Isolation and functional characterization of a novel cry gene from a native Bacillus thuringiensis strain

BERÓN, C.M.; GIARROCCO, L.; SALERNO, G. L .

Puerto Iguazú, Argentina

Congreso; XL Reunión anual de la Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular.; 2004

SAIBBM

Bacillus thuringiensis (Bt) is a gram-positive endospore forming bacterium that produces an insecticidal parasporal crystal inclusion (Cry toxins). We have isolated and characterized naturally occurring Bt strains collected in Argentina. The aim of this work was the isolation and characterization of a novel cry-type gene present in the FCC41 strain. The full-length encoding sequence (2,061 bp) was obtained from genomic DNA and using PCR methodologies including TAIL-PCR. Then, it was cloned and sequenced. The deduced protein (687 amino-acid residues) presented a 52% identity with Cry24Aa toxins. To functionally identification, the encoding sequence was expressed in a BL21 Escherichia coli strain. Cell extract from the recombinant strain showed high insecticidal activity against Aedes aegypti. Therefore, bioassays against third instar larvae of this diptera were carried out with the recombinant E. coli strain, FCC 41 strain, in comparison to the commercial Bt sp. israelensis strain, toxic against dipterans. The 50% lethal concentration (LC50) was determined by Probit analysis, showing that the novel Cry24-type protein displayed a high insecticidal activity against A. aegypti. Sequence and structural analysis of the novel Cry toxin revealed that it has conserved amino-acid residues present in other Cry proteins active against dipterans, like Cry2Aa, Cry4Aa, Cry10Aa and Cry11Aa toxins.