CLONING AND CHARACTERIZATION OF A CYTOLYTIC AND MOSQUITOCIDAL DELTA-ENDOTOXIN FROM BACILLUS-THURINGIENSIS SUBSP JEGATHESAN

A cytolytic toxin gene encoding a 30.1-kDa Cyt2Bb1 toxin protein from B. thuringiensis subsp. jegathesan was cloned employing a limited-grow th PCR screening method with forward and reverse oligonucleotide prime rs designed from N-terminal amino acid sequences of native and trypsin -cleaved protein, respectively. The expressed protein showed little cr oss-reactivity to the antibody raised against the Cyt1Aa protein. Unli ke Cyt1Aa and Cyt2Aa expression, there was little or no visible crysta l inclusion formation under microscopic observation. The amino acid se quence alignment indicated 31 and 66% identity to Cyt1Aa and Cyt2Aa, r espectively. The sequence alignment for five known cytolytic proteins indicated three highly conserved regions, two in the loop regions betw een alpha-helices and beta-sheets and one in the loop region between b eta-sheets 5 and 6. beta-Blocks 4 to 7 are also conserved, not only st ructurally but also among the amino acids in the hydrophobic faces. Mo squitocidal activity assays indicated that the Cyt2Bb toxin had less t oxicity than Cyt1Aa and had about 600-times-lower toxicity than the wi ld-type whole toxin crystal. However, both the Cyt2Bb and the Cyt1Aa t oxin showed comparable levels of hemolytic activity.