Protease Activation of the Entomocidal Protoxin of Bacillus thuringiensis subsp. kurstakit

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1985, p /85/1737-6$2./ Copyright X 1985, American Society for Microbiology Vol. 5, No. 4 Protease Activation of the Entomocidal Protoxin of Bacillus thuringiensis subsp. kurstakit ROBERT E. ANDREWS, JR.,'* MARGARET M. BIBILOS,' AND LEE A. BULLA, JR.2 Department of Microbiology, Iowa State University, Ames, Iowa 511,1 and Department of Biochemistry, University of Wyoming, Laramie, Wyoming Received 25 April 1985/Accepted 11 July 1985 Two isolates of BaciUus thuringiensis subsp. kurstaki were examined which produced different levels of intracellular proteases. Although the crystals from both strains had comparable toxicity, one of the strains, LB1, had a strong polypeptide band at 68, 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, strain HD251 produced less than 1% of the proteolytic activity found in LB1. These proteases were primarily neutral metalloproteases, although low levels of other proteases were detected. In LB1, the synthesis of protease increased as the cells began to sporulate; however, in HD251, protease activity appeared much later in the sporulation 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,) from both strains could be prepared by cleaving the protoxin (molecular weight, 135,) with trypsin, followed by ion-exchange chromatography. The procedure described gave quantitative recovery of toxic activity, and approximately half of the total protein was recovered. Calculations show that these results correspond to stoichiometric conversion of protoxin to insecticidal toxin. The toxicities of whole crystals, soluble crystal protein, and purified toxin from both strains were comparable. Bacillus thuringiensis is a sporeforming bacterium that produces a potent insecticidal crystalline protein. The crystal has been described as containing a glycoprotein protoxin (molecular weight, 135,) that is converted to a toxin (molecular weight, 68,) after ingestion by a susceptible insect (5-8, 16). Synthesis of crystal toxin in B. thuringiensis evidently is a sporulation-associated event because crystal toxin is not found in vegetative cells and does not appear in sporulating cells until about 3 h after the onset of sporulation. In B. thuringiensis subsp. kurstaki LB1, one of the better-characterized laboratory strains, toxin content becomes maximal about 5 h after sporulation is initiated (1). Apparently, crystal toxin synthesis is controlled at the transcriptional level, because crystal toxin-specific mrna appears only in the cells during the exact time at which de novo crystal toxin synthesis is occurring (2). Several workers have used proteases to activate the protoxin of B. thuringiensis. These proteases came primarily from the midguts of insects or from commercial enzyme preparations such as trypsin. However, the methods described in the literature for making this conversion are, at best, inefficient (6, 16). Although several workers have implicated the proteases of B. thuringiensis in the variation of protoxin size observed between the various toxigenic strains (9), no data have been presented to show that these proteases might also convert protoxin to the insecticidal toxin. A diverse group of proteolytic activities is produced by bacilli, and several of these enzymes are synthesized by the cell during the early stages of sporulation (12, 18). The proteases associated with B. thuringiensis have been described in several reports, but no central agreement has been reached regarding protease properties and characteristics. * Corresponding author. t Journal paper no. J (project 2673) of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA Several reports suggest that the crystal-associated protease(s) is of the serine, metallo, and sulfhydryl classes (8-1). Proteases have been proposed to be a factor during the attack of a pathogen on the insect midgut (4), but there has been no demonstration that proteases of B. thuringiensis function in this capacity. A B. thuringiensis protease may, however, play a role in inactivating the defense mechanism of the insect (11). Despite the lack of a proven role for the proteases in B. thuringiensis, their synthesis is interesting because they are produced very early in the sporulation cycle, and they interfere with biochemical studies of sporulating bacteria. The crystal toxin of B. thuringiensis subsp. kurstaki is composed primarily of the glycoprotein protoxin; when whole crystals are solubilized by either reducing or