JOURNAL OF BACTERIOLOGY, Sept. 1968, p. 713-720Copyright © 1968 American Society for Microbiology

Vol. 96, No. 3Printed in U.S.A.

Immunological Homology Between Crystal and

Spore Protein of Bacillus thuringiensisF. P. DELAFIELD,l H. J. SOMERVILLE,2 AND S. C. RITTENBERG

Department ofBacteriology, University of California, Los Anlgeles, California 90024

Received for publication 12 June 1968

Spore suspensions containing about 0.3% crystals and crystal suspensions con-

taining about 0.1% spores were obtained from cultures of Bacillus thuringiensisby extraction with a two-phase system. Both preparations were tested for the pres-

ence of contaminating material from vegetative cells and were judged to be clean.Solutions of spore protein were obtained by extracting broken spores with phos-phate buffer followed by extraction with either alkali- or urea-mercaptoethanol.The alkali spore or urea spore extracts had the same isoelectric point as crystalprotein solubilized with these reagents. An antiserum prepared against alkalicrystal solution precipitated alkali or urea spore extracts and crystal solutions butnot phosphate spore extracts or extracts of whole cells. Lines of identity betweenspore and crystal precipitates were observed by using the Ouchterlony double-diffusion technique. Absorption of the antiserum with an excess of urea spore ex-

tract caused a disappearance of the precipitin bands originating from the spore

protein and the homologous bands from the crystal protein. The results suggestthat the crystal and endospore contain one or more common proteins.

The species Bacillus thuringiensis denotes agroup of aerobic sporeformers that character-istically develop a massive intracellular crystalwhen they sporulate. It has been known since1915 (4) that these bacteria cause the death oflepidopteran larvae, but not until 1955 were thecrystals isolated and shown to be the toxic factor(2, 9). Since then, many studies have been madeon the insecticidal properties of B. thuringiensis(10), but relatively few investigations have beenconcerned with the possible physiological sig-nificance of the crystals to the bacteria which formthem. This report presents preliminary observa-tions bearing on that question.The crystal is an octahedron composed of

spherical subunits in cubic close packing (11, 12).It is insoluble in water but dissolves readily inalkali and remains in solution after neutralization(9). The resulting solution contains mainly pro-tein, with only traces of phosphorous and carbo-hydrate. All of the common amino acids arepresent and may be distributed among severalpolypeptide chains (14, 15). No enzymatic ac-tivity has yet been attributed to the dissolvedcrystal.

1 Present address: Department of Medical Micro-biology and Immunology, University of California,Los Angeles 90024.

2 Present address: Department of Bacteriology,University of California, Berkeley 94720.

The crystal first appears as a minute granulenear the forespore, enlarges as the spore develops,and reaches its full size as the spore becomesmature (22). The crystal is synthesized fromamino acids formed by protein turnover withinthe sporangium (17), a conclusion supported bythe observation that crystal antigen is not foundin the cell prior to sporulation (18). Furthermore,it has been asserted that crystal antigen is absentfrom the spore (18).The foregoing observations imply that the crys-

tal is composed of unique protein whose synthesisis fortuitously coincident with sporulation. Analternate possibility is that the crystal accumulatesas a result of unregulated synthesis of sporeprotein. We have therefore reinvestigated therelationship of the crystal to the spore, to deter-mine whether or not they contain common proteincomponents.

MATERIALS AND METHODS

Organtisms. Most of the reported experiments weredone with a strain of B. thurinigiensis var. alesti aniduzedoubly resistant to penicillin (1,000 units/ml) andstreptomycin (250 ug/ml). The strain was derived bystandard procedures from B. thurinigiensis var. alestianzduze (EAS 0-24-3) obtained from E. A. Steinhaus.

Cuiltivatiot,. Bacteria were spread uniformly onplates of the following medium: 8.0 g of NutrientBroth (Difco), 20.0 g of agar, 0.08 g of CaC12, 0.05 gof MnCl2 4H20, 0.005 g of ZnSO4 7H20, and 0.005 g

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of CuSO4 5H20, in I liter of distilled water. Afterincubation for 72 hr at 30 C, growth and sporulationwere virtually complete, and nearly all of the sporangiahad lysed.

