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Bacillus thuringiensis δ-endotoxin: An improved technique for the separation of crystals from spores
JOURNAL
OF INVERTEBRATE
PATHOLOGY
29,
2%23
1 (1977)
f3aciRus ~~u~i~g~e~sis&Endotoxin: An Improved Technique for the Separation of Crystals from Spores pension was centrifuged at 10,000 rpm for 10 min, and the pellet was resuspended in 1 M NaCl + 0.01% Triton X-100. The procedure was repeated once more. The crude spore-crystal suspension (60 rn~~) was sonicated until completely disaggregated. Five to twenty milligrams of the suspension was layered onto 12 ml of a 50-80% Renografin-76 gradient (solutions of Renografin-76 were diluted with water, v/v, and checked with a refractometer). The samples were centrifuged in an SW 40 rotor at 28,OOOg~~ for 30 min at YC (Fig. I). Ahquots from the spore and crystal bands were collected using a syringe with a 23-gauge needle inserted in the side of the tube. The refractive index of each sample was determined, and the percentage of Renografin-76 was calculated from a standard curve. For all nine varieties of B. thzuingiensis, crystals banded between 60 and 65%, and spores between 70 and 75%. A modification of the above procedure allowed the separation of up to 300 mg of
A previously reported purification of crystals from spores and debris using isopycnic centrifugation in a CsCl gradient (P. G. Fast, J. Znvertebr, Pathoi. 20, 139140, 1972) proved to have limited apphcation. Changes in either the fermentation medium or the variety of Bacilhu thuringien,si.s resulted in severe slumping of both spores and crystals causing streaming and contamination of the bands. We report here a more widely applicable technique for the separation of spores from crystals based on reported separations of sporulating cultures and spore mutants using Renografin-76% (H. Tamir and C. Gilvarg, .Z. ZM. Chew. 241, 1085-1090, 1946; H. 0. Halvorson and A. Swanson, ZE “Spores IV,” L. L. Campbell, ed., pp. 121-132, 1969). Using Renografin76 we have readily purified crystals from mne B. thu~~ng~ensi~ varieties grown in various media in flasks or fermentors. Spores and crystals were harvested by making the spent medium 1 M with NaCl and 0.01% with Triton X-100, The sus-
CRYSTAL S
SPORE3 FIG, 1. Separation of spores. cryst&, continuous 50-8096 Renografin gradients, alesti,
dawnstadiensis,
morrisoni,
uizawai,
and debris from nine varieties of Baci&s thurj~gje~s~~ in from left to right: galMue, sotto, entomocidw, tobwrtfzi, and kurstaki. Upper bands: debris and germinated spores;
middle bands: crystals: lower bands: spores. ’ Avaikble
from E. R. Squibb and Sons. 230
Copyright 63 1977 by Academic Press. Inc. All righIs of reproduction in any form resewed,
ISSN iXtZ2-201 I
231
NOTES
‘? 1 CRYSTALS
J FIG. 2. Separation of spores, crystals, and debris from Bacillus thuringiensis var. kurstaki on a discontinuous gradient. Upper band; germinated spores, debris; lower band: crystals. Spores have formed a pellet on the bottom of the tube (not shown in this figure).
crude culture in suspension per tube. A spore and crystal suspension in 25 ml of 60% Renografin-76 (determined from the refractive index of the final solution) was displaced with 10 ml of 70% Renografin-76 and centrifuged at 22,000gaV at 5°C for 1 hr in an SW 27 rotor (Fig. 2). Purification using the above discontinuous gradients was demonstrated on crystals from B. alesti, aizawi, thuringiensis var. galleriae, and kurstaki. Recovery of crystals from a crude spore-crystal suspension was determined to be 57% by measuring the total amount of 0.1 N NaOH-soluble protein in both preparations. Before purification, B. thuringiemis var. kurstaki cultures contained -1.4 x IO7
viable spores/mg, determined by plating on nutrient agar. After two discontinuous gradient centrifugations, the resulting crystal preparation contained 4.7 x 105 viable spores/mg. Purified crystals were dried at 1lVC for 3 hr and weighed, and protein was measured by the FolinCiocalteau method (J. L. Bailey, “Techniques in Protein Chemistry,” Elsevier, New York, 1969). Protein measured by this procedure accounted for the total dry weight of the crystal sample: The detection limit of this analysis is less than 1%. The viable spore count was found to be 3% of the original level in the spore-crystal crude, equivalent to 0.009% by weight. Purified viable spores could also be recovered from either of the above procedures. A spore preparation collected from a single run on a discontinuous gradient yielded 5.05 x 10g viable spores/mg. This preparation showed 4% by weight of alkalisoluble protein, presumed to be crystal contamination and spore coat protein. IPRI Contribution
No. 308.
