JOURNAL OF BACTERIOLOGY, Sept. 1968, p. 721-726Copyright @ 1968 American Society for Microbiology

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

Biochemical Homology Between Crystal and SporeProtein of Bacillus thuringiensis

H. J. SOMERVILLE,1 F. P. DELAFIELD,2 AND S. C. RITIENBERGDepartment ofBacteriology, University of California, Los Angeles, California 90024

Received for publication 12 June 1968

The crystalline inclusion of Bacillus thuringiensis, dissolved in 8 M urea contain-ing 10% 2-mercaptoethanol and dialyzed to pH 8.3 to 8.5, was compared with a

fraction obtained by the same extraction procedure from spores broken by dryrupture. The two fractions behaved similarly on chromatography with SephadexG-100 and diethylaminoethyl cellulose. The preparations behaved identically on

acrylamide gel electrophoresis at pH 12 and pH 9.5. Further, peptide maps of thetwo fractions obtained after digestion with trypsin were almost superimposable.Amino acid analyses of the crystal and spore fraction were closely similar; dis-crepancies are attributed to contamination of the spore extract with small amountsof other proteins. It is concluded that a significant portion of the spore protein isidentical with the crystal protein.

In the preceding paper (2), the hypothesis wasadvanced that the parasporal crystal of Bacillusthuringiensis results from uncontrolled synthesisof some essential component of the endospore.Preliminary evidence, mainly immunological,was presented suggesting that the crystal andspore of B. thuringiensis var. alesti contain oneor more proteins in common. The present studieswere carried out with more definite biochemicalprocedures to test critically the indicated iden-tity of spore and crystal protein and to arrive atsome approximation of the levels of the possiblycommon components.

MATERIALS AND METHODS

Preparation of spores and crystals. A strain of B.thuringiensis var. alesti, resistant to penicillin and tostreptomycin, was used in this investigation. The iso-lation of this strain, procedures for its maintenanceand growth, and procedures for the separation andpurification of spores and crystals have been describedpreviously (2). The spore and crystal preparationsused for these studies were at least 99.5% pure in rela-tion to one another, and their contamination withvegetative cells or with solubilized components of cellswas below detectable levels (2).

Extraction of spores and solubilization of crystals.The detailed procedures for the preparation of phos-phate-spore and urea-spore extracts have been given

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

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

(2). The extracts were thoroughly dialyzed at 5 Cagainst either 0.01 M NaHCO3 or 0.01 M tris (hy-droxymethyl)aminomethane (Tris)-hydrochloride buf-buffer, pH 8.3 to 8.5.

Crystals were dissolved directly in the urea-mer-captoethanol reagent and dialyzed as above. Thesmall amount of insoluble material remaining afterdialysis, which consisted almost entirely of contami-nating spores, was removed by centrifugation.

Acrylamide-gel electrophoresis. Acrylamide-gel col-umns (7.5 or 5% acrylamide, final concentration)were prepared according to the method of Hjerten etal. (6). No spacer or sample gels were used. The gelswere polymerized in 0.375 M Tris-chloride (pH 8.3).Tris (0.05 M)-glycine (0.4 M) buffer (pH 9.3 to 9.5)was generally used for electrophoresis which wascarried out for 40 to 60 min at room temperaturewith a current of 3 ma/tube. In some experiments, thegels were equilibrated with 0.01 N NaOH-0.05 M KClbuffer (pH 12) by electrophoresis at room temper-ature for 30 to 60 min at 3 ma/tube. When the pH 12buffer was used, the apparatus was cooled with iceand electrophoresis of the sample was for 40 to 50min at a current of 8 ma/tube.The samples, initially in either 0.01 M Tris-chloride

(pH 8.5) or in 0.01 M NaHCO3, were processed in oneof three ways and then layered above the gels: (i) di-luted with 60% sucrose (w/v) to a final concentrationof 30 to 50%; (ii) diluted with 10 M urea (pH 8.5) to aconcentration of about 8 M a few minutes before ap-plication to the gel; or (iii) diluted with an equal vol-ume of 0.25 M NaOH-0.125 M KCl (14) and, afterstanding for 60 to 90 sec at room temperature, furtherdiluted (1: 1) with 60% sucrose. The method of Leboyet al. (11 was used to apply two samples to the samecolumn for direct comparison. After completion ofelectrophoresis, the gels were removed from the glass

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supporting tubes and stained either with Amido Black(15) for 1 to 24 hr, or with Coomassie Brilliant Blue[1%c(w/v) in 10% (v/v) acetic acid] for 10 to 15 min.In each case, destaining was accomplished electro-phoretically with 10% (v/v) acetic acid as electrolyte.