Separationi of spores anid crystals. Autolyzed cul-tures were scraped from the surface of 250 petri dishesand were suspended in 1 M NaCI-0.02 M potassiumphosphate buffer (pH 7.0) containing 0.01l' Triton-X-100. The suspension was filtered through cheese-cloth to remove small pieces of agar and was centri-fuged. The sediment was washed repeatedly withfresh portions of the same solution until only traces ofmaterial absorbing at 260 nm remained in the super-natant liquid. The particles were then washed once in0.2 M NaCI-0.004 M phosphate buffer (pH 7.0)-0.01 6X0 Triton-X-100 and once in 0.01 %c, Triton-X-100,and were suspended in water. Residual cells were thenremoved from the suspension by extracting five timesin 1.5 liters of Phase Mixture I of Sacks and Alderton(19).

The remaining crystals and spores were centrifugedand washed three times in 0.02 M phosphate buffer(pH 7.0) -0.01 ', c Triton-X-100. The suspension, in 182ml of the same buffer, was added to a cylindricalseparatory funnel containing 105 g of a 20%': (w/w)aqueous solution of sodium dextran sulfate 500(Pharmacia Inc., New Market, N.J.), 13.2 g of solidpolyethylene glycol 6000 (Union Carbide), 3.3 ml of 3M phosphate buffer (pH 7.0), and 7.5 g of NaCl. Aftershaking to dissolve the solids, the volume was ad-justed to 600 ml by adding a well-shaken solution ofthe same composition, but without bacterial parti-cles. The complete mixture was shaken vigorouslyand placed at 5 C for 30 min. The mixture separatedinto two phases, the upper phase rich in polyethyleneglycol and the lower phase rich in sodium dextransulfate. This two-phase system was empiricallydeveloped from methods described by Albertsson (1);a similar one was independently described by Good-man et al. (7).

The upper phase contained most of the spores ini-tially present, but very few crystals. The lower phasecontained a mixture of crystals and spores. The upperphase was drawn off and centrifuged. The super-natant solution was poured back into the separatoryfunnel and the extraction was repeated. Spores in thefirst two upper phases constituted the bulk of thoseinitially present and were saved; spores subsequentlyextracted from the lower phase were discarded. Afterthe tenth extraction, the crystals in the lower phasewere virtually free of spores and were collected bycentrifugation. The spores and crystals were bothwashed five times in cold distilled water. The sporeswere stored at -20 C as freeze-dried powders. Thecrystals were stored at -5 C as suspensions in water.

Enumerationz of crystals and spores. Crystals andspores are both refractile and of roughly equal size.Since the crystals have pointed apices, they may easilybe differentiated from the oval spores by phase con-trast microscopy. Differential counts were made in aPetroff-Hauser counting chamber, with suspensionscontaining not less than 108 particles per ml.

Fractionation of crystals antd spores. Freeze-driedspores were ballistically disintegrated (20). A ball

bearing weighing 180 mg, 50 mg of spore powder, and50 mg of 50-mesh NaCl were shaken for 10 min in thecup of a Toothmaster Hi Speed Amalgamator (Tooth-master Co., Racine, Wisc.). The spores, by micros-copy judged to be nearly all broken, were extractedtwice with 0.02 M potassium phosphate buffer (pH6.8) for 1 hr at 0 C. The supernatant solutions werepooled; they are referred to as "phosphate spore"extract.

The residue remaining after phosphate extractionwas reextracted twice for 1 hr at 23 C and centrifugedfor 15 min at 15,000 X g with either 0.1 N NaOH or8 M urea-10%; g3-mercaptoethanol (pH 8.5) as thesolvent. The pooled solutions, referred to as "alkalispore" or "urea spore" extracts, respectively, and thephosphate spore extracts were thoroughly dialyzed at5 C against 0.01 M tris(hydroxymethyl)aminomethane(Tris)-chloride buffer (pH 8.2). The solutions wereclarified, if necessary, by centrifugation and frozenuntil needed.The crystals were solubilized directly with 0.1 N

NaOH or with the urea-mercaptoethanol reagent.The solutions were dialyzed and clarified as describedabove to give "alkali crystal" or "urea crystal" solu-tions, respectively.

Vegetative cells were harvested during exponentialgrowth in Nutrient Broth. They contained no visiblecrystals or spores. The procedures for extracting thecells were the same as those used for the spores.

