APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1985, p. 737-7420099-2240/85/100737-06$02.00/0Copyright X 1985, American Society for Microbiology

Protease Activation of the Entomocidal Protoxin of Bacillusthuringiensis subsp. kurstakit

ROBERT E. ANDREWS, JR.,'* MARGARET M. BIBILOS,' AND LEE A. BULLA, JR.2

Department of Microbiology, Iowa State University, Ames, Iowa 50011,1 and Department ofBiochemistry, University ofWyoming, Laramie, Wyoming 820712

Received 25 April 1985/Accepted 11 July 1985

Two isolates of BaciUus thuringiensis subsp. kurstaki were examined which produced different levels ofintracellular proteases. Although the crystals from both strains had comparable toxicity, one of the strains,LB1, had a strong polypeptide band at 68,000 molecular weight in the protein from the crystal; in the other,HD251, no such band was evident. When the intraceUlular proteases in both strains were measured, strainHD251 produced less than 10% of the proteolytic activity found in LB1. These proteases were primarily neutralmetalloproteases, although low levels of other proteases were detected. In LB1, the synthesis of proteaseincreased as the cells began to sporulate; however, in HD251, protease activity appeared much later in thesporulation cycle. The protease activity in strain LB1 was very high when the ceUs were making crystal toxin,whereas in HD251 reduced proteolytic activity was present during crystal toxin synthesis. The insecticidal toxin(molecular weight, 68,000) from both strains could be prepared by cleaving the protoxin (molecular weight,135,000) with trypsin, followed by ion-exchange chromatography. The procedure described gave quantitativerecovery of toxic activity, and approximately half of the total protein was recovered. Calculations show thatthese results correspond to stoichiometric conversion of protoxin to insecticidal toxin. The toxicities of wholecrystals, soluble crystal protein, and purified toxin from both strains were comparable.

Bacillus thuringiensis is a sporeforming bacterium thatproduces a potent insecticidal crystalline protein. The crys-

tal has been described as containing a glycoprotein protoxin(molecular weight, 135,000) that is converted to a toxin(molecular weight, 68,000) after ingestion by a susceptibleinsect (5-8, 16). Synthesis of crystal toxin in B. thuringiensisevidently is a sporulation-associated event because crystaltoxin is not found in vegetative cells and does not appear insporulating cells until about 3 h after the onset of sporula-tion. In B. thuringiensis subsp. kurstaki LB1, one of thebetter-characterized laboratory strains, toxin content be-comes maximal about 5 h after sporulation is initiated (1).Apparently, crystal toxin synthesis is controlled at thetranscriptional level, because crystal toxin-specific mRNAappears only in the cells during the exact time at which denovo crystal toxin synthesis is occurring (2).

Several workers have used proteases to activate theprotoxin ofB. thuringiensis. These proteases came primarilyfrom the midguts of insects or from commercial enzymepreparations such as trypsin. However, the methods de-scribed in the literature for making this conversion are, atbest, inefficient (6, 16). Although several workers haveimplicated the proteases of B. thuringiensis in the variationof protoxin size observed between the various toxigenicstrains (9), no data have been presented to show that theseproteases might also convert protoxin to the insecticidaltoxin.A diverse group of proteolytic activities is produced by

bacilli, and several of these enzymes are synthesized by thecell during the early stages of sporulation (12, 18). Theproteases associated with B. thuringiensis have been de-scribed in several reports, but no central agreement has beenreached regarding protease properties and characteristics.

* Corresponding author.t Journal paper no. J-11623 (project 2673) of the Iowa Agriculture

and Home Economics Experiment Station, Ames, IA 50011.