Peptide maps. Maps were prepared from samplesoxidized by performic acid (16) and hydrolyzed withtrypsin. Acid-washed glassware was used throughout.The spore or crystal extracts (5 to 20 mg of protein in1 to 5 ml of 0.01 M Tris-chloride, pH 8.3) were di-alyzed exhaustively against distilled water, transferredto a small beaker, and evaporated to dryness underreduced pressure at room temperature. Formic acid(1.8 ml) and hydrogen peroxide (30% H202, 0.2 ml)were added to the residue which was stirred with aglass rod. After 15 min at room temperature, the solu-tion was diluted with water (about 5 ml) and evapo-rated as before. Water (1 ml) was added and theevaporation was repeated twice. The residue waswashed with acetone and stored in a desiccator overCaCI2.

About 2.5 to 3 mg of the resulting material wassuspended in 2 to 5 ml of 0.01 M triethylamine. ThepH was adjusted to 8.0 to 8.5, and trypsin (1 to 2%, byweight) was added as an aqueous solution. The mix-ture was stirred slowly at 37 C and the pH was main-tained at 8.0 to 8.5 by periodic addition of 0.05 M tri-ethylamine. In some cases, the progress ofthe digestionwas followed by maintaining apH of 8.5 with an auto-titrator (Radiometer Co., Copenhagen) or by estima-tion of the ninhydrin-positive material in the suspen-sion (3). After 6 to 8 hr, the reaction was stopped byadjusting the pH to approximately 2.2 with 1 to 2drops of formic acid. The resulting suspension wasevaporated as before. The residue was taken up in afew microliters of formic acid, which dissolved all ofthe crystal and the bulk of the spore hydrolysate, andwas applied as a )4-inch (1.91 cm) strip to a sheet ofWhatman 3 MM paper. The peptides were separatedby descending chromatography (butanol: aceticacid:water, 4:1:5, v/v) for 16 hr, followed by elec-trophoresis in pyridine-acetic acid buffer, pH 3.7 (8),for 45 min. The electrophoresis was carried out at2,500 v by use of a horizontal water-cooled apparatussimilar to that described by Gross (5). After drying,the sheets were stained by dipping in the ninhydrin-collidine stain of Canfield and Anfinsen (1), and thecolor was developed by heating at 70 C for 15 to 20min.

Amino acid analysis. This was carried out in aSpinco amino acid analyzer by using standard pro-cedures (17). All hydrolyses were accomplished at 110C in 6 N HCI in sealed, evacuated tubes with eitherlyophilized crystals and spores or extracts of sporeswhich had been dialyzed exhaustively against distilledwater and then lyophilized. The tryptophan content ofsolubilized crystal was determined by the procedure ofGoodwin and Morton (4).

Protein was determined by the method of Lowryet al. (13) with crystalline bovine serum albumin as astandard or from the ratio of absorbance, OD 280nm: OD 260 nm (10). These two methods gave al-most identical results for the crystal protein.

