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.

1 . A nucleic acid sequence comprising, in the following order, 
 a  B. thuringiensis  cyt 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.    
     
     
         2 . The method of  claim 1 , wherein the  B. thuringiensis  promoter is selected from the group consisting of cyt1Aa1, cyt1Aa2, cyt1Aa3, and cyt1Aa4.  
     
     
         3 . The nucleic acid of  claim 2 , wherein the  B. thuringiensis  promoter is a cyt1Aa1 promoter.  
     
     
         4 . The nucleic acid of  claim 1 , having a BtI promoter and a BtII promoter, wherein the BtI promoter and the BtII promoter are overlapping.  
     
     
         5 . An expression vector comprising a nucleic acid of  claim 1 .  
     
     
         6 . An expression vector comprising a nucleic acid of  claim 2 .  
     
     
         7 . An expression vector comprising a nucleic acid of  claim 3 .  
     
     
         8 . An expression vector comprising a nucleic acid of  claim 4 .  
     
     
         9 . A host cell comprising an expression vector of  claim 5 .  
     
     
         10 . A host cell comprising an expression vector of  claim 6 .  
     
     
         11 . A host cell comprising an expression vector of  claim 7 .  
     
     
         12 . A host cell comprising an expression vector of  claim 8 .  
     
     
         13 . A host cell of  claim 9  further comprising a cry11A 20 kD protein.  
     
     
         14 . A host cell of  claim 10  further comprising a cry11A 20 kD protein.  
     
     
         15 . A host cell of  claim 11  further comprising a cry11A 20 kD protein.  
     
     
         16 . A host cell of  claim 12  further comprising a cry11A 20 kD protein.  
     
     
         17 . A host cell of  claim 9 , wherein the cell is a  B. thuringiensis  cell.  
     
     
         18 . A host cell of  claim 10 , wherein the cell is a  B. thuringiensis  cell.  
     
     
         19 . A host cell of  claim 11 , wherein the cell is a  B. thuringiensis  cell.  
     
     
         20 . A host cell of  claim 12 , wherein the cell is a  B. thuringiensis  cell.  
     
     
         21 . A method of creating a recombinant bacterium, said method comprising the steps of: 
 (a) transforming the recombinant bacterium with a gene 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 gene in the host cell;    whereby expression of said gene 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 gene.    
     
     
         22 . The method of  claim 21 , wherein the recombinant bacterium is selected from the group consisting of  B. thuringiensis, B. sphaericus , and a member of a  Bacillus  species other than Bti or Bs.  
     
     
         23 . A method of increasing toxicity of a  B. thuringiensis  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 gene in the bacterium;    whereby expression of said gene 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 gene.    
     
     
         24 . A method for suppressing resistance to  Bacillus sphaericus  binary toxin, said method comprising expressing a  Bacillus thurengiensis israelisis  (Bti) Cyt1Aa1 protein in a  B. thuringiensis  cell expressing said binary toxin