Cyt1Ca from Bacillus thuringiensis subsp. israelensis: production in Escherichia coli and comparison of its biological activities with those of other Cyt-like proteins

• DOI: 10.1099/mic.0.28981-0
• Publication Date: 2006-08-31T20:32:03Z
• Authors: Robert Manasherob; Mark Itsko; Nadine Sela-Baranes; Eitan Ben-Dov; Colin Berry; Shmuel Cohen; Arieh Zaritsky
• Keywords: Life Cycle Stages; 0303 health sciences; Bacillus thuringiensis Toxins; Bacterial Toxins; Bacillus thuringiensis; Protein Structure, Tertiary; 3. Good health; Endotoxins; Hemolysin Proteins; 03 medical and health sciences; Bacterial Proteins; Aedes; Genes, Bacterial; Larva; Escherichia coli; Animals; Pest Control, Biological

The larvicidal activity ofBacillus thuringiensissubsp.israelensisagainst dipteran larvae is determined by four major polypeptides of the parasporal crystalline body produced during sporulation. Cyt1Aa shows the lowest toxicity when used alone but is the most synergistic with any of the other proteins. The sequence of the plasmid pBtoxis, which contains all the toxin genes in this subspecies, revealed a newcyt-like coding sequence namedcyt1Ca. In addition to the Cyt-like region, the predicted Cyt1Ca contained an extra domain at the C terminus, which appeared to be aβ-trefoil carbohydrate-binding motif, as found in several ricin-like toxins. The gene was PCR-amplified from pBtoxis and cloned in several vectors, allowing high-level expression inEscherichia coli. Cyt1Ca was purified by nickel-nitrilotriacetic acid affinity chromatography, characterized, and its biological activity was determined. Toxicity against larvae ofAedes aegyptiof Cyt1Ca in recombinantE. colicells was compared with that of Cyt1Aa and Cyt2Ba, and the ability of these proteins to enhance the activity of Cry4Aa was assessed. Although Cyt2Ba appeared able to interact with Cry4Aa, no activity for Cyt1Ca was observed, even when produced in truncated form. Furthermore, in contrast to Cyt1Aa, Cyt1Ca did not lyse sheep erythrocytes, and it was not bactericidal to the host cell.