Several reports suggest that the crystal-associatedprotease(s) is of the serine, metallo, and sulfhydryl classes(8-10). Proteases have been proposed to be a factor duringthe attack of a pathogen on the insect midgut (4), but therehas been no demonstration that proteases of B. thuringiensisfunction in this capacity. A B. thuringiensis protease may,

however, play a role in inactivating the defense mechanismof the insect (11). Despite the lack of a proven role for theproteases in B. thuringiensis, their synthesis is interestingbecause they are produced very early in the sporulationcycle, and they interfere with biochemical studies ofsporulating bacteria.The crystal toxin of B. thuringiensis subsp. kurstaki is

composed primarily of the glycoprotein protoxin; whenwhole crystals are solubilized by either reducing or denatur-ing agents or by mild alkali several smaller proteins are

observed (20). These smaller components apparently areproducts of proteolytic activity that degrades the 135,000-molecular-weight protoxin molecule. One such product is a

68,000-molecular-weight polypeptide that has been pur-ported to be the ultimate toxin of B. thuringiensis (6, 7).Because there has been some disagreement over theprotoxin-to-toxin conversion (13, 16), we further investi-gated the involvement of protease action on native crystalsto determine if protoxin conversion is facilitated byproteolytic enzymes. In doing so, we utilized an isolate ofB.thuringiensis subsp. kurstaki with decreased proteolyticactivity and compared it with a parent laboratory strain thatpossesses normal levels of proteolytic activity. Also, we

describe a toxin purification procedure that rendersstoichiometric conversion of protoxin to toxin.

MATERIALS AND METHODS

Bacterial strains and growth conditions. B. thuringiensissubsp. kurstaki (LB1) was isolated from Dipel (AbbottLaboratories, North Chicago, Ill.), and was maintained in thelaboratory on brain heart infusion agar slants. Strain HD251

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738 ANDREWS ET AL.

was provided by H. T. Dulmage, U.S. Department ofAgriculture, Brownsville, Tex. Spores and crystals from eachsubspecies of B. thuringiensis subsp. kurstaki were grown inliquid GYS medium (17) and harvested by centrifugation;spores were separated from crystals on NaBr gradients asdescribed by Ang and Nickerson (3).Growth and protease assay. When sporulating cells were to

be assayed for protease production, growth was as describedby Andrews et al. (1, 2). The cells from 100 ml of culturewere harvested each hour during the sporulation cycle,washed in sodium azide (0.2 mg/ml), and then frozen at-20°C. Sporulation was followed by phase-contrast micros-copy.When protease was to be extracted from the washed

pellets, the frozen cells were suspended in 5 ml of 10 mMTris hydrochloride (pH 8.3) containing 0.2 mg of sodiumazide per ml and sonicated for four 30-s bursts. Microscopicexamination of the cells after sonication of the preparationsshowed that >99% of the vegetative cells and the mothercells from both isolates were lysed; however, the spores andforespores did not appear to be broken. After centrifugationat 15,000 x g for 10 min, the enzyme-containing supernatantwas used for the protease assays. A 60-,ul sample of theprotease suspension was incubated in 1 ml of 25 mM Trishydrochloride (pH 8.3) containing 3 mg of azoalbumin(Sigma Chemical Co., St. Louis, Mo.) per ml for 2 h at 30°C.After incubation, 4 ml of 5% trichloroacetic acid was added,the protein was allowed to precipitate for 1 h at roomtemperature, the protein was removed by centrifugation at20°C for 20 min at 4,000 x g, and then 1 ml of 10 N NaOHwas added to the supernatant. Protease activity, expressedas absorbance units of trichloroacetic acid-soluble material,was determined at 440 nm. The effect of several inhibitors onprotease activity was tested. EDTA, a chelating agent, andphenylmethylsulfonyl fluoride and diisopropylfluorophosph-ate, serine protease inhibitors, were assayed separately byadding the appropriate inhibitor to give a final concentrationof 10 mM.

Effect of pH on protease activity. Assays to determine theoptimum pH for protease activity were conducted by incu-bating the protease sample and substrate in buffered solu-tions which were of differing pH. For pH 5.0 and 6.0, sodiumacetate buffers were used; for pH 7.0 to 9.0, Tris hydrochlo-ride buffers were used. At pH 10.0, a sodium carbonate-sodium bicarbonate buffer was used. In several experiments,Tris hydrochloride and phosphate buffers or Tris hydrochlo-ride and carbonate or phosphate and acetate buffers wereused at the same pH. Under these conditions, it appearedthat only the pH, not the buffer composition, effectedenzyme activity.