denaturing agents or by mild alkali several smaller proteins are observed (2). These smaller components apparently are products of proteolytic activity that degrades the 135,- molecular-weight protoxin molecule. One such product is a 68,-molecular-weight polypeptide that has been purported to be the ultimate toxin of B. thuringiensis (6, 7). Because there has been some disagreement over the protoxin-to-toxin conversion (13, 16), we further investigated the involvement of protease action on native crystals to determine if protoxin conversion is facilitated by proteolytic enzymes. In doing so, we utilized an isolate of B. thuringiensis subsp. kurstaki with decreased proteolytic activity and compared it with a parent laboratory strain that possesses normal levels of proteolytic activity. Also, we describe a toxin purification procedure that renders stoichiometric conversion of protoxin to toxin. MATERIALS AND METHODS Bacterial strains and growth conditions. B. thuringiensis subsp. kurstaki (LB1) was isolated from Dipel (Abbott Laboratories, North Chicago, Ill.), and was maintained in the laboratory on brain heart infusion agar slants. Strain HD251 Downloaded from on September 22, 218 by guest

2 738 ANDREWS ET AL. was provided by H. T. Dulmage, U.S. Department of Agriculture, Brownsville, Tex. Spores and crystals from each subspecies of B. thuringiensis subsp. kurstaki were grown in liquid GYS medium (17) and harvested by centrifugation; spores were separated from crystals on NaBr gradients as described by Ang and Nickerson (3). Growth and protease assay. When sporulating cells were to be assayed for protease production, growth was as described by Andrews et al. (1, 2). The cells from 1 ml of culture were harvested each hour during the sporulation cycle, washed in sodium azide (.2 mg/ml), and then frozen at -2 C. Sporulation was followed by phase-contrast microscopy. When protease was to be extracted from the washed pellets, the frozen cells were suspended in 5 ml of 1 mm Tris hydrochloride (ph 8.3) containing.2 mg of sodium azide per ml and sonicated for four 3-s bursts. Microscopic examination of the cells after sonication of the preparations showed that >99% of the vegetative cells and the mother cells from both isolates were lysed; however, the spores and forespores did not appear to be broken. After centrifugation at 15, x g for 1 min, the enzyme-containing supernatant was used for the protease assays. A 6-,ul sample of the protease suspension was incubated in 1 ml of 25 mm Tris hydrochloride (ph 8.3) containing 3 mg of azoalbumin (Sigma Chemical Co., St. Louis, Mo.) per ml for 2 h at 3 C. After incubation, 4 ml of 5% trichloroacetic acid was added, the protein was allowed to precipitate for 1 h at room temperature, the protein was removed by centrifugation at 2 C for 2 min at 4, x g, and then 1 ml of 1 N NaOH was added to the supernatant. Protease activity, expressed as absorbance units of trichloroacetic acid-soluble material, was determined at 44 nm. The effect of several inhibitors on protease activity was tested. EDTA, a chelating agent, and phenylmethylsulfonyl fluoride and diisopropylfluorophosphate, serine protease inhibitors, were assayed separately by adding the appropriate inhibitor to give a final concentration of 1 mm. Effect of ph on protease activity. Assays to determine the optimum ph for protease activity were conducted by incubating the protease sample and substrate in buffered solutions which were of differing ph. For ph 5. and 6., sodium acetate buffers were used; for ph 7. to 9., Tris hydrochloride buffers were used. At ph 1., a sodium carbonatesodium bicarbonate buffer was used. In several experiments, Tris hydrochloride and phosphate buffers or Tris hydrochloride and carbonate or phosphate and acetate buffers were used at the same ph. Under these conditions, it appeared that only the ph, not the buffer composition, effected enzyme activity. Protein techniques. The purified crystals were solubilized in Ellis buffer and treated with trypsin as described by Lilley et al. (16). After proteolytic digestion, the material was dialyzed into 2 mm NaH2PO4 and separated by ionexchange 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). Bioassays were done by using neonate larvae of the cabbage looper Tricoplusia ni in a manner similar to that described by Schesser et al. (19). Mosquito Insect bioassays. bioassays were done on second-instar larvae of Aedes aegypti as described by Tyrell et al. (21). Immunological techniques. Antibody against purified crystals from each strain was prepared in rabbits, and