BACILLUS-THURINGIENSIS PROTOXIN - LOCATION OF TOXIC BORDER AND REQUIREMENT OF NONTOXIC DOMAIN FOR HIGH-LEVEL IN-VIVO PRODUCTION OF ACTIVE TOXIN

Insecticidal crystal proteins, or protoxins, of Bacillus thuringiensis are composed of two domains, an amino-terminal half essential for tox icity, and a carboxy-terminal half with an as yet unassigned function. To define the boundary of the two domains, sequential termination cod ons were introduced from the 3'-end of the DNA sequence encoding the t oxic domain of the 1155-residue cry1A(b) gene product. The mutated and the intact genes were placed under the control of the Escherichia coi l inducible promoter PrecA, and toxicity of the cell extracts was dete rmined using silkworm larvae. Under non-induced conditions, in which t he gene products accumulated to a limited degree, mutations encoding 6 06 amino acid residues or more were toxic, whereas those encoding 605 residues or less were non-toxic. Comparison of the toxicities and the levels of the toxic proteins suggested that the mutant proteins had co mparable activity to that of the intact protoxin. Furthermore, the non -toxic protein seemed to be unstable in the extracts. To investigate t he roles of the non-toxic domain, the mutant proteins were overproduce d in both E. coil and B. thuringiensis. The intact and the mutated gen es carrying natural promoters were introduced into acrystalliferous B. thuringiensis. Upon induction of PrecA in E. coil, and upon sporulati on in B. thuringiensis, there was a large accumulation of gene product s which formed inclusion bodies. The inclusion bodies of the intact pr otoxin were active, whereas those of the mutant proteins were inactive . Inclusion bodies of the intact protein could be solubilized in alkal i, whereas the mutant inclusion bodies were insoluble. Since solubiliz ation under alkaline conditions in the insect midgut is considered to be the first step of toxic action, the non-toxic domain is required to direct the synthesis of inclusion bodies as an active soluble form.