Serology. Rabbits were immunized with vegetativecells, purified whole crystals, or with alkali crystalsolution centrifuged at 60,000 X g for 1 hr to removeresidual spores. Vegetative cells and whole crystalswere injected intravenously at 1.0 mg (dry weight)daily for 5 days. The alkali crystal solution wasmixed with an equal volume of Freund's completeadjuvant (Hyland Laboratories, Inc., Los Angeles,Calif.), and a total of 1.0 ml, containing 5.0 mg ofprotein, was injected subcutaneously into the footpadsand dorsal skin of rabbits. Three weeks after theinitial course of injections, each rabbit received 10.0mg of the appropriate antigen intravenously. Oneweek later, each rabbit was bled from the heart.

Precipitation reactions in agar-gel were studied bythe double-diffusion technique of Ouchterlony (6).The gel consisted of 1.0% Difco Agar in 0.077 Mbarbital buffer (pH 8.2) containing 0.025% Merthio-late. The agar was poured into plastic boxes to adepth of 2 mm, and after solidification was drilledwith 20-,uliter wells spaced 8 mm apart. The wells werefilled with solutions of antigen or antiserum, and theboxes were incubated in a moist atmosphere at 5 C for4 days before examination.Enzyme determinations. Catalase activity was esti-

mated in the Warburg apparatus at 30 C. The side armcontained 0.2 ml of a suspension of intact sporeswhich, after thermal equilibration, was mixed with 1.9ml of 0.066 M phosphate buffer (pH 7.0) containing 14,umoles of H202 per ml. Activity, measured as theinitial linear rate of 02 evolution in microliters of gasper hr per mg (dry weight) of spores, was proportionalto the amount of spores tested.

Alanine racemase activity was assayed by measur-ing the uptake of oxygen with a Clark electrode at 30 C

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with L-(+)-alanine as substrate and an excess ofD-amino acid oxidase (Boehringer). The assay mixturecontained 0.9 ml of 0.01 M Tris-chloride buffer (pH8.2), 0.1 ml of 0.1 M L-alanine, and 0.1 ml of the oxi-dase solution. After thermal equilibration, 0.1 ml ofdiluted intact spore suspension was added to start thereaction, and oxygen uptake was recorded. Under theconditions of the assay, the rate of oxygen uptake,which was proportional to the number of spores used,was a direct measure of D-alanine formation.

Protein determinations. Protein was usually esti-mated by the method of Lowry et al. (16). Since Tris-chloride buffer interferes with protein determinationsby this method, protein in Tris buffer was estimatedspectrophotometrically by the method ofWarburg andChristian (23). Determination of crystal protein bythis method agreed closely with that obtained by theLowry method in the absence of Tris. By eithermethod, with bovine serum albumin as standard, theratio of crystal protein to crystal dry weight was closeto 1.4.

Miscellaneous methods. Phosphorous was esti-mated by the method of Chen et al. (5), and carbo-hydrate by the anthrone procedure with glucose as astandard. Calcium and magnesium were determinedby flame photometry.

RESULTS

Separation of crystals and spores. By using theprocedures described, overall recovery of bothspores and crystals, based on their initial contentsin the harvested autolysate, approximated 70%(Table 1). The final spore preparation (step 4A)contained about 0.3% crystal and the crystalpreparation about 0.1% spores (step 4B).

Cleanliness of crystals and spores. Although thepurified suspensions were microscopically homo-geneous, their possible contamination withcellular debris was examined, since such debrismight give the false impression that spores andcrystals share common components. Purifiedcrystal suspensions contained, by weight, less

than 0.001 % phosphorous, less than 0.05% car-

bohydrate, and less than 0.02% calcium andmagnesium. Dissolved crystal protein had an

absorption ratio (OD28o nll OD260 nm) of 1.5.This value is consistent with a pure proteinpreparation and is, in fact, the highest yet re-

ported for soluble crystal preparations.The spores of B. thuringiensis, washed 15 to 20

