylpenicillin, 1.0 0.2.sg/ml (cells lysed)

Benzylpenicillin, 0.1 0.7pg/Ml

Spore peptide, 1.5 0.7;sg/ml

Spore peptide, 0.7 0.5jg/ml

Sporulating None 0.7

"International unit per milliliter inducing system.In. up

I.M0* nce

. moliFrACIgOS sIusius 1.S all

FiG. 4. Chromatography of inducing material onBiogel P-150. Ordinate is optical density.

sporulation, are present in the peptidoglycansalong with traces of most other amino acids.Sulfur-containing amino acids were not found.Absence of tryptophan was inferred from lack ofoptical activity at 280 nm in unhydrolyzed sam-ples.The activity of two fractions was compared in

inducing ability with that of two penicillins(Table 8). The molar concentration of each ofthe fractions was roughly estimated from theapproximate molecular weights assigned on thebasis of the elution volume of the fractions fromthe gel and their sedimentation behavior. Ac-cording to these estimates, on a molar basisYo to M as much peptidoglycan as penicillin isneeded for fivefold stimulation of #-lactamaseinduction. Although much more than fivefoldinduction is observed when more inducer is used,the smallest amount giving significant inductionwas purposely chosen for this comparison. Itshould be noted that these fractions, based on

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weights of material required for induction,represent a minimum of 250- to 500-fold purifica-tion compared to the crude extract (supernatantfluid obtained after treatment of cells in Branson20-kc Sonifier).The peptidoglycan-rich inducing material,

in the form in which it was put on the BiogelP150 column, was also fractionated by sedi-mentation through a sucrose gradient as shownon Fig. 5. When the data obtained in this experi-ment are compared with the gel filtration studies(Fig. 4), the following properties are found tobe identical: (i) three components are discerned;(ii) significant induction activity resides in thetwo faster-moving (larger) components; (iii)the sedimentation properties of the three com-ponents are consistent with exclusion of onlythe largest from Biogel P60 and inclusion of allthree on Biogel P150. Although the ninhydrintest is a convenient measure of free amino groups,it is likely to reduce the apparent amounts oflarge polymers and over-exaggerate small poly-peptides. Comparative estimates of the relativeamounts of material in each peak are thereforenot possible. The one tube displacement between

TABLE 7. Molar ratios offraction constituents

FractionConstituent

Muramic acid.. 0 2.78 1.4Total hexosamine.. 0 7.48 4.1Aspartate (NH2)". 4.6 0.57 0.8Threonine...... 2.8 +c +cSerine. 13.8 + +Glutamate (NH2). 10.7 1.0 1.0Proline.. 1.9 i +Glycine.. 13.9 1.25 0.63Alanineb.... 7.4 0.81 1.85Cysteine.... 0 0 0Valine... 2.9 + +Methionine.. 0 0 0Diaminopimelic acid. 1.0 0.79 1.23Isoleucine.... 1.5 + +Leucine.. 2.9 + ±Tyrosine. 1.0 4 +Phenylalanine... 2.0 + +Hydroxylysine .............. 0 0 ±Ornithine/diaminobutyricacid.. 0 A +Lysine... 1.0 + 0.67Histidine. 1.0 i +Arginine....................1.0 0 0Inducer of ,B-lactamase + +

a Virtually all as D-stereoisomer.b 35% A 10% as D-stereoisomer.c (+) Amino acids found in lesser amount; sum

of all amino acids so designated < 10% of fraction.

TABLE 8. Minimum concentration of inducer forfivefold,6-lactamase inductiona

Inducer Concn (X)

Benzylpenicillin. 1 X 10-8Methicillin..... 3 X 10-8Fractionlb. 1.5 X 10-9 (0.1 sg)Fraction IIb. 3 X 10-9 (0.1 ,ug)Fraction Ilb..... >»10-' (no induction with

40.0 jg)

Organism used for induction was Bacilluscereus 569. Fractions I, II, and III refer to frac-tions shown in Fig. 4.bAssumed molecular weights based on cali-

brated gel exclusion volumes and analysis usingproteins as molecular weight markers. These arehighly conservative estimates because of assym-etry of peptidoglycans involved.

Position of Pesities of11is marker 4ts marker

c31-.c 4j.- .94i !g

.k.a0 .

4!- ax;, z!: 0z :

Position of12S marker

I

I

A1

p-

* * 1.78

FiG. 5. Sedimentation of 1-lactamase inducer. Or-dinate is expressed in Perret Units; to convert tointernational units, divide by 60. Solid line, ,B-lactamaseinduced; dotted line, ninhydrin value of fraction.

the center peak of ninhydrin activity and induc-tion may be due to the method of collection of0.2-ml fractions by drops and the pooling of twoindividual sedimentation-tube samples beforeassay.

