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Evidence of the Importance of the Met115 for Bacillus thuringiensis subsp. israelensis Cyt1Aa Protein Cytolytic Activity in Escherichia coli

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Abstract

Cyt1Aa is a cytolytic toxin, found together with the delta-endotoxins in Bacillus thuringiensis subsp. israelensis parasporal insecticidal crystals. The latter are used as an environmental friendly insecticide against mosquitoes and black flies. Contrary to Cry delta-endotoxin, the mode of action of Cyt1Aa is not completely understood. In the absence of direct structural data, a novel mutated cyt1Aa gene was used to obtain indirect informations on Cyt1Aa conformation changes in the lipid membrane environment. A mutated cyt1Aa gene named cyt1A97 has been isolated from a B. thuringiensis israelensis strain named BUPM97. The nucleotide sequence predicted a protein of 249 amino acids residues with a calculated molecular mass of 27 kDa. Both nucleotide and amino acid sequences similarity analysis revealed that cyt1A97 presents one amino acid different from the native cyt1Aa gene. This mutation was located in the helix α C corresponding to a substitution of Met115 by a Thr. The heterologous expression of the cyt1A97 and another cyt1Aa-type gene called cyt1A98, not affected by such mutation used as control, was performed in Escherichia coli. It revealed that the mutated Cyt1A97 protein was over produced as inclusion bodies showing a very weak toxicity to E. coli contrarily to Cyt1A98 that stopped E. coli growth. Hence, hydrophobic residue Met at position 115 of Cyt1Aa should play a very important role for the maintenance of the structure and cytolytic functions of Cyt1Aa.

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Abbreviations

B :

Bacillus

E :

Escherichia

PCR:

Polymerase Chain Reaction

Cyt:

Cytolytic toxin

LB:

Luria-Bertani medium

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Acknowledgements

We thank Mrs. Najeh Belguith-Ben Hassen for her technical assistance. This work was supported by grants from the Tunisian Ministère de l’Enseignement Supérieur de la Recherche Scientifique et de la Technologie et du développement des compétences.

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Authors and Affiliations

  1. Laboratory of Biopesticides, Centre of Biotechnology of Sfax, P.O. Box‘K’, 3038, Sfax, Tunisia

    Raida Zribi Zghal, Hana Trigui & Samir Jaoua

  2. Laboratory of Enzymes and Metabolites of Prokaryotes, Centre of Biotechnology of Sfax, P.O. Box‘K’, 3038, Sfax, Tunisia

    Mamdouh Ben Ali

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  1. Raida Zribi Zghal
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  2. Hana Trigui
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  3. Mamdouh Ben Ali
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  4. Samir Jaoua
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Correspondence to Samir Jaoua.

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Zghal, R.Z., Trigui, H., Ali, M.B. et al. Evidence of the Importance of the Met115 for Bacillus thuringiensis subsp. israelensis Cyt1Aa Protein Cytolytic Activity in Escherichia coli . Mol Biotechnol 38, 121–127 (2008). https://doi.org/10.1007/s12033-007-9015-6

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