A purification and some properties of an insecticidal exotoxin from Bacillus thuringiensis Berliner

Abstract

An insecticidal exotoxin from Bacillus thuringiensis var. thuringiensis (Berliner) has been purified. The efficiency of each stage of the purification has been ascertained and the yield of toxic material estimated by means of a quantitative bioassay. It is shown that the exotoxin is an adenine derivative substituted at position 9 and having a molecular weight of approximately 825. It can be dephosphorylated enzymically or chemically under conditions that define the exotoxin as a phosphomonoester. This results in loss of toxicity, both to insects and to mice. Spectroscopic and kinetic data are presented which suggest that a beta-ribofuranosyl moiety may be attached to the adenine. Glucose and allomucic acid have been positively identified as hydrolysis fragments from the exotoxin. These results are discussed and compared with the results of others on similar (or possibly identical) compounds.

Vip3A is responsible for the potency of Bacillus thuringiensis 9816C culture supernatant against Helicoverpa armigera and Spodoptera exigua

“…This is an insecticidal adenine-nucleotide analogue with a molecular weight of 701 Da (Bond et al, 1969). Toxicity of the b-exotoxin is probably due to inhibition of DNA-directed RNA polymerases by competition with ATP, inhibiting the synthesis of RNA (Sebesta and Horska, 1970;Vankova, 1978).…”

An improved bioassay for the detection of Bacillus thuringiensis β-exotoxin

“…b-exotoxin is a secreted thermostable adenine nucleotide analogue of low molecular weight (701 Da) that is toxic to a wide range of insect species (Bond et al, 1969;de Barjac and Dedonder, 1965;Farkas et al, 1969). Levinson et al (1990), using high-performance liquid chromatography (HPLC), described two types of b-exotoxin (types I and II).…”

“…Although other insects bioassay systems could be used, fly (Musca domestica) bioassays may be superior for the detection of b-exotoxin activity (see discussion in Levinson et al (1990)). Ohba et al (1981) reported a qualitative fly bioassay based on filter paper impregnated with autoclaved culture supernatant, but most fly bioassays are performed by mixing autoclaved culture supernatant with diet ingredients that are added to support larval growth and development (Bond and Boyce, 1971;Bond et al, 1969;Ignoffo and Gard, 1970;Johnson et al, 1998;Levinson et al, 1990). Although there is not a generally accepted or standard fly bioassay, it is noteworthy that two WHO guidelines (WHO, 1999(WHO, , 2007 state that detection of b-exotoxin should be done using the diet-based methods of Bond and Boyce (1971).…”

“…For the milk-yeast-tissue-based diets (D1, D2, D3) a diet suspension was dispensed into cups (50 ml plastic) containing strips (±10 mm wide) of 1.5 facial tissues (19 Â 21 cm sheets; Shoprite Checkers, Brackenfell). The diet suspension ingredients per cup were: D1 -1.25 g full-cream milk powder (MP; Clover, Roodepoort), 0.2 g instant dry yeast (IDY; Anchor Yeast, Industria), 10 ml sdH 2 O; D2 -1.25 g MP, 0.6 g IDY, 10 ml sdH 2 O; D3 -1.25 g MP, 1.0 g IDY, 10 ml sdH 2 O. D1-D3 were based on the diet in the quantitative method of Bond et al (1969).…”

“…MP-IDY-agar-based diets (D7, D8, D9), which were based on the diet used in the semi-quantitative method of Bond et al (1969), contained 1.25 g MP but differed in the amount of IDY (D7, 0.2 g; D8, 0.6 g; D9, 1.0 g). The diets were prepared by mixing cooled (55°C), autoclaved, molten agar (0.2 g agar, 10 ml sdH 2 O) with IDY-MP suspensions and dispensing into cups.…”