crystal toxin concentrations were determined by using rocket immunoelectrophoresis as described by Andrews et al. (1). APPL. ENVIRON. MICROBIOL. RESULTS When compared with an equal amount of soluble crystal toxin from strain LB1, toxin from strain HD251 did not appear to contain a protein band migrating at a molecular weight of 68, on SDS-PAGE (Fig. 1). Purified crystals from both strains were equally toxic when bioassayed against the larvae of the cabbage looper, and neither isolate was toxic when assayed against larvae of the mosquito A. aegypti (data not shown). The lack of a protein migrating at a molecular weight of 68, was unusual for lepidopterantoxic strains of B. thuringiensis, and we became interested in understanding why this particular isolate was different. Because we believe that toxin is created by proteolytic cleavage of protoxin in the insect, we began to examine the proteases produced by these two isolates of Bacillus thuringiensis subsp. kurstaki. Growth and protease activity of strain LB1 are shown in Fig. 2B. Protease activity in this isolate during vegetative growth, if present, was not detected; measurable activity first appeared at To (defined as the end of vegetative growth; about 3 h after inoculation of the culture) and remained high throughout sporulation. Maximum proteolytic activity first appeared at about T3 (3 h) of sporulation. There was a distinct drop in protease activity during T3 to T4, which corresponds to the period when crystal toxin dominates cellular protein synthesis in strain LB1 (2). This drop in protease activity was followed by another maximum which occurred at about T7, corresponding to the period when the cells have completed crystal toxin synthesis. The protease activity in strain HD251 was greatly reduced during the early period of sporulation but did appear later in the sporulation cycle (Fig. 2A). Inasmuch as protease-deficient isolates of B. subtilis often do not sporulate efficiently, it was necessary to determine whether strain HD251 was defective in its ability to sporulate or form toxic crystals. Figure 3 shows the growth, sporulation, and crystal toxin formation for both strains of B. thuringiensis subsp. kurstaki. In both strains, vegetative growth was completed and the cells entered sporulation by 3 h (To). Crystal toxin antigen, measured by rocket immunoelectrophoresis, first appeared in the cells of both isolates about 6 h after inoculation (T4) and reached its maximum rate of synthesis by T5. All the crystal toxin present in a fully sporulated culture was present by about T7. Complete, phase-light spores first appeared in the cells at about T7. By FIG. 1. SDS-PAGE of solubilized crystals from B. thuringiensis subsp. kurstaki isolates LB1 and HD251. Whole crystals that had been purified on NaBr gradients were electrophoresed on a 7 to 17% polyacrylamide gradient. Lane A, LB1; lane B, HD251. Molecular weights (x 13) are shown on the right. Downloaded from on September 22, 218 by guest

3 VOL. 5, 1985 PROTEASE ACTIVATION OF ENTOMOCIDAL PROTOXIN 739 T8 approximately 5% of the cells contained a complete spore, and by Tg, >95% of all cells contained a phase-light spore. Evidently, growth, spore formation, and crystal toxin production were not influenced by the reduction in protease production found in strain HD251. Because the protease activity in the two strains is vastly different during the period of crystal toxin synthesis and.3 12 * 6 because this difference may account for the different pro- 1 teins found in whole crystals from the two strains, we believed that it was of interest to begin to characterize these / enzymes. The proteases present in cells at T4 were chosen. because (i) proteases present in the cell at earlier stages 4 co should not affect the crystal toxin proteins, and (ii) after WIW 2 about T5, the crystal toxin proteins should be almost exclusively in crystals and therefore less susceptible to proteolytic ** o9c degradation. The activity measured at T4 was an intracellular c protease or was tightly bound to the cells because when a T4 e 4 culture was centrifuged and washed twice with 1 M NaCl, -5 / the activity remained in the pellet (Table 1). Most of the / proteolytic activity found in both LB1 and HD251 was of the 3I metallo class (Fig. 4), because almost all the protease 3 activity was inhibited by EDTA but not by phenylmethyl- l sulfonyl fluoride. The use of diisopropylfluorophosphate as a -2 s 4 protease inhibitor gave results similar to those observed with 6 phenylmethylsulfonyl fluoride (data not shown). The prote i 2 x T 1 I I 12 Time thr) 1 16 FIG. 3. Growth, crystal toxin formation, and sporulation in B. * 5 _ + ; thuringiensis subsp. kurstaki isolates HD251 (A) and LB1 (B). Symbols:, growth measured by A6N;, crystal antigen measured l 2 by rocket immunoelectrophoresis; *, presence of phase-light spores measured by phase-contrast microscopy. The arrow indicates the 3 approximate occurrence of To. X2 _/ 1. 