Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Bacteria or actinomycetales
Patent
1998-07-02
2000-08-01
Carlson, Karen Cochrane
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Bacteria or actinomycetales
424 932, 4352525, A01N 6300, A61K 4800, C12N 120
Patent
active
060963069
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to novel strains of Bacillus thuringiensis, cesticidal compositions employing them as well as the use of these strains for the expression of proteins of interest.
Bacillus thuringiensis (Bt) is a Gram-positive bacterium which produces proteins having insecticidal properties, especially against larvae of a large number of insects. These bacteria, possibly after inactivation, are used in pesticidal compositions intended to combat insects harmful to crops or vectors of disease, especially mosquitoes.
At present, Bt serotype 3a 3b especially is used against crop pests, and the serotype H14 is used to destroy mosquito larvae.
The proteins with pesticidal activity produced by Bacillus thuringiensis are called .delta.-endotoxins and are produced abundantly during sporulation. They accumulate in the form of parasporal crystalline inclusions, and can represent up to 25% of the dry weight of the sporulated cells.
Numerous genes of .delta.-endotoxins have been cloned, sequenced and classified in five groups and in various subgroups on the basis of sequence homologies and of the toxicity spectrum. The corresponding genes are called cry genes.
Formulations based on Bacillus thuringiensis have been used as biopesticides for close to 30 years under different trade names. The use of Bacillus thuringiensis as a biological control agent has numerous advantages with respect to chemical pesticides; in fact, it has a narrow and very specific host spectrum and it is without effect on the insects which are not targets and it is without unfavorable effect on vertebrates or on the environment.
However, the slight persistence of the .delta.-endotoxins in the environment and the presence of spores in the formulations represent two disadvantages for the marketing of products based on Bacillus thuringiensis. In order to resolve these two problems, it has been proposed in the patent application EP-192 319 to encapsulate the toxins in cell membranes, in particular using Pseudomonas fluorescens-type cells expressing the Cry1Ac toxin, or alternatively in the patent application PCT W094/25612, by expressing the Cry1IIA toxin in an affected nonsporulating mutant in the spo0A gene. This latter strategy is possible because the mode of expression of the cryIIIA gene is different from the mode of expression of other cry genes, in fact this cryIIIA gene is expressed from a promoter whose activation is independent of all the genes involved in the initiation of sporulation or of the factors involved in sporulation.
In Bacillus thuringiensis, the sporulation is dependent on the expression of two sigma factors respectively called sigma [.sigma.]35 and sigma [.sigma.]28; account having been taken of their great homology with the sigma (.sigma.)E and sigma (.sigma.)K factors in Bacillus subtilis, it is this latter terminology which will be used below, in the same way as the corresponding genes will be called sigE and sigK.
The present invention relates to a strain of Bacillus thuringiensis which expresses .sigma.E but does not sporulate or sporulates little or does not produce viable spores.
The present invention is based on the demonstration of the fact that a mutant of Bacillus thuringiensis which expresses sigE and which does not express sigK produces a quantity of toxins virtually identical to the corresponding wild strain, but, on the other hand, does not sporulate or does not produce viable spores.
This is particularly the case when the strain is a sigK.sup.- strain.
The construction of such strains has two advantages: 1) avoiding the dissemination of spores into the environment during treatment with biopesticides; 2) increasing the persistence of the toxins in the environment because of their encapsulation.
Tests have shown that a Bt strain not expressing the sigma K gene sigK.sup.-) was capable of accumulating a quantity of toxins equivalent to the source strain, while not producing spores. It was capable of producing the virtual totality of the toxins encoded by the cry genes or the related genes whose expression
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Agaisse Herve
Bravo Alejandra
Lereclus Didier
Salamitou Sylvie
Sanchis Vincent
Carlson Karen Cochrane
Institut National de la Recherche Agronomique
Institut Pasteur
Srivastava Devesh
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