RESULTS

Chromatography on diethylaminoethyl (DEAE)cellulose. Both the urea-crystal and urea-sporeextracts were adsorbed on columns of DEAEcellulose in 0.01 M Tris-chloride, pH 8.3. A por-tion of the protein (fraction I) could be elutedby washing the column with 0.4 M (NH4)2CO3 in0.01 M Tris-chloride, pH 8.3. Fraction I con-tained a variable amount of protein, rangingfrom 20 to 50% of the protein initially applied tothe column. The rest of the protein (fraction II)remained bound to the column, but could all beeluted by 0.1 N NaOH. Fraction I and fractionII were concentrated by freeze-drying, redissolvedin a small amount of urea-mercaptoethanolreagent, and dialyzed against Tris buffer. Whenrechromatographed on DEAE-cellulose, eitherfraction gave rise to the same two fractions asinitially observed. Both fraction I and fractionII, as well as the four fractions derived from themby rechromatography, contained material pre-cipitable by antiserum against alkali-solublecrystal protein. From these observations, it wasinferred that the urea-crystal and urea-sporeextracts consist partially or entirely of protein(s)capable of reversible aggregation. Attempts toobtain stable solutions of disaggregated peptideswere not successful, and further efforts to com-pare the extracts by column chromatographywere therefore abandoned. Accordingly, al-though both the urea-crystal and urea-spore ex-tracts behaved similarly in these experiments,the fractionation was not sufficiently detailed orreproducible to warrant conclusions about theiridentity.

Sephadex filtration. Both the dissolved crystaland the urea extract of spores were excludedfrom Sephadex G-100 and G-200. The crystalsolution, in 0.01 M Tris-chloride (pH 8.3 to 8.5),was excluded from G-100 under a variety of con-ditions, including the presence of Cleland'sreagent (0.02% dithiothreitol), sodium dodecylsulfate (0.1%), or 8M urea. Filtration throughG-100 was used to free the urea-spore extracts ofsmall amounts of retained material of lowmolecular weight, and to remove a 260-nmabsorbing fraction, which adsorbed to the geland was eluted after the total volume of thepacked column. Spore extracts purified in thisway were used in the subsequent comparisons.

Disc electrophoresis. No stainable materialentered the 7.5% gel from crystal or spore ex-tracts which had received either no pretreatment(procedure i, above) or which had been treatedwith 8 M urea (procedure ii). At a gel concentra-tion of 5%, both crystal solution and urea-spore

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extract, applied in 8 M urea, gave a single bandentering the gel (Fig. la), although much of theprotein was still excluded. Without the ureatreatment, the band was very faint or absent.The material which entered the gel was extremelymobile, moving closely behind the band formedby a tracking dye (bromophenol blue). The bandhad the same mobility from both crystal andspore, and they coelectrophoresed when samplesof both were applied to a single polyacrylamidecolumn (Fig. la).

Samples of crystal or spore protein dissociatedwith alkali (procedure iii) entered a 7.5% gel,and little or no stainable material remained atthe gel surface after electrophoresis at pH 12.Both crystal and spore extracts consistentlygave a major single band (Fig. lb) under theseconditions, although other, much fainter, bandswere observed in both preparations when theamount of protein applied to the column wasrelatively large (greater than 10 jig of protein).It was difficult to obtain reproducible mobilities,probably because of the low buffering capacity ofthe liquid phase and the relatively high currentused. Nevertheless, the major bands were indis-tinguishable when samples of both crystal andspore were electrophoresed on a single column(Fig. ib).

Tryptic maps. Preliminary experiments showedthat crystal extracts were resistant to digestion bytrypsin even when alkylated with iodoacetic acid.However, material which had been oxidized with

............ ..~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.DLts . .,~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.......

FIG. 1. Disc electrophoresis of solubilized crystaland urea-mercaptoethanol extract of spores. (a) pH9.5, 5% acrylamide; left to right, spore (ca. 20 jg ofprotein), crystal (ca. 20 jug), crystal and spore (ca. 8 jigeach) in split gel; stained with Amido Black. (b) pH12.1, 7.5% acrylamide; left to right, spore (8 jig),crystal (2 jig), spore (4 jig), and crystal (I jig) in splitgel; stained with Coomassie Blue.

performic acid was extensively hydrolyzed by theenzyme. When the course of hydrolysis was fol-lowed by the appearance of titratable acidity orof ninhydrin-positive material, the reaction wasabout 70% complete in 2 hr and complete after8 to 10 hr.The peptides formed from the crystal (about

2.5 mg, dry weight) were separated by two-dimensional chromatography and electrophore-sis. Although some material remained as a coreat the origin, a distinctive map was formed (Fig.2a) in which all of the ninhydrin-positive areascould be attributed to the crystal by comparisonwith a control in which trypsin had been incu-bated alone.

