Expression in Escherichia coli of a cloned crystal protein gene of Bacillus thuringiensis subsp. israelensis

Abstract

A ca. 10-kilobase (kb) HindHII fragment of plasmid DNA from Bacillus thuringiensis subsp. israelensis was cloned into plasmid pUC9 and transformed into Escherichia coli. Extracts of the recombinant strain contained a 27-kilodalton (kDa) peptide that reacted with antibodies to a 27-kDa peptide isolated from crystals produced by B. thuringiensis subsp. israelensis. Extracts of the recombinant strain were hemolytic and toxic to Aedes aegypti larvae. Full expression of the 27-kDa peptide required the presence of a ca. 0.8-kb region of DNA located 4 kb upstream from the structural gene; the 0.8-kb region could be present in cis or trans relative to the gene and apparently acted post-transcriptionally. Analysis of maxicelis showed that the 10-kb insert also coded for peptides of 67, 20, and 16 kDa; data obtained with different subclones suggest that the 20-kDa peptide is encoded in the 0.8-kb DNA region.During sporulation, Bacillus thuringiensis subsp. israelensis (hereafter referred to as Bti) produces irregular, multicomponent parasporal crystals (6,15,29). The crystals are lethal to larvae of mosquitoes and black flies (11); solubilized preparations of crystals lyse erythrocytes and cultured mosquito cells (26). Electrophoretic analyses of solubilized, denatured crystals show several peptides ranging in size from 27 to 130 kilodaltons (kDa) (29). The relationship of these peptides to each other is not known, and there are conflicting reports concerning the identity of the toxic and cytolytic peptides. A number of investigators (1,9,15,16,35,38) have presented evidence that toxicity alone or that toxicity and hemolytic and cytolytic activities are associated with a 27-kDa peptide or its 25-to 26-kDa derivative. A gene coding for a peptide with a deduced molecular mass of 27,340 daltons has been cloned in Escherichia coli (32, 35) and sequenced (32, 34). reported that extracts of recombinant E. coli strains containing the 27-kDa gene are toxic and hemolytic, whereas Waalwijk et al. (32) found that extracts of recombinant strains are hemolytic but not mosquitocidal. The latter investigators reported that mosquitocidal activity is associated with peptides of 130 and 230 kDa (31). Others (14,19) have attributed the mosquitocidal activity to a ca. 66-kDa peptide and the hemolytic activity to the 27-kDa peptide. Walfield et al. (33) and Thorne et al. (27) have cloned and sequenced a gene coding for a 72-kDa peptide and have reported that the apparent processed product of this gene, a peptide of 58 kDa, is highly toxic but not hemolytic. Yet other investigators (7) propose that toxicity resides in a 31-to 35-kDa peptide or that toxicity involves the synergistic action of two peptides (15, 37).The present communication reports studies made with a recombinant E. coli strain containing the genes coding for the 27-kDa crystal protein peptide of Bti. Extracts of the recombinant strain are toxic to mosquito larvae and are hemolytic in vitro. We also report the unexpected observation that the presence of the 27-kDa peptide, bu...

Regulation of insecticidal crystal protein production in Bacillus thuringiensis

“…2). Early studies of the expression of the cytA gene in E. coil indicated that the 20kDa protein encoded by the third orf in the crylVD operon is required for efficient CytA production (Adams et aL, 1989;McLean and Whiteley, 1987;Visick and Whiteley, 1991). This requirement for the 20 kDa protein could be bypassed by the use of E. coli host strains deficient in intracellular proteolysis (Visick and Whiteley, 1991).…”

A 20-kilodalton protein preserves cell viability and promotes CytA crystal formation during sporulation in Bacillus thuringiensis

“…Expression of this gene during sporulation is apparently driven by the cryIVD promoter, with both cryIVD and the 20-kDa gene being expressed as a single transcriptional unit (1). In fact, expression of the cytA gene in E. coli in the Enhancement of CytA production by the 20-kDa protein in E. coil (1,15,21) suggested that this protein functions similarly in the subspecies of B. thuringiensis in which it occurs naturally and that this could be tested by placing the 20-kDa protein gene under the control of a strong promoter. Our objective was to manipulate the expression of the 20-kDa protein and thereby contribute to our knowledge of this protein's function and its effect on CytA crystal formation.…”

“…However, several recent studies have shown that efficient production of CytA in Escherichia coli requires the presence of a 20-kDa protein that is coded for by a gene located immediately downstream from the cryIVD gene (1,7,15,21). Expression of this gene during sporulation is apparently driven by the cryIVD promoter, with both cryIVD and the 20-kDa gene being expressed as a single transcriptional unit (1).…”

Effect of a 20-kilodalton protein from Bacillus thuringiensis subsp. israelensis on production of the CytA protein by Escherichia coli

“…In Escherichia coli, both the cytA gene and the gene for a 20-kDa protein must be present for the production of significant amounts of CytA (1,29). In BTI, the 20-kDa protein gene is located on the same plasmid as cytA but about 4 kb upstream; its product is present in small amounts in crystals and is synthesized beginning about 2 h after the start of sporulation, concurrent with CytA synthesis (1).…”

“…The inclusion is composed of multiple components that are distinguishable by electron microscopy (7,22), and electrophoresis indicates that four major polypeptide species are present, in addition to several minor peptides (9,22,28). The most prominent crystal protein, a 27-kDa polypeptide, has been cloned (29,41,44), and its gene has recently been designated cytA (21). The CytA protein is unlike the other known B. thuringiensis toxins both in its amino acid sequence (21) and in its mechanism of action.…”