times in the course of purification, evolved gasfrom H202 at a rate equivalent to 153 ,lliters of02 per hr per mg (dry weight). This catalaseactivity was largely thermolabile, being rapidlydestroyed at 70 C. However, a fraction of theactivity was stable to heating for at least 15 min,so that a bimodal curve of thermal inactivationwas observed. As estimated from this curve, 30%of the total activity is heat-stable (Table 2), and,accepting the interpretation of other workers,would be an integral part of the spore. The restof the activity, or 70%, is heat-labile and couldbe cellular enzyme. To discover whether or notany of the catalase activity could be solubilized,as should be the case were it simply adsorbedenzyme, the spores were subjected to ultrasonicvibration under conditions described by Bergerand Marr (3). The treatment disintegrated few ifany of the spores, but removed the exosporiaand associated alanine racemase from more than85% of them. However, even after washing thestripped spores four more times in distilledwater, the amounts of heat-labile and heat-stable catalase were unchanged (Table 2). Ac-cordingly the heat-labile component appears tobe part of the spore rather than an adsorbedcellular enzyme.

Heat-shocked (at 75 C for 10 min) spores

were incubated at 2 mg (dry weight) per ml of0.02 M phosphate buffer (pH 7.0) containing 1.2mM glucose, 6 mM L-(+)-alanine, and 0.012 mM

TABLE 1. Separationi of crystals and spores

Total particles remaining after each stepSeparation step

Spores Crystals Cells

1. Harvest and filtration 4.25 X 1012 4.20 X 1012 0.08 X 1012

2. Preliminary washings 3.10 X 1012 3.60 X 1012 0.08 X 1012

3. Removal of cells(A) Polyethylene glycol-rich phase 2.79 X 1012 3.25 X 1012 Undetectable

4. Separation of crystals and spores(A) Polyethylene glycol-rich phasea 2.93 X 1012 0.01 X 1012 Undetectable(B) Sodium dextran sulfate-rich phaseb 0.003 X 1012 2.90 X 1012 Undetectable

a Combined harvest from first two extractions.b Harvest after 10th extraction.

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TABLE 2. Removal of exosporia and enzymesfrom intact sporesa

Alanine Catalaseracemase activity

Spores (percentage of (micromoles of (microliters ofLength initial) D-alanine per 02 per min

of min per 3X per 3 X 10'°oscilla- 1010 spores) spores)

tion

(min)Ref rac- Phase- With Sedimenexos- Total - Labile Stabletile dark pra table

0 99 1 85 12.7 3,2101,38080 89 11 15 12.0 1.4 2,9701,290

a Spores were treated in a 10-kc Raytheon sonicoscillator containing 3 X 1015 spores (30 mg, dryweight) in 50 ml of 0.02 M phosphate buffer (pH6.8) at 5 C under an atmosphere of N2. Sampleswere withdrawn at intervals, and the fraction ofspores with exosporia was determined by phasecontrast microscopy. The first-order rate constantfor removal of exosporia was 0.01 min-'. Alanineracemase and catalase activity of the intact sporeswas determined before and after the ultrasonictreatment. The treated spores were then washedfour times in cold distilled water, and their alanineracemase and catalase activities were again deter-mined. By direct count, less than 2%7o of the initialspores were lost during the experiment; this num-ber is not noted in the table, as its significance isquestionable.

adenosine. After shaking for 4 hr at 30 C, thesuspension was stained with 0.5% aqueousmethylene blue and examined microscopically.Judging from the absence of swollen, blue spores,there was no germination under these conditions.In a parallel experiment, in which five times asmuch glucose, alanine, and adenosine was used,62% of the spores germinated. Omission of glu-cose from the concentrated mixture reduced thenumbers germinating to 43 7. Since germinationunder the latter condition is clearly far fromoptimal, 30 mM L-alanine and 0.06 mm adenosineapproximate the minimal germination require-ments for these spores. Negligible germination(less than 10%1") occurred when either alanine oradenosine was omitted from the concentratedincubation mixture.

Fractioncition of the spores. The course of frac-tionation of ballistically disintegrated, freeze-dried spores is shown in Table 3. The total pro-tein solubilized in this experiment is somewhathigher than usual. More typically, of the totalprotein, about 25%c is solubilized by phosphatebuffer and an additional 25%0 by either alkali orurea-mercaptoethanol. These solutions probablycontain nucleic acid as well as protein, becausethe ratio of their optical density at 280 nm tothat at 260 nm is low.