Note added in proof: A publication (E. S. Sharpe, K. W. Nickerson, L. A. Bulla, and J. R. Aronson, Appl Microbio/. 30, 1052-1053, 1975) using O-100% Renograhn gradients to separate spores and crystals came to our attention after this manuscript was submitted. R. MILNE D. MURPHY P. G. FAST Insect Pathology Research Institute Environment Canada Canadian Forestry Service P.O. Box 490 Sault Ste. Maries Ontario, Canada Received February 6, 1976
OF INVERTEBRATE
PATHOLOGY
29,
2%23
1 (1977)
f3aciRus ~~u~i~g~e~sis&Endotoxin: An Improved Technique for the Separation of Crystals from Spores pension was centrifuged at 10,000 rpm for 10 min, and the pellet was resuspended in 1 M NaCl + 0.01% Triton X-100. The procedure was repeated once more. The crude spore-crystal suspension (60 rn~~) was sonicated until completely disaggregated. Five to twenty milligrams of the suspension was layered onto 12 ml of a 50-80% Renografin-76 gradient (solutions of Renografin-76 were diluted with water, v/v, and checked with a refractometer). The samples were centrifuged in an SW 40 rotor at 28,OOOg~~ for 30 min at YC (Fig. I). Ahquots from the spore and crystal bands were collected using a syringe with a 23-gauge needle inserted in the side of the tube. The refractive index of each sample was determined, and the percentage of Renografin-76 was calculated from a standard curve. For all nine varieties of B. thzuingiensis, crystals banded between 60 and 65%, and spores between 70 and 75%. A modification of the above procedure allowed the separation of up to 300 mg of
A previously reported purification of crystals from spores and debris using isopycnic centrifugation in a CsCl gradient (P. G. Fast, J. Znvertebr, Pathoi. 20, 139140, 1972) proved to have limited apphcation. Changes in either the fermentation medium or the variety of Bacilhu thuringien,si.s resulted in severe slumping of both spores and crystals causing streaming and contamination of the bands. We report here a more widely applicable technique for the separation of spores from crystals based on reported separations of sporulating cultures and spore mutants using Renografin-76% (H. Tamir and C. Gilvarg, .Z. ZM. Chew. 241, 1085-1090, 1946; H. 0. Halvorson and A. Swanson, ZE “Spores IV,” L. L. Campbell, ed., pp. 121-132, 1969). Using Renografin76 we have readily purified crystals from mne B. thu~~ng~ensi~ varieties grown in various media in flasks or fermentors. Spores and crystals were harvested by making the spent medium 1 M with NaCl and 0.01% with Triton X-100, The sus-
CRYSTAL S
SPORE3 FIG, 1. Separation of spores. cryst&, continuous 50-8096 Renografin gradients, alesti,
dawnstadiensis,
morrisoni,
uizawai,
and debris from nine varieties of Baci&s thurj~gje~s~~ in from left to right: galMue, sotto, entomocidw, tobwrtfzi, and kurstaki. Upper bands: debris and germinated spores;
middle bands: crystals: lower bands: spores. ’ Avaikble
from E. R. Squibb and Sons. 230
Copyright 63 1977 by Academic Press. Inc. All righIs of reproduction in any form resewed,
ISSN iXtZ2-201 I
231
NOTES
‘? 1 CRYSTALS
J FIG. 2. Separation of spores, crystals, and debris from Bacillus thuringiensis var. kurstaki on a discontinuous gradient. Upper band; germinated spores, debris; lower band: crystals. Spores have formed a pellet on the bottom of the tube (not shown in this figure).
crude culture in suspension per tube. A spore and crystal suspension in 25 ml of 60% Renografin-76 (determined from the refractive index of the final solution) was displaced with 10 ml of 70% Renografin-76 and centrifuged at 22,000gaV at 5°C for 1 hr in an SW 27 rotor (Fig. 2). Purification using the above discontinuous gradients was demonstrated on crystals from B. alesti, aizawi, thuringiensis var. galleriae, and kurstaki. Recovery of crystals from a crude spore-crystal suspension was determined to be 57% by measuring the total amount of 0.1 N NaOH-soluble protein in both preparations. Before purification, B. thuringiemis var. kurstaki cultures contained -1.4 x IO7
viable spores/mg, determined by plating on nutrient agar. After two discontinuous gradient centrifugations, the resulting crystal preparation contained 4.7 x 105 viable spores/mg. Purified crystals were dried at 1lVC for 3 hr and weighed, and protein was measured by the FolinCiocalteau method (J. L. Bailey, “Techniques in Protein Chemistry,” Elsevier, New York, 1969). Protein measured by this procedure accounted for the total dry weight of the crystal sample: The detection limit of this analysis is less than 1%. The viable spore count was found to be 3% of the original level in the spore-crystal crude, equivalent to 0.009% by weight. Purified viable spores could also be recovered from either of the above procedures. A spore preparation collected from a single run on a discontinuous gradient yielded 5.05 x 10g viable spores/mg. This preparation showed 4% by weight of alkalisoluble protein, presumed to be crystal contamination and spore coat protein. IPRI Contribution
No. 308.
Note added in proof: A publication (E. S. Sharpe, K. W. Nickerson, L. A. Bulla, and J. R. Aronson, Appl Microbio/. 30, 1052-1053, 1975) using O-100% Renograhn gradients to separate spores and crystals came to our attention after this manuscript was submitted. R. MILNE D. MURPHY P. G. FAST Insect Pathology Research Institute Environment Canada Canadian Forestry Service P.O. Box 490 Sault Ste. Maries Ontario, Canada Received February 6, 1976