Protein techniques. The purified crystals were solubilizedin Ellis buffer and treated with trypsin as described by Lilleyet al. (16). After proteolytic digestion, the material wasdialyzed into 20 mM NaH2PO4 and separated by ion-exchange chromatography as described by Bulla et al. (6).Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) was done as described by Andrews et al. (2).

Insect bioassays. Bioassays were done by using neonatelarvae of the cabbage looper Tricoplusia ni in a mannersimilar to that described by Schesser et al. (19). Mosquitobioassays were done on second-instar larvae of Aedesaegypti as described by Tyrell et al. (21).Immunological techniques. Antibody against purified crys-

tals from each strain was prepared in rabbits, and crystaltoxin concentrations were determined by using rocket im-munoelectrophoresis as described by Andrews et al. (1).

RESULTSWhen compared with an equal amount of soluble crystal

toxin from strain LB1, toxin from strain HD251 did notappear to contain a protein band migrating at a molecularweight of 68,000 on SDS-PAGE (Fig. 1). Purified crystalsfrom both strains were equally toxic when bioassayedagainst the larvae of the cabbage looper, and neither isolatewas toxic when assayed against larvae of the mosquito A.aegypti (data not shown). The lack of a protein migrating ata molecular weight of 68,000 was unusual for lepidopteran-toxic strains of B. thuringiensis, and we became interested inunderstanding why this particular isolate was different.Because we believe that toxin is created by proteolyticcleavage of protoxin in the insect, we began to examine theproteases produced by these two isolates of Bacillusthuringiensis subsp. kurstaki.Growth and protease activity of strain LB1 are shown in

Fig. 2B. Protease activity in this isolate during vegetativegrowth, if present, was not detected; measurable activityfirst appeared at To (defined as the end of vegetative growth;about 3 h after inoculation of the culture) and remained highthroughout sporulation. Maximum proteolytic activity firstappeared at about T3 (3 h) of sporulation. There was adistinct drop in protease activity during T3 to T4, whichcorresponds to the period when crystal toxin dominatescellular protein synthesis in strain LB1 (2). This drop inprotease activity was followed by another maximum whichoccurred at about T7, corresponding to the period when thecells have completed crystal toxin synthesis. The proteaseactivity in strain HD251 was greatly reduced during the earlyperiod of sporulation but did appear later in the sporulationcycle (Fig. 2A).Inasmuch as protease-deficient isolates of B. subtilis often

do not sporulate efficiently, it was necessary to determinewhether strain HD251 was defective in its ability to sporulateor form toxic crystals. Figure 3 shows the growth, sporula-tion, and crystal toxin formation for both strains of B.thuringiensis subsp. kurstaki. In both strains, vegetativegrowth was completed and the cells entered sporulation by 3h (To). Crystal toxin antigen, measured by rocket immuno-electrophoresis, first appeared in the cells of both isolatesabout 6 h after inoculation (T4) and reached its maximum rateof synthesis by T5. All the crystal toxin present in a fullysporulated culture was present by about T7. Complete,phase-light spores first appeared in the cells at about T7. By

200

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45FIG. 1. SDS-PAGE of solubilized crystals from B. thuringiensis

subsp. kurstaki isolates LB1 and HD251. Whole crystals that hadbeen purified on NaBr gradients were electrophoresed on a 7 to 17%polyacrylamide gradient. Lane A, LB1; lane B, HD251. Molecularweights (x 103) are shown on the right.

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PROTEASE ACTIVATION OF ENTOMOCIDAL PROTOXIN

T8 approximately 50% of the cells contained a completespore, and by Tg, >95% of all cells contained a phase-light

0.100

spore. Evidently, growth, spore formation, and crystal toxinproduction were not influenced by the reduction in proteaseproduction found in strain HD251.Because the protease activity in the two strains is vastly

different during the period of crystal toxin synthesis and 0.3 120 * 60because this difference may account for the different pro- 100teins found in whole crystals from the two strains, we 0 2 80 40

believed that it was of interest to begin to characterize these /0enzymes. The proteases present in cells at T4 were chosen

.

