Transcriptional Regulation of thecryIVDGene Operon from Bacillus thuringiensissubsp.israelensis

TheCryIVDproteinisinvolvedintheoveralltoxicityoftheBacillusthuringiensissubsp.israelensisparasporal inclusions and is one of the four major components of the crystals. Determination of the DNA sequence indicatedthatthecryIVDgeneisthesecondgeneofanoperonwhichincludesthreegenes.Thefirstoneencodes a 19-kDa polypeptide and has sequence homology with the orf1 gene of the Bacillus thuringiensis cryIIA and cryIICoperons.ThesecondandthirdgeneshavealreadybeenidentifiedandencodetheCryIVDcrystalprotein and the P20 polypeptide, respectively. The promoter region was located by deletion analysis, and the 5*end of the mRNA was determined by primer extension mapping. Transcription of thecryIVDgene inB. thuringiensis subsp. israelensis strains is induced 9 h after the beginning of sporulation. Sequence analysis indicated two potential promoters, a strong one and a weak one, recognized respectively by the RNA polymerase associated with the s 35 or the s 28 factor ofB. thuringiensis(s E and s K ofBacillus subtilis, respectively). Transcriptional lacZfusion integrated in single copy into the chromosome of variousB. subtilissporulation mutants confirmed the s E dependence ofcryIVDgene transcription. Bacillus thuringiensisis a gram-positive soil bacterium which produces large amounts of entomocidal proteins that accumulate in the cytoplasm to form crystals. Bacillus thuringiensis subsp.israelensisinclusions are toxic to mosquito and blackfly larvae,whicharevectorsofseveraltropicaldiseases(18,39). B. thuringiensis subsp. israelensis crystals are composed of four major polypeptides with molecular masses of about 125, 135, 68, and 28 kDa, referred to as CryIVA, CryIVB, CryIVD, and CytA, respectively (21); their corresponding genes are located on a large conjugative resident plasmid with a size of 72 MDa (for a review, see reference 27). Synergistic interactions between the crystal components are responsible for the high toxicity of B. thuringiensis subsp. israelensis (15, 46). Various proteinsseemtobeinvolvedinthecrystal’sbiosynthesis,which is coordinated with the sporulation process and is highly efficient (20% of the total cell proteins). A 20-kDa protein (P20) has been shown to increase CryIVD and CytA production in Escherichia coli by a posttranslational stabilization (40), although its role inB. thuringiensissubsp.israelensisis subject to controversy (12, 47). Most of the clonedB. thuringiensistoxin genes are monocistronic, although the two major toxin genes of B. thuringiensis subsp. thompsoni and the B. thuringiensis subsp.kurstaki cryIIAandcryIICtoxin genes are organized in operons (8, 44, 45). ThecryIIAandcryIICgenes are associated with two other genes,orf1andorf2. Recently, Crickmore et al. (14) have shown that cloning the orf1 and orf2 genes of the cryIIA operon with the cryptic cryIIB protein gene allows the synthesis of CryIIB inclusions. The deletion of orf1 has no effect on the production of the CryIIA or CryIIC toxins in B. thuringiensis (13, 45). However, crystallization of CryIIA requires the presence of the orf2 gene product, which is composed of a head-to-tail repetition of the same polypeptide fragment (13). Both the P20 and Orf2 polypeptides may act as