8 ase activity found in LB1 had a ph optimum at about ph 7.,lo.1 (Fig. 5). This observation indicated that the proteolytic of the neutral.1 _ _ 4 activity found in LB1 at T3 to T5 is typical o1% proteases found in the genus Bacillus (14). 8 To demonstrate t that the protoxin of HD251 and LB1 could S *5 < J, be converted to a 68,-molecular-weight toxin, the solubil- _Ba ized crystals from both isolates were treated with trypsin and 1 16:@ then purified by ion-exchange chromatography. Figure 6 gr_shows an ion-exchange chromatogram obtained when trypoi sin-treated crystal toxin from HD251 was adsorbed to a DEAE-cellulose column and eluted with a to.5 M NaCl.5 \ fl 2 gradient. The insert (Fig. 6) shows solubilized crystals, O/3 trypsin-treated solubilized crystals, and purified toxin rated by SDS-PAGE. The sepa- same techniques can be used to 2 8 purify the insecticidal toxin from LB1 (data not shown). Downloaded from on September 22, 218 by guest O _ 4 TABLE 1. Protease activity of B. thuringiensis subsp. kurstaki LB1 and HD251 cells before and after washing in 1 M NaCl Cell source Treatment Activity' -55, I a I I I I HD251 Unwashed.17 Washed.15 I Time (hr) FIG. 2. Growth and protease activity of B. thuringiensis subsp. kurstaki isolates HD251 (A) and LB1 (B). Cell extract (6,u) was added to each reaction mixture that contained azoalbumin. Symbols:, acid-soluble material;, growth. The arrow indicates the approximate occurrence of To. LB1 Unwashed.215 Washed.21 a Activity expressed as A4Q. Sonication of cells from both isolates at this stage (T4) resulted in breakage of >99%o of the cells as observed by phasecontrast microscopy.

4 74 ANDREWS ET AL. v 1r 49 - o I I I I A I I.3 I I l Time (hr) Time (hr) FIG. 4. Effect of protease inhibitors on proteases from B. thuringiensis subsp. kurstaki isolates HD251 (A) and LB1 (B). Symbols:, EDTA; A, phenylmethylsulfonyl fluoride; U, no treatment. The arrow indicates the approximate occurrence of To. By using the techniques described herein, the toxin from HD251 and from LB1 could be purified with essentially quantitative recovery of toxic activity and stoichiometric conversion of toxin (molecular weight, 68,) from protoxin (molecular weight, 135,) (Table 2). The toxin purified from both strains was approximately twice as toxic as the protein from solubilized crystals and approximately four times as toxic as are whole crystals. The method can be used to prepare milligram quantities of insecticidal toxin from B. thuringiensis subsp. kurstaki in 2 days. DISCUSSION The method described for toxin purification provides much larger quantities of insecticidal toxin then previous methodologies have allowed. By these techniques, 5 mg of purified insecticidal toxin can be prepared in as few as 2 days. This advance should lead to a more detailed physical characterization of the toxin and facilitate a better understanding of the mode of action of the toxin. Despite a number of reports that describe the protoxic nature of the crystal toxin subunit, this model is still under some question because of the very poor efficiencies with which toxin can be purified from whole crystals. The quantitative conversion of protoxin to toxin achieved in this study strongly supports the model proposed for the protoxic nature of the crystal toxin. When crystals of either strain of B. thuringiensis subsp. kurstaki were solubilized in alkali, they became about twice as toxic when assayed against 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 the crystals with trypsin, followed by dialysis, reduced the protein concentration by about 5%, with no reduction in the total toxic activity of the preparation. Ion-exchange chromatography increased the purity of the material but had little, if any, effect on the toxic activity. Calculated on the basis of total protein content, the toxin is roughly twice as toxic as the solubilized crystal. Because solubilized crystal is mostly protoxin (molecular weight, 135,) and because the molecular weight of the toxin (68,) is roughly half that of the protoxin, the molar toxicities of the toxin and protoxin are about equal. If the 135,-molecular-weight molecule was a dimer, one would expect that