because (i) proteases present in the cell at earlier stages 40 co

should not affect the crystal toxin proteins, and (ii) after WIW 20about T5, the crystal toxin proteins should be almost exclu-

sively in crystals and therefore less susceptible to proteolytic co9**degradation. The activity measured at T4 was an intracellular cprotease or was tightly bound to the cells because when a T4 e 4culture was centrifuged and washed twice with 1 M NaCl, 0-5 /0the activity remained in the pellet (Table 1). Most of the /proteolytic activity found in both LB1 and HD251 was of the 3IImetallo class (Fig. 4), because almost all the protease 0 3activity was inhibited by EDTA but not by phenylmethyl- l00sulfonyl fluoride. The use of diisopropylfluorophosphate as a 0-2 s 40protease inhibitor gave results similar to those observed with 60phenylmethylsulfonyl fluoride (data not shown). The prote- 40 20

01i 20

x 100 0 1 2 3 4 5 6 78 T 10 I I 12Time thr)

1 0- 16 FIG. 3. Growth, crystal toxin formation, and sporulation in B.

thuringiensis subsp. kurstaki isolates HD251 (A) and LB1 (B).

Symbols: 0, growth measured by A6N; 0, crystal antigen measured0 *5 _ + ; l 2 by rocket immunoelectrophoresis; *, presence of phase-light spores

measured by phase-contrast microscopy. The arrow indicates the0 3 approximate occurrence of To.

0X2 _/ 1. 8

ase activity found in LB1 had a pH optimum at about pH 7.0,lo (Fig. 5). This observation indicated that the proteolytic

0.1 _ | _ 4 activity found in LB1 at T3 to T5 is typical of the neutral

o1% proteases found in the genus Bacillus (14).8 | | tTo demonstrate that the protoxin of HD251 and LB1 could

S *05 < J , be converted to a 68,000-molecular-weight toxin, the solubil-_Ba ized crystals from both isolates were treated with trypsin and10 _ _ _ _ _ _ _ __ 16:@ then purified by ion-exchange chromatography. Figure 60gr_shows an ion-exchange chromatogram obtained when tryp-

oI sin-treated crystal toxin from HD251 was adsorbed to a

DEAE-cellulose column and eluted with a 0 to 0.5 M NaCl0.5| \ fl 2 gradient. The insert (Fig. 6) shows solubilized crystals,

O/3 trypsin-treated solubilized crystals, and purified toxin sepa-rated by SDS-PAGE. The same techniques can be used to

0 2 8 purify the insecticidal toxin from LB1 (data not shown).

0O _ 4 TABLE 1. Protease activity of B. thuringiensis subsp. kurstakiLB1 and HD251 cells before and after washing in 1 M NaCl

-055, I a I I I I

I 2 3 4 5 6 7 8 9 10

Time (hr)FIG. 2. Growth and protease activity of B. thuringiensis subsp.

kurstaki isolates HD251 (A) and LB1 (B). Cell extract (60 ,u) wasadded to each reaction mixture that contained azoalbumin. Sym-bols: 0, acid-soluble material; 0, growth. The arrow indicates theapproximate occurrence of To.

Cell source Treatment Activity'

HD251 Unwashed 0.017Washed 0.015

LB1 Unwashed 0.215Washed 0.210

a Activity expressed as A4Q. Sonication of cells from both isolates at thisstage (T4) resulted in breakage of >99%o of the cells as observed by phase-contrast microscopy.

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740 ANDREWS ET AL.

0o15

0 12

0

0

009

0 06

003

0

0 2

0v1r

49

0

00-0

015

005

0-05

l 2 3 4 5 6 7

Time (hr)8 9 90

4 5 6 7Time (hr)

FIG. 4. Effect of protease inhibitors on proteases from B.thuringiensis subsp. kurstaki isolates HD251 (A) and LB1 (B).Symbols: 0, EDTA; A, phenylmethylsulfonyl fluoride; U, no treat-ment. The arrow indicates the approximate occurrence of To.

By using the techniques described herein, the toxin fromHD251 and from LB1 could be purified with essentiallyquantitative recovery of toxic activity and stoichiometricconversion of toxin (molecular weight, 68,000) from protoxin(molecular weight, 135,000) (Table 2). The toxin purifiedfrom both strains was approximately twice as toxic as theprotein from solubilized crystals and approximately fourtimes as toxic as are whole crystals. The method can be usedto prepare milligram quantities of insecticidal toxin from B.thuringiensis subsp. kurstaki in 2 days.