Digests of urea-spore preparations (afterparallel incubation) consistently gave nearlyidentical fingerprints with crystal when exam-ined by the two-dimensional system (Fig. 2b).The spore map did show more streaking on thebase line in the direction of electrophoresis.However, of the approximately 20 spots whichcould be clearly detected around the periphery ofthe crystal map, at least 16 were consistentlypresent in the spore map and the others may havebeen present in small amounts. Some spots,notably the two farthest removed from theorigin, varied considerably in intensity in differ-ent digests prepared from the same material.From the number of peptides observed and thearginine-lysine content, a molecular weight ofthe order of 30,000 can be calculated for theprotein, assuming a single polypeptide chain.Amino acid analyses. The relative amounts of

the amino acids found in the dissolved crystaland spore extracts are presented in Table 1. Cal-culation from the analysis shows that theseamino acids account for 100.4% of the dryweight of the crystal taken for analysis. Otherunidentified ninhydrin-positive compounds weredetected in the hydrolysates of whole spores andphosphate extracts of spores but not in hydroly-sates of urea-spore extracts. Because of thepresence of contaminating compounds in thesepreparations, no attempt has been made to cor-relate the amino acid composition from sporeextracts with dry weights. The amino acid com-position of the crystal is strikingly similar to thatof the urea extracts of spores and shows a markedparallel in the content of nearly all the individualamino acids. The composition of whole spores,although similar, is not nearly so close to that ofthe crystals, and that of the phosphate extract ofspores differs markedly from both the crystaland the other spore preparations.

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b

m .

FIG. 2. Fingerprints of tryptic digests ofcrystal (ca. 2.5 mg ofprotein) and spore (3 mg) preparations.

DIscussioNIn this and the preceding paper (2), evidence

has been presented supporting the relationship ofthe crystal to a protein fraction from brokenspores of B. thuringiensis var. alesti. The solu-bilized crystal and the protein of the urea-mer-captoethanol extract of spores behave almostidentically by all of the criteria used to assesstheir relationship. The crystal, dissolved ineither alkali or urea-mercaptoethanol, and thecorresponding alkali- or urea-spore extract showmarked similarities in their solubilities as a func-tion of pH (2). In addition, crystal solution andspore extracts show homologous precipitin bandswhen examined by the Ouchterlony techniqueusing rabbit antiserum prepared against alkali-dissolved crystal protein (2). The urea-sporeextract and the urea-crystal solution behavealmost identically on electrophoresis in acryla-mide gel under two different sets of conditions.Further, peptide maps prepared of tryptic digestsof the two preparations are almost superim-posable and reproducibly so.Although only sequence analysis can provide

ultimate proof of identity, the results indicatethat the crystal and urea-spore extract containclosely similar proteins. Further, the overallsimilarity of the amino acid analyses of the dis-solved crystal and the urea-spore extract suggestsan almost quantitative identity between them,and it seems reasonable to conclude that the twopreparations are substantially the same. Thisportion of the spore has been shown (2) to com-prise approximately 25%7, of the protein and about12'C of the dry weight. About 50% of the spore

protein remained as insoluble material afterextraction with urea-mercaptoethanol and it ispossible that this residue contains more of theshared material.The two preparations may have components

which are not common to both, because eithersolution could contain material which is notsubject to tryptic digestion or material whichdoes not enter acrylamide gel under any of theconditions used. Also, there are differences intheir amino acid analyses, e.g., in their contentsof glycine and alanine. These can be attributedto contamination of the urea-spore extract withother material, possibly from the exosporium.Alternatively, should the crystal have severalpolypeptide subunits (12), the spore extract andthe crystal may contain different proportions ofthe shared components.The protein of the crystal from B. thuringiensis

var. thuringiensis has been dissociated almostentirely into a form of low molecular weightwith a sedimentation coefficient of 0.83S, andhas been shown to have several polypeptidechains by N-terminal analysis (12). In the presentwork, results with acrylamide gel electrophore-sis, at pH 12, indicate that the crystal (and spore)protein must have been dissociated into a frac-tion of low molecular weight. The observationthat, at this pH, the bulk of the protein enters a7.5% gel as a single band suggests that, if thereare several components, they must have identi-cal mobilities and, consequently, must be ofsimilar size and charge. The entry of a band into5% gel at pH 9.3, which is excluded from 7.5%gel under the same conditions, indicates a com-