DISCUSSIONA cellular material capable of inducing -

lactamase in B. cereus with an efficiency at leastequal to that of penicillins has been isolated. Itsavailability at different stages of the growth cyclemay help to explain the great variability in thelevels of ,-lactamase in uninduced cultures.Upon characterization, the inducing activitywas found to be associated with either of twosoluble peptidoglycans similar in compositionto those isolated from sporulating or germinatingB. cereus cultures, or from spores. This materialis often referred to as spore peptide (32). In thework of Strange and Powell (42), the sporepeptide having sedimentation properties similar

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to inducing fraction II was found to comprise75% of the spore peptide found in germinationexudates. The analyses of fractions reported herewere for materials derived from sporulatingcultures, and fraction II represented 41% of thesoluble peptidoglycan. Powell and Strange foundthat their two peptidoglycans had different aminoacid-to-amino sugar ratios. This was also truewhen fractions I and II, described in this study,were compared.Normal derepression of ,-lactamase closely

follows release of soluble peptidoglycan duringnormal sporulation in inducible strains of B.cereus. Even constitutive strains (569/H and 5/B)which are unresponsive to pencillins duringvegetative growth exhibit increased levels of,-lactamase during sporulation compared tothe levels in these strains in early to mid-logphase. Such constitutive strains have alreadybeen shown to exhibit increases in ,-lactamaseproduction when grown at 42 C (so-calledthermal derepression; 6, 48). In the early literaturethere are several reports that ,B-lactamase couldbest be isolated from 4- to 7-day-old cultures(5, 12). This increased production of ,-lactamaseduring sporulation was found for all wild-typestrains of B. cereus, including a laboratory strainpreviously thought not to produce B-lactamase(cf. Tables 4 and 6). The production of at leastsome ,B-lactamase activity in all wild-type strainsof B. cereus has been previously described (39).Even in S. aureus (which does not sporulate),,B-lactamase induction has been shown to increasein the stationary phase of growth in responseto 2, 2'-carboxyphenylbenzoyl-6-aminopenicilloicacid, a compound having minimal antibioticactivity (23).The interference with cell wall metabolism by

penicillins, purportedly analogues of peptido-glycans (e.g., 43, 46), and induction of 84-lac-tamase by peptidoglycans suggest that peptidogly-can fragments may accumulate subsequent to pen-icillin action. There is some evidence for solublepeptidoglycan accumulating subsequent to peni-cillin treatment in S. aureus (H. J. Rogers,personal communication). Thus, the antibioticcould be acting indirectly in inducing systems.Several studies support this hypothesis for B.cereus, e.g., (-lactamase is derepressed duringautolysis (24) or protoplast formation (38, 45)but cannot be induced by penicillin in washedprotoplasts. Further, induction of (3-lactamase bypenicillin occurs only after a 15 to 30-min lag,whereas, in other induction systems, new enzymeappears after a lag of only 2 to 3 min. This hy-pothesis of ,3-lactamase induction as a conse-quence of penicillin action could also explainhow apparent nonspecificity of induction could

be due to the accumulation of cellular peptido-glycan. Saz and Lowery (36, 37) founid that anumber of 1-amino acid-containing peptidesinduced ,-lactamase. Of additional interest is thefinding by Lowery and Saz (unpublished data)that colistin, which inhibits cell wall autolysisin B. cereus, did not induce (3-lactamase syn-thesis, whereas very similar peptides did induce.A cell wall enzyme which mediates rod-sphereconversion in Arthrobacter is induced by amaterial having some of the properties of apeptidoglycan (7). This is the only previouslyreported instance where peptidoglycan is knownto induce an enzyme involved in cell wall me-tabolism.The data presented herein and other data to be

presented subsequently are also consistent withthe possibility of a function for ,B-lactamase incell wall metabolism.

ACKNOWLEDGMENTS

This work was supported by Public Health Service researchgrants Al 06497 and TI Al 298 from the National Institutes ofAllergy and Infectious Diseases.

One of us (J.H.O.) is the recipient of an National Institutesof Health Predoctoral Research Fellowship (T-GM 37267).

The assistance of J. R. Daniels, National Institute of DentalResearch, in the final steps of purification and analysis, of thepeptidoglycan inducer is gratefully acknowledged. We thankC. L. Wittenberger, National Institute of Dental Research, foraid in determining 1-lactic acid.