DISCUSSIONThe method described for toxin purification provides

much larger quantities of insecticidal toxin then previousmethodologies have allowed. By these techniques, 5 mg ofpurified insecticidal toxin can be prepared in as few as 2days. This advance should lead to a more detailed physicalcharacterization of the toxin and facilitate a better under-standing of the mode of action of the toxin. Despite a numberof reports that describe the protoxic nature of the crystaltoxin subunit, this model is still under some question be-cause of the very poor efficiencies with which toxin can bepurified from whole crystals.The quantitative conversion of protoxin to toxin achieved

in this study strongly supports the model proposed for theprotoxic nature of the crystal toxin. When crystals of eitherstrain of B. thuringiensis subsp. kurstaki were solubilized inalkali, they became about twice as toxic when assayedagainst the cabbage looper. The reason for this is not clear,but essentially the same results were obtained by Bulla et al.

(7) using larvae of the tobacco hornworm. Treatment of thecrystals with trypsin, followed by dialysis, reduced theprotein concentration by about 50%, with no reduction in thetotal toxic activity of the preparation. Ion-exchange chroma-tography increased the purity of the material but had little, ifany, effect on the toxic activity. Calculated on the basis oftotal protein content, the toxin is roughly twice as toxic as

the solubilized crystal. Because solubilized crystal is mostlyprotoxin (molecular weight, 135,000) and because the mo-

lecular weight of the toxin (68,000) is roughly half that of theprotoxin, the molar toxicities of the toxin and protoxin areabout equal. If the 135,000-molecular-weight molecule was adimer, one would expect that the molar toxicity of the toxinwould be about half of that obtained by using solubilizedcrystals (which are mostly protoxin). These observationsstrongly support the protoxin-toxin model. Further, inas-much as very little if any protein at a molecular weight of68,000 was found in HD251, the high toxicity of this isolateis difficult to explain without using the protoxin-toxin model.

This study has shown that although proteins found inwhole crystals may differ among various strains of B.thuringiensis, these differences may not exclusively reflectdifferences in the genetic information responsible for synthe-sis, but may be caused by other metabolic differences as

well. Disagreement with the protoxin-toxin model for thecrystal toxin has centered on the argument that the toxinmolecule synthesized by B. thuringiensis has a molecularweight of 68,000 and that the 135,000-molecular-weight bandis an unusually stable dimer. The data contained hereinsuggest that the initial transcript has a molecular weight of135,000 and that the presence of the activated toxin reportedin most strains of B. thuringiensis (20) results fromproteolytic cleavage of the protoxin during its synthesis.

Support for this hypothesis can be derived from at leastfour lines of evidence. (i) The fact that very little toxin(molecular weight, 68,000) can be found in strain HD251suggests that this dimer would have to be extremely stable inthe presence of the SDS, 2-mercaptoethanol, and ureapresent in the SDS gel sample buffer. (ii) The observationthat the toxin is readily created with proteases suggests thata break in the polypeptide chain is required to create toxin.This would not be expected if the 135,000-molecular-weightmolecule was a dimer. (iii) The toxicity data suggest that onemolecule of protoxin gives rise to one molecule of toxin. Ifthe putative protoxin was a dimer, two molecules of toxin

4

%.,

.7 -

-6

.5-

.4

.3-

.2-

.1 I I I I I- IS 6 7 8 9 10

pHFIG. 5. Effect of pH on the activity of the protease activity from

T4 cells of B. thuringiensis subsp. kurstaki isolate LB1.

