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

Abstract

The larvicidal activity of Bacillus thuringiensis subsp. israelensis against 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 new cyt-like coding sequence named cyt1Ca. In addition to the Cyt-like region, the predicted Cyt1Ca contained an extra domain at the C terminus, which appeared to be a β-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 in Escherichia coli. Cyt1Ca was purified by nickel-nitrilotriacetic acid affinity chromatography, characterized, and its biological activity was determined. Toxicity against larvae of Aedes aegypti of Cyt1Ca in recombinant E. coli cells 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.

Cyt1Aa Toxin: Crystal Structure Reveals Implications for Its Membrane-Perforating Function

“…It is twice the size of Cyt toxins and is predicted to display the structure of a two-domain fusion protein: an N-terminus that resembles Cyt toxins and a C-terminus that is similar to the receptor binding domain of ricin-B lectin. 17 The C-truncated Cyt1Ca domain (homologous to Cyt1Aa) remains nontoxic, 44 indicating that the C-terminus is not responsible for its lack of toxicity.…”

Bacillus thuringiensis subsp. israelensis and Its Dipteran-Specific Toxins

“…medellin [96]. Cyt2Ba is synergistic with Cry4Aa, Cry4Ba or Cry11Aa [97,98] and with the B sphaericus binary toxin [96]; it may thus contribute to the overall toxicity of Bti .…”

“…Cyt1Ca is encoded by sequence of 1575 bp (525 amino acids) [98,99]. Its N-terminal half is 52% identical to Cyt1Aa, and at the C terminus it contains an extra domain, which appears to be a β -trefoil carbohydrate-binding motif, similar to the receptor binding domain of ricin-B lectin type found in several ricin-like toxins [99].…”

“…Transcripts of cyt1Ca were detected, but Cyt1Ca has not been found [100]; the reason may include instability of the transcript or the protein and failure in message translation. Neither mosquito larvicidal activity nor other biological function has been reported for Cyt1Ca [98,99]. The lack of activity of Cyt1Ca may be related to its inability to undergo a certain conformational change due to its lack of flexibility [101].…”

Antibacterial activity of Cyt1Aa from Bacillus thuringiensis subsp. israelensis

“…The encoding genes are located on a large plasmid (128 kb) called pBtoxis (Ben-Dov et al, 1999;Berry et al, 2002). The plasmid contains two other cyt genes, cyt2Ba and cyt1Ca, in addition to cyt1Aa (Guerchicoff et al, 1997;Li et al, 1996;Manasherob et al, 2006). Cyt1Aa (249 amino acids; 27 kDa) is the major component (45-50 %) of the d-endotoxin and was the first cytolytic toxin to be isolated and comprehensively characterized (Bourgouin et al, 1986).…”