Insecticidal bacteria, and methods for making and using them

Abstract

The invention relates to the discovery that nucleic acid sequences comprising a BtI or BtII promoter, or a combination of a BtI and a BtII promoter, a bacterial STAB-SD sequence, and a sequence encoding proteins of the B. sphaericus (“Bs”) binary toxin and expressed in B. thuringiensis (“Bt”) or Bs cells results in production of Bs binary toxin at least 10 times that of untransformed Bs cells. The invention provides nucleic acid sequences, expression vectors, host cells, and methods of increasing the toxicity of an insecticidal bacterium by transforming the bacterium with a nucleic acid sequence of the invention. Further, the invention relates to the discovery that the Cyt1Aa1 protein of Bt subspecies israelensis (“Bti”) reverses resistance to Bs binary toxin in Bs-resistant mosquitoes. The invention provides Bs cells expressing Bti Cyt1Aa1 protein, and methods of reversing resistance to Bs binary toxin by co-administering the Cyt1Aa1 protein with Bs binary toxin.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS [0001] This is a continuation of U.S. application Ser. No. 09 / 639,576, filed Aug. 14, 2000, the contents of which are incorporated herein by reference.STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT [0002] Not applicable BACKGROUND OF THE INVENTION [0003] Despite advances in medical science and new drugs, malaria, filariasis, dengue and the viral encephalilitides remain important diseases of humans, with an estimated 2 billion people worldwide living in areas where these are endemic (The World Health Report—1999, World Health Organization, Geneva, Switzerland (1999)) The causative agents of these diseases are transmitted by mosquitoes, and therefore disease control methods have relied heavily on broad spectrum chemical insecticides to reduce mosquito populations. However, chemical insecticide usage is being phased out in many countries due the development of insecticide resistance in mosquito popul...

AI Technical Summary

Benefits of technology

[0016] In yet another set of embodiments, the invention provides a method for increasing toxicity of a B. sphaericus cell, said method comprising transforming the cell with a nucleic acid sequence comprising, in the following order, a B. thuringiensis promoter selected from the group consisting of a BtI promoter, a BtII promoter, and a combination of a BtI and a BtII promoter, a bacterial STAB-SD sequence, a ribosome binding site, and a sequence encoding one or both proteins of a B. sphaericus binary toxin; and expressing said nucleic acid sequence in the host cell; whereby expression of said nucleic acid sequence enhances production of B. sphaericus binary toxin as compared to production of B. sphaericus binary toxin in a wild-type B. sphaericus cell that is not transformed with said nucleic acid sequence. The bacterial STAB-SD sequence can be selected from the group consisting of GAAAGGAGG (SEQ ID NO:1), GAAGGGGGG (SEQ ID NO:2), GAGGGGGGG (SEQ ID NO:3), GAAAGGGGG (SEQ ID NO:4), GAAAGGAGG (SEQ ID NO:5), and GAAAGGGGT (SEQ ID NO:6). The B. thuringiensis promoter can be a cry promoter, or can be selected from the group consisting of cry1Aa1, cry1Aa2, cry1Aa3, cry1Aa4, cry1Aa5, cry1Aa6, cry1Ba1, cry1Ba2, cry1Ca1, cry1Ca2, cry1Ca3, cry1Ca4, cry1Ca5, cry1Ca6, cry1Ca7 cry1Fa1, cry1Fa2, cyt1Aa1, cyt1Aa2, cyt1Aa3, and cyt1Aa4. In a preferred embodiment, the B. thuringiensis promoter is a cyt1Aa1 promoter.

Problems solved by technology

Furthermore, many governments restrict use of these chemicals because of concerns over their effects on the environment, especially on non-target beneficial insects, and vertebrates through contamination of food and water supplies.

These products have achieved moderate commercial success, but their high cost and lower efficacy compared to many chemical pesticides prevents them from being used more extensively in many developing countries.

Attempts to combine the advantages of Bs and Bt in other manners have also apparently not proven commercially useful.

Simply growing cultures of Bs and Bt and then combining the two organisms, for example, is not effective because the spores of the two organisms are considered to form too large a proportion of the resulting mix in proportion to the weight of the toxins to provide adequate toxicity.

Commercial development of new biopesticides is costly, in part because of EPA regulations requiring extensive testing, and margins are low relative to, for example, pharmaceutical agents.

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