Solubility of extract protein. The solubility ofprotein in urea crystal and urea spore extractswas examined as a function of pH (Fig. 1).Both extracts have a fairly low isoelectric point

TABLE 3. Fractionation of ballisticallydisintegrated sporesa

Course of fractionation

Initial dry weightTotal protein initially presentTotal protein dissolved by phos-phate (OD 280/260 nm)

Phosphate-insoluble proteindissolved by urea-mercapto-ethanol (OD 280/260 nm)

Phosphate-insoluble proteindissolved by NaOH

Residual insoluble proteinTotal protein recovered

Batch 1

mg

200.0108.0

27.0(0.59)

52.0(0.70)

43.6122.6

Batch 2

mg

200.0106.5

24.3(0.60)

42.339.2

105.8

a See Methods for composition of solvents.

901

0w 80

CL 700LU

aL 60zLUIo 500

J 40

0I- 30

0

20

z 10CLU0i

- 3 4 5 6 7 8pH

FIG. 1. Solubility oj protein in urea crystal (0),urea spore (0), alnd phosphate spore (A) extracts as afunictioni of pH.

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at pH 4.6, a value in good agreement with pre-vious observations for solubilized crystal protein(9). The shapes of the curves are very similar inthe vicinity of the isoelectric point but less so atthe extremes of pH studied. The solubility curvefor phosphate spore extract is distinctly different.

Immunological comparison of spore and crystalextracts. Antisera were taken from rabbitsimmunized with (i) whole cells harvested duringexponential growth and containing, as judged bymicroscopy, no spores or crystals; (ii) purifiedwhole crystals; (iii) alkali-soluble crystal proteinfrom which residual contaminating spores wereremoved by centrifugation at 60,000 x g for60 min. Alkali was used to prepare this immuno-gen, since it would extract considerably lessprotein than urea-mercaptoethanol from wholespores if they were present. This immunogenrepresents the purest solution of crystal proteinthat we have achieved. Seven antigen solutionswere tested with these antisera: phosphate spore,urea spore, alkali spore, phosphate cell, urea cell,alkali crystal, and urea crystal.The specificity of each antiserum was exam-

ined by double immunodiffusion in agar-gel witheach of the seven soluble antigens; the presenceor absence of precipitin band formation isshown in Table 4. Antiserum to whole crystalswas nonspecific and precipitated all of the anti-gens tested. In contrast, antiserum to solublecrystal protein was specific in the sense that itprecipitated spore and crystal protein extractedwith urea-mercaptoethanol or alkali but did notprecipitate either of the vegetative cell extracts orthe phosphate spore extract. This immunologicalpattern confirms a previous report that crystalantigen is not found in the vegetative cell (18).It also suggests that spore and crystal may shareone or more similar antigens. Finally, antiserum

TABLE 4. Precipitin patterns in double diffusionplate tests

Antigens

Rabbit sera Cells SporesCrystals(Alkali

Urea Phos- Alkali Phos- or ureaextact phate or urea phate extract)ra extract extract extract

Anti-whole 4 +a + + +crystal

Anti-soluble _b - + - +crystal

Anti-whole + + + + -cell

a One or more precipitin lines, +.I No precipitin lines observed, -.

2

1

3

1 cm

FIG. 2. Precipitini patternsformed by urea-mercapto-ethanol extracts of crystal (1) and spore (3) with anti-serum to soluble crystal protein (2). Antiserum wasused without dilution; 200 ,ug of protein was added toeach antigen well.

to vegetative cells precipitates cell and sporeextracts but not crystal extracts. This observa-tion supports the conclusion that the crystals arerelatively free of cellular components. The pre-cipitation of the spore extracts by this serum canbe attributed to the known fact that spores arepartially composed of vegetative cell proteins.A detailed comparison of the precipitin pat-

terns formed by the urea-mercaptoethanol ex-tracts of spore and crystal with antiserum tosoluble crystal protein is shown in Fig. 2. Themost rapidly moving component from eachextract forms a continuous precipitate withoutspur formation and, therefore, appears to becommon to both spore and crystal. The moreslowly moving spore antigen forms a precipitatethat may be continuous with a component of theintermediate crystal band, but the precipitate isso faint that it is difficult to be certain. Theunique band under the crystal well contains, tojudge from its density, most of the crystal pro-tein.The precipitin bands shown in Fig. 2 are not

formed if normal rabbit serum is substituted forthe immune serum or if the immune serum isabsorbed with an excess of soluble crystal pro-tein. Absorption of the antiserum with an excessof urea spore extract causes a disappearance ofthe precipitin bands originating from the sporewell and of their homologues from the crystalwell.