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TABLE 1. Amino acid analysis of crystals, spores,and spore fractions of Bacillus thuringiensis

var. alesti

Urea-mercal- Phos-

Amino acidsa Crys- Crys- to- Whole phatetalsb talsc ethanol soec extractextract prs of

of sporescsporesc

Lysine 0.24 3.1 4.3 5.8 3.0Histidine 0.16 2.1 2.1 2.3 1.4Ammonia 1.18Arginine 0.48 6.2 5.1 3.9 3.4Aspartic acid 0.93 12.1 11.0 9.8 7.1Threonine 0.47 6.1 6.9 6.2 5.1Serine 0.55 7.1 5.7 5.1 3.8Glutamic acid 0.90 11.7 10.3 11.1 13.5Proline 0.36 4.7 4.7 4.2 3.2Glycine 0.57 7.5 9.2 11.2 10.6Alanine 0.41 5.4 8.3 11.2 14.9Cysteine 0.12 1.5 1.3 2.1 TraceValine 0.60 7.9 7.8 6.9 6.3Methionine 0.07 0.9 1.8 3.0 3.2Isoleucine 0.45 5.9 5.7 4.9 3.8Leucine 0.63 8.1 7.9 6.7 5.6Tyrosine 0.36 4.7 3.3 2.8 2.2Phenylalanine 0.39 5.1 5.6 4.5 10.3Tryptophand 0.10

a Amino acids were determined by using aSpinco amino acid analyzer. The composition ofthe crystal was calculated from a series of hydroly-ses for 24, 48, 72, and 96 hr with extrapolation tozero-time where necessary.

I Micromoles per milligram (dry weight) ofprotein. Overall recovery, 100.4%.

c Micromoles per 100 ,umoles of total aminoacids.

d Determined by the method of Goodwin andMorton (5).

ponent of molecular weight of the order of 1 to2 million. The presence of this band in gels fromboth spore and crystal suggests that, after disso-ciation with urea-mercaptoethanol and duringthe subsequent dialysis, a portion of the proteinreaggregates to form an ordered or preferredpolymer in a manner analogous to that shown tooccur in the unfolding and reconstitution ofribonuclease (18).

Although 8 M urea alone causes a reversibledecrease in light-scattering of a crystal suspen-sion associated with swelling of the individualcrystals, neither this reagent nor mercapto-ethanol alone is sufficient to cause dissolution ofthe crystal at pH 8 to 9, indicating the involve-ment of both covalent disufide linkages andnoncovalent bonds in the crystal structure.Exposure of the dialyzed, dissolved crystal to8 M urea before electrophoresis may be sufficient

to counteract association of aggregates caused bynoncovalent bonds, but would not affect anycovalent disulfide linkages formed during removalof the reducing agent after the initial solubiliza-tion. That the urea-treated solution is excludedfrom 7.5% gel suggests that intermoleculardisulfide linkages may be involved in the struc-ture of the aggregate entering the 5% gel andpossibly in the crystal structure. By analogy,massive organized aggregates of the protein maybe present in the spore.The behavior of the protein on chromatogra-

phy on DEAE cellulose and on Sephadex canalso be attributed, at least in part, to aggrega-tion. The results of chromatography on DEAEcellulose suggest that there may be severalaggregates. Since the presence of nonproteinmaterial in the spore extract could influence thecharacter of the aggregates, differences in aggre-gation could account for the lack of extensiveimmunological homology between spore andcrystal solutions (2) which is, as yet, unexplained.The crystal protein appears to comprise a

