Russell S. Travers

Effects of Bacillus thuringiensis var. kurstaki δ-endotoxin on isolated lepidopteran mitochondria☆

Abstract An investigation was undertaken to determine the effects of Bacillus thuringiensis var. kurstaki δ-endotoxin on mitochondria isolated from Bombyx mori midgut epithelium. Using manometric and colorimetric techniques, the investigation revealed that toxic polypeptides had stimulatory effects on mitochondria oxygen uptake and inhibitory effects on ATP production. These results indicated that B. thuringiensis δ-endotoxin could act as an uncoupler of oxidative phosphorylation. Loss of ATP production caused by the action of the δ-endotoxin would lead to metabolic imbalance and possible cell death.

Degradation of the parasporal crystal produced by Bacillus thuringiensis var. kurstaki

Abstract Degradation products of the parasporal crystals of Bacillus thuringiensis var. kurstaki obtained by treatment with alkali, gut juice from larvae of Bombyx mori, and various plant and mammalian enzymes were compared for elution pattern, approximate molecular weight (MW), and toxicity. The results indicated that with alkaline treatment the most toxic extract was obtained with 0.05–0.1 M NaOH. Toxicity was found associated mainly with a protein peak of 230,000 MW although other toxic peaks were found in the tailing. Heat-treated midgut juice from larval B. mori gave similar results. After digestion of parasporal crystals with clarified midgut juice, five peaks causing toxicity and having MW of approximately 235,000, 67,000, 30,200, 5000, and 1000, respectively, were identified. Treatment of B. thuringiensis δ-endotoxin with α-chymotrypsin gave peaks causing mortality of approximate MW 235,000, 34,000, 5000, and 1000. Trypsin, pronase, carboxypeptidase, and enterokinase digests of the B. thuringiensis δ-endotoxin gave toxic components ranging from 235,000 to 30,000 MW. The protein protoxin molecules are digested to give small toxic subunits that may be of practical value for structural determinations and for molecular mode of action studies.

Extrachromosomal DNA in Bacillus thuringiensis var. kurstaki, var. finitimus, var. sotto, and in Bacillus popilliae.

Abstract Four entomopathogenic bacteria contained extrachromosomal deoxyribonucleic acid (DNA) molecules of various sizes. Bacillus thuringiensis var. kurstaki contained twelve elements banding on agarose gels that ranged from 0.74 to > 50 × 106 daltons, three of which were giant extrachromosomal DNA elements. B. thuringiensis var. sotto contained one giant extrachromosomal DNA element with a molecular size of about 23.5 × 106 daltons and two lesser elements of 0.80 and 0.62 × 106 daltons. B. thuringiensis var. finitimus harbored two giant DNA elements corresponding to >50 × 106 daltons and two lesser bands with relative small size (0.98 and 0.97 × 106 daltons). B. popilliae contained no giant extrachromosomal DNA elements but did contain two smaller elements corresponding to 4.45 and 0.58 × 106 daltons. The possible use of extrachromosomal DNA elements that prove to be autonomous replicons for recombinant DNA studies is discussed.

Occurrence of resistance to neomycin and kanamycin in Bacillus popilliae and certain serotypes of Bacillus thuringiensis: Mutation potential in sensitive strains

Abstract Serotypes of Bacillus thuringiensis and the commercial strain of Bacillus popilliae were examined for their inherent resistance to antibiotics and their mutation potential in respect to neomycin and kanamycin, the presence of which would preclude the use of plasmids marked by genes for resistance to the antibiotics. Clones on initial plates were detected by the recurrence of resistance colonies at superimposable sites on serial replicaplates containing the antibiotic. Susceptible strains were selected for the determination of their antibiotic-resistance mutation potential. Three varieties of B. thuringiensis were found to be doubly resistant, seven varieties were singly resistant (Neo r ), and three other varieties, including B. popilliae , were susceptible to both antibiotics. Estimates of mutation ratios revealed that three serotypes developed no resistant mutants to either antibiotics in populations as high as 3.0 × 10 10 ; seven other serotypes developed no resistance to kanamycin in populations as high as 4.6 × 10 9 cells. Three other serotypes exhibited mutation ratios as high as 1.6 × 10 −2 . We were unable to determine the mutation ratio for B. popilliae .