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One possible source of the common precipitinbands originating from the spore extract mightbe the traces of contaminating crystal proteinthat must be solubilized when extracting thespores with urea-mercaptoethanol. This idea,however, is not correct, for spores of a mutantstrain that does not form crystals prove to haveurea-mercaptoethanol-soluble antigens precipi-table in a pattern indistinguishable from thatalready described.

DIscUSSIONTo determine whether or not crystals and

spores of B. thuringiensis share common com-ponents, it was essential to this investigationthat these particles be effectively separated fromeach other and be free of common contami-nating protein from the vegetative cells. Themethod devised for their separation, althoughunwieldy and monotonous to use, did yieldeffective separation with recovery of nearlythree-fourths of the desired particles initiallypresent in the cultures. The method had theadditional virtue of subjecting spores and crystalsto more than 20 washings, which should haverendered them free of all cellular componentsthat are not part of their intrinsic structure.Analysis of the purified suspensions supportedthis prediction. The crystal suspension con-tained a negligible amount of phosphorous andanthrone-reactive material, and it was thereforejudged to be free of nucleic acid, carbohydrate,and phospholipid. The ultraviolet absorption ofdissolved crystals was characteristic of pureprotein, and approximately 100% of the mass ofthe crystal could be accounted for as aminoacids (21). The crystals did contain traces ofcalcium and magnesium, but these metals werenot present in greater quantity than expectedfrom the level of spore contamination. Finally,antiserum to vegetative cells did not give detec-table precipitin reactions with crystal solutions.The cleanliness of the spores could not be

evaluated by direct chemical analysis. However,the spores failed to germinate unless both L-ala-nine and adenosine were provided. In this respect,they were similar to well-cleaned spores of theclosely related B. cereus (8). The spore suspen-sions had considerable catalase activity, of whichmore than half was heat-labile. Lawrence andHalvorson (13), studying intact spores of B.cereus, concluded that heat-stable catalase wasan integral part of the spore, whereas heat-labile catalase was probably a cellular enzyme onthe spore surface, from which it could be re-moved by exhaustive washing. Considering thelarge number of washings involved in purifying

the spores of B. thuringiensis and, more impor-tant, that removal of their exosporia and asso-ciated alanine racemase did not diminish theirtotal catalase activity or alter the proportion ofthe heat-labile and stable components, it appearsthat the heat-labile enzyme is a spore component.Its presence is therefore not a criterion of cellu-lar contamination in spores of B. thuringiensis.In fact, since the total catalase activity was notdecreased by removal of the exosporia, it isprobable that no cellular catalase was adsorbedon the spore surface, and by inference this wouldbe true of other cellular protein as well. Consid-ering the results of Lawrence and Halvorson(13), it appears that species or strain differencesin the heat-lability of spore catalase activityexist, and a comparative study of this questionis desirable. Although the foregoing observationsdo not prove that the spores and crystals arecompletely free of occluded components fromthe vegetative cell, they provide compellingreasons to believe that the level of contaminationis low.

Crystals are completely dissolved by alkali ora urea-mercaptoethanol solution. The samesolvents solubilize protein from broken sporesthat is not soluble in phosphate buffer, and theextracts obtained are closely similar in isoelectricpoints to the crystal solutions. These data sug-gest a common class of protein in crystal andspore. Serum from a rabbit immunized withsoluble crystal protein, from which residualspores were removed by centrifugation, pre-cipitated the urea-mercaptoethanol or alkaliextracts of crystals and spores but did not pre-cipitate the phosphate extract of spores. Sporeprotein soluble in phosphate buffer thus appearsto be both chemically and immunologicallydifferent from crystal protein.

This observation suggests an explanation forMonro's (18) earlier failure to detect antigenicsimilarity of spores and crystals. He comparedthe antigenicity of alkali-soluble crystal proteinand water-soluble spore protein obtained byultrasonic disruption of whole spores. Bothextracts were precipitated by rabbit antiserum towhole crystals. After absorption with a cell-freeextract of sporulating cells, the serum still pre-cipitated the crystal protein but no longer pre-cipitated the spore protein. Since our experi-ments suggest that phosphate buffer or water doesnot dissolve crystal-like protein in broken spores,Monro's negative results could be attributed tothe use of an inappropriate spore extract. If so,there are no experimental grounds for believingthat the crystal is composed of unique protein.

