ELUCIDATION OF THE MECHANISM OF CRYIIIA OVERPRODUCTION IN A MUTAGENIZED STRAIN OF BACILLUS-THURINGIENSIS VAR TENEBRIONIS

NB176 is a Bacillus thuringiensis mutant derived by gamma-irradiation of NB125 Bacillus thuringiensis var. tenebrionis (Krieg). It exhibits two interesting phenotypes: (i) oligosporogeny and (ii) twofold to thr eefold overproduction of the CryIIIA protein. Southern profiles of the NB176 strain showed an additional copy(s) of the cryIIIA gene located on a 4 kb HindIII fragment, in addition to the expected cryIIIA gene on a 3 kb HindIII fragment. Each cryIIIA gene-bearing HindIII fragment was cloned from NB176. The restriction map of the 3 kb HindIII fragme nt was identical to that published by Donovan and coworkers. Sequencin g of the 4 kb HindIII fragment showed no alterations in the promoter r egion of the cryIIIA gene but did show replacement of the region immed iately following the cryIIIA open reading frame with a sequence encodi ng a transposase with 50% amino acid homology to that of Tn1000. These findings suggest that the overproduction phenotype of NB176 results f rom extra copies of the cryIIIA gene produced from a transposition eve nt(s) induced or stabilized by gamma-irradiation. Integration of addit ional copies of the cryIIIA gene into the native 90 MDa plasmid of the wild-type B. thuringiensis var. tenebrionis strain resulted in strain s that made enormous crystals, many possessing greatly enhanced insect icidal activity.