large fraction of the total protein of the spore,and, since it is absent from the vegetative cell, itis not unreasonable to assume that it contributessignificantly to unique spore characteristics. Thefact that sporulating acrystalliferous (sp+ cr-)mutant strains are common, and that sp- cr+are extremely rare and show abortive sporulation(Somerville and Delafield, unpublished) suggeststhat the protein is indeed essential for sporula-tion and that crystal formation is closely linkedto sporulation but not vice versa. The extremeinsolubility of the crystal protein, its crystallinenature (7, 9), and its relatively high content ofhydrophobic amino acid residues point to astructural function for it in the spore, possibly asa major component of one (or more) of the sporecoats. The protein might function similarly inaccumulation of metals by the spore, a possi-bility suggested by its precipitation by divalentcations. The validity of these and other possiblespeculations remains to be shown. In any case,the data presented in this and in the precedingpaper are consistent with the initial hypothesisthat the formation of the crystal per se is a con-sequence of unregulated production of a normalspore protein.

ACKNOWLEDWMENTSWe thank John Goines for his assistance in the

amino acid analyses.This investigation was supported by grants GB

1225 and GB 6223 from the National Science Foun-dation. F. Delafield held a Public Health ServicePostdoctoral Fellowship, 5-F2-AI 31,369, from theNational Institute of Allergy and Infectious Dis-eases.

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LITERATURE CITED1. Canfield, R. E., and C. B. Anfinsen. 1963. Con-

cepts and experimental approaches in the de-termination ofthe primary structure of proteins,p. 311-378. In H. Neurath (ed.), The proteins,vol. 1. Academic Press, Inc., New York.

2. Delafield, F., H. J. Somerville, and S. C. Ritten-berg. 1968. Immunological homology betweencrystal and spore protein of Bacillus thuringien-sis. J. Bacteriol. 96:713-720.

3. Fasold, H., and G. Gundlach. 1963. Characteriza-tion of peptides and proteins with enzymes, p.350-362. In H. U. Bergmeyer, (ed.), Methodsof enzymatic analysis. Academic Press, Inc.,New York.

4. Goodwin, T. W., and R. A. Morton. 1946. Thespectrophotometric determination of tyrosineand tryptophan in proteins. Biochem. J. 40:628-632.

5. Gross, D. 1961. An apparatus for high-voltagepaper electrophoresis. J. Chromatog. 5:194-206.

6. Hjerten, S., S. Jerstedt, and A. Tiselius. 1965.Some aspects of the use of "continuous" and"discontinuous" buffer systems in polyacrylam-ide gel electrophoresis. Anal. Biochem. 11:219-221.

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

8. Katz, A. M., W. J. Dreyer, and C. B. Anfinsen.1959. Peptide separation of two-dimensionalchromatography and electrophoresis. J. Biol.Chem. 234:2897-2900.

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

10. Layne, F. 1957. Spectrophotometric and turbidi.metric methods for measuring proteins, p.447-454. In S. P. Colowick and N. 0. Kaplan(ed.), Methods in enzymology, vol. 3. Aca-demic Press, Inc., New York.

11. Leboy, P. S., E. C. Cox, and J. G. Flaks. 1964.The chromosomal site specifying a ribosomalprotein in Escherichia coli. Proc. Natl. Acad.Sci. U.S. 52:1367-1374.

12. Lecadet, M. M. 1966. La toxine figuree de Bacillusthuringiensis dissolution par action du thio-glycolate ou de la cysteine. Compt. Rend. 262:195-198.

13. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, andR. J. Randall. 1951. Protein measurement withthe Folin phenol reagent. J. Biol. Chem. 193:265-275.

14. Martinez, R. J., D. M. Brown, and A. N. Glazer.1967. The formation of bacterial flagella. III.Characterization of the subunits of the flagellaof Bacillus subtilis and Spirillum serpens. J.Mol. Biol. 28:45-51.

15. Ornstein, L., and B. J. Davis. 1962. Disc electro-phoresis. Preprinted by Distillation ProductsIndustries, Rochester, New York.

16. Sanger, F. 1949. Fractionation of oxidized insulin.Biochem. J. 44:126-128.

17. Spackman, D. H., W. H. Stein, and S. Moore.1958. Automatic recording apparatus for use inthe chromatography of amino acids. Anal.Chem. 30:1190-1205.

18. White, F. H., and C. B. Anfinsen. 1959. Some re-lations of structure to function in ribonuclease.Ann. N.Y. Acad. Sci. 81:515-523.

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