Transformation and expression of a cloned delta-endotoxin gene in Bacillus thuringiensis.

A shuttle vector containing the replication region of a resident plasmid of B. thuringiensis, was used to determine the conditions allowing efficient transformation of B. thuringiensis by electroporation. Using this plasmid a delta-endotoxin gene was cloned and expressed both in Escherichia coli and B. thuringiensis. It was shown that this gene was poorly expressed in the wild type situation whereas after cloning in acrystalliferous strains of B. thuringiensis large amounts of crystal protein were obtained.

Specificity of action on mosquito larvae of Bacillus thuringiensis israelensis toxins encoded by two different genes.

A 135 kDa protein gene and two open reading frames (ORF1 and ORF2) have been cloned from a large plasmid of Bacillus thuringiensis israelensis (Bourgouin et al. 1986). The Escherichia coli recombinant clones containing these genes were highly toxic to larvae of Aedes aegypti, Anopheles stephensi and Culex pipiens. From subcloning experiments it was deduced that the 135 kDa polypeptide alone was responsible for the toxic activity on both A. aegypti and An. stephensi larvae. In contrast, the presence of two polypeptides, the 135 kDa protein and the ORF1 product was required for toxicity to C. pipiens larvae. The minimal toxic fragment of the 135 kDa polypeptide has been delineated. The results indicate that a polypeptide of about 65 kDa, corresponding to an amino-terminal part of the 135 kDa protein is sufficient for toxicity. Sequence comparisons indicate that the ORF1 product may correspond to an N-terminal part of a rearranged 130 kDa protein.

[Influence of various ions in sporulation and the formation of delta-endotoxin in Bacillus thuringiensis cultures]. / Influencia de algunos iones en la esporulación y formación de delta-endotoxina en cultivos de Bacillus thuringiensis.

This paper deals with studies related to the influence of several ions on growth, spore formation and endotoxin formation by a Bacillus thuringiensis HD-1 strain commonly used for bioinsecticide production. Two basal media (4 and 5, Table 1) containing glucose, (NH4)2 SO4, phosphates and yeast extract or bacto peptone as organic nitrogen sources were supplemented with several ions in different concentrations, as shown in table 1. The experiments were conducted in 1000 ml Erlenmeyer flasks, containing 100 ml of medium, located in a rotary shaker at 30 degrees C. Several estimations were carried out, mainly biomass by optical density and colony forming units (CFU), glucose, Mg+2 and Mn+2 consumption and delta-endotoxin by a rocket immunoelectrophoretic method. The results obtained (Table 2) clearly show the importance of the addition of Ca+2, Mg+2 and Mn+2 to the basal media, because the highest values of CFU/l and delta-endotoxin (expressed as protein in g/l) were achieved in those media supplemented with the ions mentioned. It was also proved that the supplementation with Ca+2 was also essential for maintaining the thermal stability of the spores (Table 3). It can be concluded that an adequate formulation of media mainly related with the content of Mn+2, Mg+2 and Ca+2 is essential for obtaining high yields of spore crystal production of a Bacillus thuringiensis HD-1 strain.

[Possible means for increasing exotoxin synthesis by a Bacillus thuringiensis culture--the producer of bitoxibacillin]. / Vozmozhnye puti uvelicheniia sinteza ékzotoksina kul'turoi Bacillus thuringiensis--produtsentom bitoksibatsillina.

Various procedures were studied for obtaining Bacillus thuringiensis strains of serotype I which synthesized exotoxins. Mutant clones with elevated exotoxin synthesis could be selected by treating the cells with N-methyl-N-nitro-N-nitrosoguanidine. The frequency of (+) variant selection was from 17 to 12 X 10(-2). The clones of S and R types differed in the insecticide activity of the exotoxin. Its yield could be increased by optimizing the composition of growth media. The strain specificity of B. thuringiensis producing the exotoxin was assayed in terms of carbon and nitrogen requirements.

[Effect of physicochemical factors on Bacillus thuringiensis sporulation]. / Vliianie nekotorykh fiziko-khimicheskikh faktorov na prorastanie spor Bacillus thuringiensis.

Spore germination was studied in Bacillus thuringiensis subsp. galleriae. Optimal temperature regimes were selected for spore activation and initiation. The formation of spores and the rate of their subsequent germination were shown to depend on the composition of growth media in which the organism was cultivated. The paper describes changes in the fine structure of spores at different stages of their germination.

Microbial metabolites with insecticidal properties.

A screen of fungi for insecticidal activity revealed the ability of Aspergillus versicolor to make versimide, methyl-alpha-(methylsuccinimido)acrylate, a novel contact insecticide. The larvicidal activities of Alternaria tenuis and Fusarium lateritium were found to be due to tenuazonic acid and diacetoxyscirpenol, respectively. Thiolutin, cycloheximide, rubratoxin, patulin, trichothecin, an actinomycin, and scirpene-producing fungi also had insecticidal activity.