Cloning and study of the expression of a novel cry1Ia-type gene from Bacillus thuringiensis subsp. kurstaki

Abstract

Aims: Cloning and expression of a new cry1Ia-type gene of Bacillus thuringiensis. Methods and Results: PCR amplification, using gene cry1I-specific primers revealed the presence of such a gene in the strain BNS3 of Bacillus thuringiensis subsp. kurstaki. The cloning and sequencing from BNS3 of the cry1Ia-type gene, called crybns3-3, showed an open reading frame of 2160-bp, encoding a protein of 719 amino acid residues. Both nucleotide and amino acid sequences similarity analysis revealed that the crybns3-3 is a new cry1Ia-type gene, presenting several differences from the cry1Ia-type genes. The study of the expression of crybns3-3 by Northern blot and RT-PCR showed that it was transcribed. The expression of crybns3-3 under the control of BtI and BtII promoters revealed that Crybns3-3 would co-crystallize with the endogenous delta-endotoxins. Conclusions: crybns3-3 is a novel cry1Ia gene isolated from B. thuringiensis subsp. kurstaki strain BNS3. Significance and Impact of the Study: The characteristics of crybns3-3 indicate that it is a new cry1Ia-type gene. Amino acid residue substitutions presented in Crybns3-3 could be exploited for both toxicity and specificity studies. Crybns3-3 would interact and co-crystallize at least partially with the endogenous delta-endotoxins of BNS3, and then participate in the formation of the parasporal crystal inclusions.

Molecular and Insecticidal Characterization of a Cry1I Protein Toxic to Insects of the Families Noctuidae, Tortricidae, Plutellidae, and Chrysomelidae

“…Amino acid sequence analysis of Cry1Ia7 showed characteristics similar to those of other secreted proteins from the same group. However, Cry1Ia7 was different at several positions from the reported Cry1I proteins (46). Moreover, minor changes in amino acid sequences can produce important variations in the host range or insecticidal properties of these toxins.…”

“…In fact, our results indicated a significant contribution of 130-to 145-kDa proteins in the parasporal crystal that may correspond to the expression of other cry1 genes, but not cry1I, which code for proteins of approximately 80 kDa (6). This could be due to the lack of significant expression of this gene or to the fact that the protein is secreted into the medium (23,37,41,46).…”

“…Some B. thuringiensis strains also secrete insecticidal proteins during the vegetative growth phase; these are called VIP proteins (10). In addition, some cry genes, named cry1I genes (formerly cryV genes), encode proteins of around 70 to 81 kDa that do not accumulate in the crystal (5,12,23,34,35,37,39,41,43,46); these have been classified as Cry1I proteins due to their similarity with those in the Cry1 group (6). Their lack of involvement in the crystal structure has prevented these proteins from being included as active ingredients of B. thuringiensis-based insecticides.…”

“…cry1I genes are usually located approximately 500 bp downstream of other cry1 genes, but cry1I genes sometimes may not be expressed due to the lack of an upstream promoter-like sequence (23). In fact, cry1I genes are usually either silent or expressed in the vegetative phase and secreted into the growth suspension (23,37,41,46). Cry1I proteins have a broader host range than most other Cry1 proteins, and the hosts include important species of lepidopteran and coleopteran pests (43).…”

Identification and bioactivity of native strains of Bacillus thuringiensis from grain-related habitats in Turkey

“…It was also the only strain harboring nine cry1 genes including cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C, cry1D, cry1E, cry1F, and cry1G among all B. thuringiensis strains tested. Similarly, a novel B. thuringiensis strain (serovar kurstaki) isolated from Tunisian soil was reported to be toxic to lepidopteran insects including E. kuehniella due to Cry1Aa, Cry1Ac, and Cry2Aa proteins (Tounsi et al, 1999Jaoua, 2002, 2003). Similar to their results, cry1Aa and cry1Ac were the second and third most abundant genes, respectively, among the strains analyzed in the present study and some of the strains were positive for cry2 gene as well.…”