the molar toxicity of the toxin would be about half of that obtained by using solubilized crystals (which are mostly protoxin). These observations strongly support the protoxin-toxin model. Further, inasmuch as very little if any protein at a molecular weight of 68, was found in HD251, the high toxicity of this isolate is difficult to explain without using the protoxin-toxin model. This study has shown that although proteins found in whole crystals may differ among various strains of B. thuringiensis, these differences may not exclusively reflect differences in the genetic information responsible for synthesis, but may be caused by other metabolic differences as well. Disagreement with the protoxin-toxin model for the crystal toxin has centered on the argument that the toxin molecule synthesized by B. thuringiensis has a molecular weight of 68, and that the 135,-molecular-weight band is an unusually stable dimer. The data contained herein suggest that the initial transcript has a molecular weight of 135, and that the presence of the activated toxin reported in most strains of B. thuringiensis (2) results from proteolytic cleavage of the protoxin during its synthesis. Support for this hypothesis can be derived from at least four lines of evidence. (i) The fact that very little toxin (molecular weight, 68,) can be found in strain HD251 suggests that this dimer would have to be extremely stable in the presence of the SDS, 2-mercaptoethanol, and urea present in the SDS gel sample buffer. (ii) The observation that the toxin is readily created with proteases suggests that a break in the polypeptide chain is required to create toxin. This would not be expected if the 135,-molecular-weight molecule was a dimer. (iii) The toxicity data suggest that one molecule of protoxin gives rise to one molecule of toxin. If the putative protoxin was a dimer, two molecules of toxin APPL. ENVIRON. MICROBIOL..1 I I I I I- I S FIG. 5. Effect of ph on the activity of the protease activity from T4 cells of B. thuringiensis subsp. kurstaki isolate LB1. ph Downloaded from on September 22, 218 by guest

5 VOL. 5, 1985 PROTEASE ACTIVATION OF ENTOMOCIDAL PROTOXIN N a LZ FRACTION NUMBER FIG. 6. Purification of the entomocidal toxin from B. thuringiensis subsp. kurstaki HD251 with DE52. Crystal from this strain were solubilized in Ellis buffer, dialyzed against 1 mm NaHCO4, treated with trypsin, and then dialyzed against column buffer (2 mm Na2HPO4 at ph 7.5). The purified toxin was eluted with a to.5 M NaCl gradient. The inset shows soluble HD251 crystals (lane A), trypsin-treated crystals (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 no 68,-molecular-weight protein, further supports this theory. The major weakness in this argument stems from the fact that the genetic relationship between HD251 and LB1 is not excactly known. A reduced-protease mutant of LB1 would greatly strengthen this argument. We are currently working on a more complete genetic characterization of LB1. Proteolysis of cellular components in bacilli, however, is well known, and this work underscores the importance of considering proteases when working with B. thuringiensis. There has been a recent report of a mosquito toxin (molecular weight, 65,) in the crystals from B. thuringiensis subsp. kurstaki HD1 (13). LB1 is thought to be very similar to HD1. Results of this study and others (2, 21) indicate that LB1 is not toxic to mosquitoes. Because there are indications in the literature that the two strains are not identical based on different plasmid profiles (15), it is possible that only HD1 carries the mosquito-toxic factor. These results do have some important implications in understanding sporulation in B. thuringiensis. Further studies of the biochemistry and molecular biology of sporulation in B. thuringiensis will require investigations of a more TABLE 2. Potency and yield of toxic protein during purification of the entomocidal toxin from B. thuringiensis subsp. kurstaki LB1 and HD251 Treatment Protein (mg) LC5. LB1 Whole crystals Solubilized crystals Trypsin treated "Pure toxin" HD251 Whole crystals Solubilized crystals Trypsin treated "Pure toxin" a LC, 5%o lethal concentration, in nanograms of toxic product per square centimeter applied to the surface of the artificial diet, calculated by probit analysis. detailed nature about the control of transcription and translation during endospore formation and crystal toxin synthesis. Previous work has shown that the synthesis of crystal toxin is controlled, at least in part, by regulating synthesis of crystal toxin-specific