I I I I A I I .3 I I

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PROTEASE ACTIVATION OF ENTOMOCIDAL PROTOXIN

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a6

0.6

0.4

0.2

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1 3 5 7 9 11 13 15 17FRACTION NUMBER

FIG. 6. Purification of the entomocidal toxin from B. thuringiensis subsp. kurstaki HD251 with DE52. Crystal from this strain weresolubilized in Ellis buffer, dialyzed against 1 mM NaHCO4, treated with trypsin, and then dialyzed against column buffer (20 mM Na2HPO4at pH 7.5). The purified toxin was eluted with a 0 to 0.5 M NaCl gradient. The inset shows soluble HD251 crystals (lane A), trypsin-treatedcrystals (lane B), and purified toxin (lane C) separated on SDS-PAGE. OD, Optical density.

would be expected. (iv) The identification of strain HD251,which produces reduced levels of protease and little or no68,000-molecular-weight protein, further supports this the-ory. The major weakness in this argument stems from thefact that the genetic relationship between HD251 and LB1 isnot excactly known. A reduced-protease mutant of LB1would greatly strengthen this argument. We are currentlyworking on a more complete genetic characterization ofLB1. Proteolysis of cellular components in bacilli, however,is well known, and this work underscores the importance ofconsidering proteases when working with B. thuringiensis.There has been a recent report of a mosquito toxin

(molecular weight, 65,000) in the crystals from B. thuringi-ensis subsp. kurstaki HD1 (13). LB1 is thought to be verysimilar to HD1. Results of this study and others (20, 21)indicate that LB1 is not toxic to mosquitoes. Because thereare indications in the literature that the two strains are notidentical based on different plasmid profiles (15), it is possi-ble that only HD1 carries the mosquito-toxic factor.These results do have some important implications in

understanding sporulation in B. thuringiensis. Further stud-ies of the biochemistry and molecular biology of sporulationin B. thuringiensis will require investigations of a more

TABLE 2. Potency and yield of toxic protein during purificationof the entomocidal toxin from B. thuringiensis subsp. kurstaki

LB1 and HD251Treatment Protein (mg) LC50.

LB1Whole crystals 5.0 14.2Solubilized crystals 4.7 7.2Trypsin treated 2.6 4.9"Pure toxin" 2.4 4.0

HD251Whole crystals 5.0 13.8Solubilized crystals 4.9 7.3Trypsin treated 2.7 4.6"Pure toxin" 2.5 3.7a LC, 50%o lethal concentration, in nanograms of toxic product per square

centimeter applied to the surface of the artificial diet, calculated by probitanalysis.

detailed nature about the control of transcription and trans-lation during endospore formation and crystal toxin synthe-sis. Previous work has shown that the synthesis of crystaltoxin is controlled, at least in part, by regulating synthesis ofcrystal toxin-specific mRNA (2). Inasmuch as work withRNA polymerase and in vitro translation is susceptible toerror from proteases present during sporulation, the knowl-edge of the proteases produced by B. thuringiensis providedherein should aid in these studies. It is possible, for example,that HD251 could be a valuable source ofRNA polymerases,ribosomal proteins, etc., which have not been subject to asmuch proteolytic degradation.

ACKNOWLEDGMENT

This work was supported by a grant from the Iowa State UniversityAchievement Foundation.

LITERATURE CITED1. Andrews, R. E., Jr., D. B. Bechtel, B. S. Campbell, L. I.

Davidson, and L. A. BuHla, Jr. 1981. Solubility of parasporalcrystals of Bacillus thuringiensis and presence of toxic proteinduring sporulation, germination, and outgrowth, p. 174-177. InH. S. Levinson, A. L. Sonenshein, and D. J. Tipper (ed.),Sporulation and germination. American Society for Microbiol-ogy, Washington, D.C.

2. Andrews, R. E., Jr., K. Kanda, and L. A. BuUla, Jr. 1982. Invitro and in vivo synthesis of the parasporal crystal of B.thuringiensis, p. 121-130. In A. T. Ganesan, S. Chang, andJ. A. Hoch (ed.), Gene regulation in bacilli. Academic Press,Inc., New York.

3. Ang, B. J., and K. W. Nickerson. 1978. Purification of theprotein crystal from Bacillus thuringiensis by zonal gradientcentrifugation. Appl. Environ. Microbiol. 36:625-626.

4. Brandt, C. R., M. J. Adang, and K. D. Spence. 1978. Theperitrophic membrane: ultrastructural analysis and function as amechanical barrier to microbial infection in Orgyiapseudotsugata. J. Invertbr. Pathol. 32:12-20.

