During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism’s pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests. Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit.

Bacillus thuringiensis and Its Pesticidal Crystal Proteins

Insecticidal crystal proteins of Bacillus thuringiensis.

This invention relates to a reproducible system for the production of stable, genetically transformed maize cells, and to methods of selecting cells that have been transformed. One method of selection disclosed employs the Streptomyces bar gene introduced by microprojectile bombardment into embryogenic maize cells which were grown in suspension cultures, followed by exposure to the herbicide bialaphos. The methods of achieving stable transformation disclosed herein include tissue culture methods and media, methods for the bombardment of recipient cells with the desired transforming DNA, and methods of growing fertile plants from the transformed cells. This invention also relates to the transformed cells and seeds and to the fertile plants grown from the transformed cells and to their pollen.

Methods and compositions for the production of stably transformed fertile monocot plants and cells thereof

Fertile transgenic Zea mays (corn) plants which stably express recombinant DNA which is heritable are provided wherein said DNA preferably comprises a recombinant gene which encodes a seed storage protein, so that the amino acid profile of the corn is improved

Fertile transgenic corn plants

Members of the genus Bacillus are widely used as sources of industrial enzymes, fine biochemicals, antibiotics, and insecticides (for a review, see reference 27). One of these species, Bacillus thuringiensis, accounts for more than 90% of the biopesticides used today (for recent reviews on B. thuringiensis and its toxins, see references 6, 33, and 38). The entomopathogenic properties of this bacterium are due at least in part to the production of d-endotoxins that make up the crystalline inclusions characteristic of B. thuringiensis strains. In 1989, Hofte and Whiteley proposed a classification for d-endotoxins (30). They distinguished four major classes of d-endotoxins (CryI, -II, -III and -IV) and cytolysins (Cyt), found in the crystals of the mosquitocidal strains, on the basis of their insecticidal and molecular properties. The d-endotoxins belonging to each of these classes were grouped in subclasses (A, B, C. . . and a, b, c. . .) according to sequence. Generally, these proteins are toxic for lepidoptera (CryI), both lepidoptera and diptera (CryII), coleoptera (CryIII), and diptera (CryIV). These various insecticidal proteins are synthesized during the stationary phase and accumulate in the mother cell as a crystal inclusion which can account for up to 25% of the dry weight of the sporulated cells (Fig. 1). The amount of crystal protein produced by a B. thuringiensis culture in laboratory conditions (about 0.5 mg of protein per ml) and the size of the crystals (24) indicate that each cell has to synthesize 10 to 2 3 10 d-endotoxin molecules during the stationary phase to form a crystal. This is a massive production of protein and presumably occupies a large proportion of the cell machinery. Nevertheless, sporulation and the associated physiological changes proceed in parallel with d-endotoxin production. The aim of this minireview is to analyze the various mechanisms by which B. thuringiensis accumulates large quantities of toxins as biologically active protein crystals.

How does Bacillus thuringiensis produce so much insecticidal crystal protein

Bacillus thuringiensis subsp. kurstaki total DNA was digested with BglII and cloned into the BamHI site of plasmid pUC9 in Escherichia coli. A recombinant plasmid, pHBHE, expressed a protein of 135,000 daltons that was toxic to caterpillars. A HincII-SmaI double digest of pHBHE was then ligated to BglII-cut plasmid pBD64 and introduced into Bacillus subtilis by transformation. The transformants were identified by colony hybridization and confirmed by Southern blot hybridization. A 135,000-dalton protein which bound to an antibody specific for the crystal protein of B. thuringiensis was detected from the B. subtilis clones containing the toxin gene insert in either orientation. A toxin gene insert cloned into a PvuII site distal from the two drug resistance genes of the pBD64 vector also expressed a 135,000-dalton protein. These results suggest that the toxin gene is transcribed from its own promoter. Western blotting of proteins expressed at various stages of growth revealed that the crystal protein expression in B. subtilis begins early in the vegetative phase, while in B. thuringiensis it is concomitant with the onset of sporulation. The cloned genes when transferred to a nonsporulating strain of B. subtilis also expressed a 135,000-dalton protein. These results suggest that toxin gene expression in B. subtilis is independent of sporulation. Another toxin gene encoding a 130,000- to 135,000-dalton protein was cloned in E. coli from a library of B. thuringiensis genes established in lambda 1059. This gene was then subcloned in B. subtilis. The cell extracts from both clones were toxic to caterpillars. Electron microscope studies revealed the presence of an irregular crystal inclusion in E. coli and a well-formed bipyramidal crystal in B. subtilis clones similar to the crystals found in B. thuringiensis. Images

A G Shivakumar

Papers

Vegetative expression of the delta-endotoxin genes of Bacillus thuringiensis subsp. kurstaki in Bacillus subtilis.