LITERATURE CITED

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3. Barker, S. B. 1957. Preparation and colorimetric determinationof lactic acid, p. 241-246. In S. P. Colowick and N. 0.Kaplan (ed.), Methods in enzymology, vol. 3. AcademicPress Inc., New York.

4. Barker S. B., and W. H. Summerson. 1941. Cobrietricdetermination of lactic acid. J. Biol. Chem. 138:535-554.

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6. Bernstein, A., K. W. Nickerson, and R. A. Day. 1967. Thermalpenicillinase derepression and temperature dependence ofpenicillinase production inducible and constitutive strainsof Bacillus cereus. Arch. Biochem. Biophys. 119:50-54.

7. Blankenship, L. C., and R. N. Doetsch. 1961. Iniluence ofa bacterial cell extract upon the morphogeneas of Arthro-bacter ureafaciens. J. Bacteriol. 82:882-888.

8. Burton, K. 1955. D-Amino acid oxidase from kidney, p. 119-124. In S. P. Colowick and N. 0. Kaplan (ed.), Methodsin enzymology, vol. 2. Academic Press Inc., New York

9. Dische, Z., and A. Danilchenko. 1967. Modifications of twocolor reactions of hexoses with cysteine and sulfuric acid.Anal. Biochem. 21:119-124.

10. Dische, Z., and L. B. Shettles. 1948. A specific color reacdonof methylpentoses and a spectrophotometric micro-methodfor their determination. J. Biol. Chem. 175:595-603.

11. Dubois, M. K., A. Gilles, J. K. Hamilton, and P. A. Rebers.1956. Colorimetric method for determination of sugars andrelated substances. Anal. Chem. 28:350-356.

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15. Ghuysen, J.-M., D. J. Tipper, and J. L. Strominger. 1966.Enzymes that degrade bacterial cell walls, p. 685-699.In E. F. Neufeld and V. Ginsburg (ed.), Methods in en-

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muramidase of Chalaropsis species. I. Purification andcrystallization. J. Biol. Chem. 242:5586-5590.

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NRRL 569: a comparison of its properties with those of asimilarly purified penicillinase produced spontaneouslyby a constitutive mutant strain. Biochem. J. 62:391-401.

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24. LePage, S. S. J. F. Morgan, and M. E. Campbell. 1946.Production and purification of penicillinase. J. Biol. Chem.166:465-472.

25. Matsuhashi, M., C. D. Dietrich, and J. L. Strominger. 1965.Incorporation of glycine in cell wall glycopeptide in Staphy-lococcus aureus: role of SRNA and lipid intermediates.Proc. Nat. Acad. Sci. U.S.A. 54:587-594.

26. Miller, E. J., and K. A. Piez. 1966. An accelerated singlecolumn procedure for the automatic analysis of aminoacids in collagen and elaxin hydrolysates. Anal. Biochem.16:320-326.

27. Mirelman, D., and M. Sharon. 1967. Isolation and study ofthe chemical structure of low molecular weight glycopep-tides from Micrococcus lysodeikticus cell walls. J. Biol.Chem. 242:3414-3927.

28. Moore, S., and W. H. Stein. 1948. Photometric ninhydrinmethod for use in the chromatography of amino acids. J.Biol. Chem. 176:367-388.

29. Park, J. T., and A. N. Chatterjee. 1966. Membrane associatedreactions involved in bacterial cell wall mucopeptide syn-thesis, p. 466-472. In E. F. Neufeld and V. Ginsburg (ed.),Methods in enzymology, vol. 8. Academic Press Inc.,New York.

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31. Park, J. T., and R. Hancock. 1960. A fractionation procedurefor studies of the synthesis of cell-wall mucopeptide andof other polymers in cells of Staphylococcus aureus. J.Gen. Microbiol. 22:249-258.

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33. Powell, J. F., and R. E. Strange. 1956. Biochemical changesoccurring during sporulation. Biochem. J. 63:661-668.

34. Pollock, M. R. 1967. Origin and function of penicillinase:a problem in biochemical evolution. Brit. Med. J. 4:71-77.

35. Salton, M. R. J., and B. Marshall. 1959. The compositionof the spore wall and the wall of vegetative cells of Bacillussubtilis. J. Gen. Microbiol. 21:415-420.

36. Saz, A. K., and D. L. Lowery. 1964. Staphylococcal penicil-linase. II. Non-penicillin-like cyclic peptides as inducersof, and substrates for, the enzyme. Biochem. Biophys.Res. Commun. 15:525-529.

37. Saz, A. K., and D. L. Lowery. 1965. Non-penicillin-likepeptides as inducers of and substrates for Bacillus cereus

569 penicillinase. Biochem. Biophys. Res. Commun. 19:102-107.

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