Insecticidal bacteria, and methods for making and using them

A method of enhancing production of B. sphaericus binary toxin in a host bacterial cell, said method comprising:

• a. transforming the host 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 Bt 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
• b. 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, and wherein said cell further expresses a 20 kD product of a cry11A gene.

A method of increasing toxicity of a B. thuringiensis a bacterium to a mosquito, said method comprising the steps of:

• (a) transforming said bacterium 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 a B. sphaericus binary toxin;
• and,
• (b) expressing said nucleic acid sequence in the bacterium;
• 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, and wherein said bacterium further comprises a 20 kD product of the cry11A gene.

A method for increasing toxicity of a B. sphaericus cell to an insect, said method comprising

• (a) 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
• (b) 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.

A method of enhancing production of B. sphaericus binary toxin in a host B. sphaericus cell, said method comprising:

• a. transforming the host cell with a nucleic acid sequence comprising, in the following order,
• (i) 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,
• (ii) a bacterial STAB-SD sequence,
• (iii) a ribosome binding site, and
• (iv) a sequence encoding one or both proteins of a B. sphaericus binary toxin;
• and
• b. 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.