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The δ-endotoxin of Bacillus thuringiensis var. sotto: A toxic low molecular weight fragment
JOtX3SAL
OF
lN\‘EHTEl3I
PATHOLOGY
16,
465 ( 1970)
NOTES The
a-Endotoxin of Bacillus A Toxic Low Molecular
Recently it has been reported that the d-endotosin of varieties of Bacillus thuringiensis when dissolved by various means will vield fragments that retain toxicity for larvae of Lepidoptera (for review see M. Rogoff and A. A. Yousten, Ann. Rev. Microbiol. 23, 357-386, 1969). The smallest fragment so far reported had a molecular weight “below 5000” based on gel filtration with standards of known molecular weight. WC report a toxic fragment with an apparrnt molecular weight of between 500 and 1000. Parasporal inclusions (so-called crystals) of B. thuringiensis var. sotto were dissolved in 0.04 hl NaOH containing 0.2 hl 6-mercaptoethanol. The mcrcaptoethanol was rcmoved by washing with 0.05 h1 carbonatcbicarbonate buffer ( IIII 9.5) containing 0.001 hr EDTA over a filter that retains molcculcs with molecular weight grcatcr than 1000 (URII-2 Diaflo nrembranc filter, Amicon Corp., Lexington, Alass.). The dissolved protciu was recovered from above the filter. It was then incubated for 4 hr at 37” C with 1 m,g’ml of chymotrypsin at ]jH 9.5 The incubate was filtered through a UM-2 menibranc filter and the filtrate collected. Toxicit!, of this filtrate was deterinincd by placing 0.1 ml in the crop of fifthinstar silkworm (Bomb~yr nzori) by means of a glass capillar!- ( l-111Drummond R4icrocap) fitted to an Agla micrometer syringe. Mortalitv was recorded after 2 hr.
tkuringiensis var. sotto: Weight Fragment The UM-2 filtrates prepared by this procedure wcrc toxic to fifth-instar silkworm larvae as described. Incubation of dissolved crvstals with trvpsin at pH 7.5 or chymotrypsin at other hydrogen ion concentrations vielded less or no toxic material. To$ic UM-2 filtrate prepared in this I~IIIner passesthrough a Pellicon PSAC menibrane ( Millipore Ltd., Montreal, Canada ) which, although manufactured bv a different process, also retains molecules with molecular weights greater than 1000. The toxic moiety could be concentrated nrld partially purified by. lowering the !rH of the, UM-2 filtrate to 6 with HCl and filtering through a UM-05 filter. The toxin did not pass through although some uv-absorbing material did. At this pH, molecules with molecular weights > 500 are retained. These data suggest that the toxic frag. ment obtained by chvmotryptic digcstiort has a molecular weight between SO0 and 1000.
Contribution
No. 168 from
this
lnstitntc
I?. G. FAST T. -4. ANWS
insect Pathology Research Institute Department of Fisheries and ForestrJy P.O. Box 490 Sault Ste. Marie, Ontario, Canada Received June 22, 1970
OF
lN\‘EHTEl3I
PATHOLOGY
16,
465 ( 1970)
NOTES The
a-Endotoxin of Bacillus A Toxic Low Molecular
Recently it has been reported that the d-endotosin of varieties of Bacillus thuringiensis when dissolved by various means will vield fragments that retain toxicity for larvae of Lepidoptera (for review see M. Rogoff and A. A. Yousten, Ann. Rev. Microbiol. 23, 357-386, 1969). The smallest fragment so far reported had a molecular weight “below 5000” based on gel filtration with standards of known molecular weight. WC report a toxic fragment with an apparrnt molecular weight of between 500 and 1000. Parasporal inclusions (so-called crystals) of B. thuringiensis var. sotto were dissolved in 0.04 hl NaOH containing 0.2 hl 6-mercaptoethanol. The mcrcaptoethanol was rcmoved by washing with 0.05 h1 carbonatcbicarbonate buffer ( IIII 9.5) containing 0.001 hr EDTA over a filter that retains molcculcs with molecular weight grcatcr than 1000 (URII-2 Diaflo nrembranc filter, Amicon Corp., Lexington, Alass.). The dissolved protciu was recovered from above the filter. It was then incubated for 4 hr at 37” C with 1 m,g’ml of chymotrypsin at ]jH 9.5 The incubate was filtered through a UM-2 menibranc filter and the filtrate collected. Toxicit!, of this filtrate was deterinincd by placing 0.1 ml in the crop of fifthinstar silkworm (Bomb~yr nzori) by means of a glass capillar!- ( l-111Drummond R4icrocap) fitted to an Agla micrometer syringe. Mortalitv was recorded after 2 hr.
tkuringiensis var. sotto: Weight Fragment The UM-2 filtrates prepared by this procedure wcrc toxic to fifth-instar silkworm larvae as described. Incubation of dissolved crvstals with trvpsin at pH 7.5 or chymotrypsin at other hydrogen ion concentrations vielded less or no toxic material. To$ic UM-2 filtrate prepared in this I~IIIner passesthrough a Pellicon PSAC menibrane ( Millipore Ltd., Montreal, Canada ) which, although manufactured bv a different process, also retains molecules with molecular weights greater than 1000. The toxic moiety could be concentrated nrld partially purified by. lowering the !rH of the, UM-2 filtrate to 6 with HCl and filtering through a UM-05 filter. The toxin did not pass through although some uv-absorbing material did. At this pH, molecules with molecular weights > 500 are retained. These data suggest that the toxic frag. ment obtained by chvmotryptic digcstiort has a molecular weight between SO0 and 1000.
Contribution
No. 168 from
this
lnstitntc
I?. G. FAST T. -4. ANWS
insect Pathology Research Institute Department of Fisheries and ForestrJy P.O. Box 490 Sault Ste. Marie, Ontario, Canada Received June 22, 1970