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Comparison of urea crystal and urea sporeprotein by double immunodiffusion showedpartial antigenic homology. The homologousbands are not formed if the anticrystal serum isabsorbed with spore extract, and no bandsappear if it is absorbed with spore extract.Despite this evidence for homology, the bulk ofthe crystal protein, judging from the density ofthe various precipitin bands, appears to be anti-genically unique. The significance of the homol-ogy is therefore not perfectly clear.The partial homology can be explained in at

least four different ways. Three of these wouldattribute it to contamination. That is, the uniqueprecipitin band could represent native crystalantigen, and the homologous bands couldoriginate from (i) extrinsic cellular antigenscommon to both suspensions; (ii) crystals inthe spore suspension; or (iii) spores in the crystalsuspension.The first possibility is rendered unlikely by

the apparent purity of the spore and crystalpreparations as well as by the failure of crystalantiserum to precipitate cellular antigens. Possi-bilities (ii) and (iii) are unlikely because of theinsensitivity of our immunodiffusion technique,which did not detect antigens at less than 10 Agof protein per well. Since 200 ,ug of protein wasadded to the antigen wells, a minimum of 5%contamination of spore protein in the crystalextract, or vice versa, would have been requiredfor formation of detectable bands due to thiscause. Calculations based on data in Tables 1and 2 indicate that the antigen solutions couldcontain, at maximum, no more than 1 % of suchcontaminating protein.P Alternatively, the homology could be the con-sequence of common protein(s) intrinsic to thespore and crystal. To test the validity of thisfourth possibility, a more definitive biochemicalstudy of the spore and crystal solutions wasundertaken and is reported in the followingpaper (21).

ACKNOWLEDGMENTSWe thank Gareth Wyn Jones, who did the calcium

and magnesium analyses, and H. Downs for generaltechnical assistance.

This investigation was supported by grants GB 1225and GB 6223 from the National Science Foundation.F. Delafield held a Public Health Service PostdoctoralFellowship, 5-F2-AI 31,369, from the NationalInstitutes of Allergy and Infectious Diseases.

LITERATURE CITED

1. Albertsson, P. A. 1960. Partition of cell particlesand macromolecules. John Wiley & Sons, Inc.,New York.

2. Angus, T. A. 1956. Association of toxicity withprotein crystalline inclusions of Bacillus sottoIshiwata. Can. J. Microbiol. 2:122-131.

3. Berger, J. A., and A. G. Marr. 1960. Sonic dis-ruption of spores of Bacillus cereus. J. Gen.Microbiol. 22:147-157.

4. Berliner, E. 1915. Uber die Schlaffsucht derMehlmottenraupe (Ephestia kuhniella Zell.) undihren Erreger Bacillus thuringiensis n sp. Z.angew. Entomology 2:29-56.

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

6. Crowle, A. J. 1960. Immunodiffusion. AcademicPress, Inc., New York.

7. Goodman, N. S., R. J. Gottfried, and M. H.Rogoff. 1967. Biphasic system for separation ofspores and crystals of Bacillus thuringiensis. J.Bacteriol. 94:485-487.

8. Halvorson, H., and B. Church. 1957. Biochemis-try of spores of aerobic bacilli with specialreference to germination. Bacteriol. Rev. 21:112-131.

9. Hanney, C. L., and P. C. Fitz-James. 1955. Theprotein crystals of Bacillus thuringiensis Ber-liner. Can. J. Microbiol. 1:694-710.

10. Heimpel, A. M. 1967. A critical review of Bacillusthuringiensis var thuringiensis Berliner andother crystalliferous bacteria. Ann. Rev.Entomol. 12:287-322.

11. Holmes, K. C., and R. E. Monro. 1965. Studies onthe structure of parasporal inclusions fromBacillus thuringiensis. J. Mol. Biol. 14:572-581.

12. Labaw, L. W. 1964. The structure of Bacillusthuringiensis Berliner crystals. J. Ultrastruct.Res. 10:66-75.

13. Lawrence, N. L., and H. 0. Halvorson. 1954.Studies on the spores of aerobic bacteria. J.Bacteriol. 68:334-337.

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