5. Buila, L. A., Jr., D. B. Bechtel, K. J. Kramer, I. Shethna, A. I.Aronson, and P. C. Fitz-James. 1980. Ultrastructure, physiologyand biochemistry of Bacillus thuringiensis. Crit. Rev. Micro-biol. 8:147-204.

6. Bulla, L. A., Jr., L. I. Davidson, K. J. Kramer, and B. I. Jones.1979. Purification of the insecticidal toxin from the parasporalcrystal of Bacillus thuringiensis. Biochem. Biophys. Res. Com-mun. 91:1123-1130.

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7. BuIla, L. A., Jr., K. J. Kramer, D. J. Cox, B. L. Jones, and L. I.Davidson. 1981. Purification and characterization of theentomocidal protoxin of Bacillus thuringiensis. J. Biol. Chem.256:3000-3004.

8. Bulla, L. A., Jr., K. J. Kramer, and L. I. Davidson. 1977.Characterization of the entomocidal parasporal crystal of Bacil-lus thuringiensis. J. Bacteriol. 130:375-383.

9. Chestukhina, G. G., I. A. Zalunin, L. I. Kostina, T. S. Kotova,S. P. Kattrukha, L. A. Lyublinskya, and V. M. Stepanov. 1978.Proteinases bound to crystals of Bacillus thuringiensis.Biokhimiya 43:857-864.

10. Chestukhina, G. G., I. A. Zalunin, L. I. Kostina, T. S. Kotova,S. P. Kattrukha, and V. M. Stepanov. 1980. Crystal formingproteins of Bacillus thuringiensis. Biochem. J. 187:457-465.

11. Dalhammar, G., and H. Steiner. 1984. Characterization ofinhibitor A, a protease from Bacillus thuringiensis which de-grades attacins and cecropins, two classes of antibacterialproteins in insects. Eur. J. Biochem. 139:247-253.

12. Doi, R. H. 1972. Role of proteases in sporulation. Curr. Top.Cell. Regul. 6:1-20.

13. lizuka, T., and T. Yamamoto. 1983. Possible location of themosquitocidal protein in the crytsal preparation of Bacillusthuringiensis subsp. kurstaki. FEMS Microbiol. Lett.19:187-190.

14. Keay, L., and B. S. Wildi. 1970. Proteases of the genus Bacillus.I. Neutral Proteases. Biotechnol. Bioeng. 22:179-212.

15. Kronstad, J. W., H. E. Schnepf, and H. R. Whiteley. 1983.Diversity of locations for Bacillus thuringiensis crystal proteingenes. J. Bacteriol. 154:419-428.

16. Lilley, M., R. N. Ruffell, and H. J. Somerville. 1980. Purificationof the insecticidal toxin in crystals of Bacillus thuringiensis. J.Gen. Microbiol. 1:1-11.

17. Nickerson, K. W., and L. A. Bulla, Jr. 1974. Physiology ofsporeforming bacteria associated with insects: minimal nutri-tional requirements for growth, sporulation, and parasporalcrystal formation of Bacillus thuringiensis. Appl. Microbiol.28:124-128.

18. Roitsch, C. A., and J. H. Hageman. 1983. Bacillopeptidase F:two forms of a glycoprotein serine protease from Bacillussubtilis 168. J. Bacteriol. 155:145-152.

19. Schesser, J. H., K. J. Kramer, and L. A. Bulla, Jr. 1977.Bioassay for homogeneous parasporal crystal of Bacillusthuringiensis using the tobacco hormworm, Manduca sexta.Appl. Environ. Microbiol. 33:878-880.

20. Tyrell, D. J., L. A. Bulia, Jr., R. E. Andrews, Jr., K. J. Kramer,L. I. Davidson, and P. Nordin. 1981. Comparative biochemistryof entomocidal parasporal crystals of selected Bacillus thuringi-ensis strains. J. Bacteriol. 145:1052-1062.

21. Tyrell, D. J., L. I. Davidson, L. A. Bulla, Jr., and W. A.Ramoska. 1979. Toxicity of parasporal crystals of Bacillusthuringiensis subsp. israelensis to mosquitoes. Appl. Environ.Microbiol. 38:656-658.

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