mrna (2). Inasmuch as work with RNA polymerase and in vitro translation is susceptible to error from proteases present during sporulation, the knowledge of the proteases produced by B. thuringiensis provided herein should aid in these studies. It is possible, for example, that HD251 could be a valuable source of RNA polymerases, ribosomal proteins, etc., which have not been subject to as much proteolytic degradation. ACKNOWLEDGMENT This work was supported by a grant from the Iowa State University Achievement Foundation. LITERATURE CITED 1. Andrews, R. E., Jr., D. B. Bechtel, B. S. Campbell, L. I. Davidson, and L. A. BuHla, Jr Solubility of parasporal crystals of Bacillus thuringiensis and presence of toxic protein during sporulation, germination, and outgrowth, p In H. S. Levinson, A. L. Sonenshein, and D. J. Tipper (ed.), Sporulation and germination. American Society for Microbiology, Washington, D.C. 2. Andrews, R. E., Jr., K. Kanda, and L. A. BuUla, Jr In vitro and in vivo synthesis of the parasporal crystal of B. thuringiensis, p In A. T. Ganesan, S. Chang, and J. A. Hoch (ed.), Gene regulation in bacilli. Academic Press, Inc., New York. 3. Ang, B. J., and K. W. Nickerson Purification of the protein crystal from Bacillus thuringiensis by zonal gradient centrifugation. Appl. Environ. Microbiol. 36: Brandt, C. R., M. J. Adang, and K. D. Spence The peritrophic membrane: ultrastructural analysis and function as a mechanical barrier to microbial infection in Orgyia pseudotsugata. J. Invertbr. Pathol. 32: Buila, L. A., Jr., D. B. Bechtel, K. J. Kramer, I. Shethna, A. I. Aronson, and P. C. Fitz-James Ultrastructure, physiology and biochemistry of Bacillus thuringiensis. Crit. Rev. Microbiol. 8: Bulla, L. A., Jr., L. I. Davidson, K. J. Kramer, and B. I. Jones Purification of the insecticidal toxin from the parasporal crystal of Bacillus thuringiensis. Biochem. Biophys. Res. Commun. 91: Downloaded from on September 22, 218 by guest

6 742 ANDREWS ET AL. 7. BuIla, L. A., Jr., K. J. Kramer, D. J. Cox, B. L. Jones, and L. I. Davidson Purification and characterization of the entomocidal protoxin of Bacillus thuringiensis. J. Biol. Chem. 256: Bulla, L. A., Jr., K. J. Kramer, and L. I. Davidson Characterization of the entomocidal parasporal crystal of Bacillus thuringiensis. J. Bacteriol. 13: Chestukhina, G. G., I. A. Zalunin, L. I. Kostina, T. S. Kotova, S. P. Kattrukha, L. A. Lyublinskya, and V. M. Stepanov Proteinases bound to crystals of Bacillus thuringiensis. Biokhimiya 43: Chestukhina, G. G., I. A. Zalunin, L. I. Kostina, T. S. Kotova, S. P. Kattrukha, and V. M. Stepanov Crystal forming proteins of Bacillus thuringiensis. Biochem. J. 187: Dalhammar, G., and H. Steiner Characterization of inhibitor A, a protease from Bacillus thuringiensis which degrades attacins and cecropins, two classes of antibacterial proteins in insects. Eur. J. Biochem. 139: Doi, R. H Role of proteases in sporulation. Curr. Top. Cell. Regul. 6: lizuka, T., and T. Yamamoto Possible location of the mosquitocidal protein in the crytsal preparation of Bacillus thuringiensis subsp. kurstaki. FEMS Microbiol. Lett. 19: Keay, L., and B. S. Wildi Proteases of the genus Bacillus. I. Neutral Proteases. Biotechnol. Bioeng. 22: APPL. ENVIRON. MICROBIOL. 15. Kronstad, J. W., H. E. Schnepf, and H. R. Whiteley Diversity of locations for Bacillus thuringiensis crystal protein genes. J. Bacteriol. 154: Lilley, M., R. N. Ruffell, and H. J. Somerville Purification of the insecticidal toxin in crystals of Bacillus thuringiensis. J. Gen. Microbiol. 1: Nickerson, K. W., and L. A. Bulla, Jr Physiology of sporeforming bacteria associated with insects: minimal nutritional requirements for growth, sporulation, and parasporal crystal formation of Bacillus thuringiensis. Appl. Microbiol. 28: Roitsch, C. A., and J. H. Hageman Bacillopeptidase F: two forms of a glycoprotein serine protease from Bacillus subtilis 168. J. Bacteriol. 155: Schesser, J. H., K. J. Kramer, and L. A. Bulla, Jr Bioassay for homogeneous parasporal crystal of Bacillus thuringiensis using the tobacco hormworm, Manduca sexta. Appl. Environ. Microbiol. 33: Tyrell, D. J., L. A. Bulia, Jr., R. E. Andrews, Jr., K. J. Kramer, L. I. Davidson, and P. Nordin Comparative biochemistry of entomocidal parasporal crystals of selected Bacillus thuringiensis strains. J. Bacteriol. 145: Tyrell, D. J., L. I. Davidson, L. A. Bulla, Jr., and W. A. Ramoska Toxicity of parasporal crystals of Bacillus thuringiensis subsp. israelensis to mosquitoes. Appl. Environ. Microbiol. 38: Downloaded from on September 22, 218 by guest