MOLECULAR-CLONING AND CHARACTERIZATION OF A NOVEL MOSQUITOCIDAL PROTEIN GENE FROM BACILLUS-THURINGIENSIS SUBSP FUKUOKAENSIS

A novel mosquitocidal protein gene, cry20Aa, was cloned from Bacillus thuringiensis subsp. fukuokaensis (H-3a: 3d: 3e). The gene product, Cr y20Aa, was naturally truncated and had a molecular mass of 86,138 Da. The Cry20Aa protein possessed five conserved sequence blocks, as do mo st other insecticidal Cry toxins. However, an amino acid comparison of Cry20Aa with other mosquitocidal toxins, including Cry4A, Cry4B, Cry1 0A, Cry11A, and Cry11B, demonstrated that Cry20Aa was quite different from other toxins except for the conserved blocks. The N terminus of C ry20Aa was, however, homologous to the N termini of Cry4A and Cry10A. Interestingly, an inverted repeat (IR1) sequence in the open reading f rame of the cry20Aa gene caused incomplete expression of Cry20Aa. When this internal IR1 sequence was altered with no change of amino acid s equence, acrystalliferous B. thuringiensis cells transformed with cry2 0Aa gene dramatically produced crystal inclusions. However, the intact 86-kDa Cry20Aa protein is highly labile, and it is rapidly degraded t o polypeptides of 56 and 43 kDa. To increase expression of the cry20Aa gene, the p20 chaperonelike protein and the cyt1Aa promoter were util ized. While p20 did not increase Cry20Aa expression or stability, chim eric constructs in which the cry20Aa gene was under control of the cyt 1Aa promoter overexpressed the Cry20Aa protein in acrystalliferous B. thuringiensis. The expressed Cry20Aa protein showed larvicidal activit y against Aedes aegypti and Culex quinquefasciatus. However, the mosqu itocidal activity was low, probably due to rapid proteolysis to inacti ve 56- and 43-kDa proteins.