Universidad de Guanajuato Campus Irapuato-Salamanca, División Ciencias de la Vida, Departamento de Ingeniería en Alimentos, Irapuato, Guanajuato, México.
Appl Environ Microbiol. 2012 Mar;78(6):2005-12. doi: 10.1128/AEM.06750-11. Epub 2012 Jan 13.
The cry19A operon of Bacillus thuringiensis subsp. jegathesan encodes two proteins, mosquitocidal Cry19A (ORF1; 75 kDa) and an ORF2 (60 kDa) of unknown function. Expression of the cry19A operon in an acrystalliferous strain of B. thuringiensis (4Q7) yielded one small crystal per cell, whereas no crystals were produced when cry19A or orf2 was expressed alone. To determine the function of the ORF2 protein, different combinations of Cry19A, ORF2, and the N- or C-terminal half of Cry1C were synthesized in strain 4Q7. Stable crystalline inclusions of these fusion proteins similar in shape to those in the strain harboring the wild-type operon were observed in sporulating cells. Comparative analysis showed that ORF2 shares considerable amino acid sequence identity with the C-terminal region of large Cry proteins. Together, these results suggest that ORF2 assists in synthesis and crystallization of Cry19A by functioning like the C-terminal domain characteristic of Cry protein in the 130-kDa mass range. In addition, to determine whether overexpression of the cry19A operon stabilized its shape and increased Cry19A yield, it was expressed under the control of the strong chimeric cyt1A-p/STAB-SD promoter. Interestingly, in contrast to the expression seen with the native promoter, overexpression of the operon yielded uniform bipyramidal crystals that were 4-fold larger on average than the wild-type crystal. In bioassays using the 4th instar larvae of Culex quinquefasciatus, the strain producing the larger Cry19A crystal showed moderate larvicidal activity that was 4-fold (95% lethal concentration [LC(95)] = 1.9 μg/ml) more toxic than the activity produced in the strain harboring the wild-type operon (LC(95) = 8.2 μg/ml).
苏云金芽孢杆菌亚种 jegathesan 的 cry19A 操纵子编码两种蛋白质，杀蚊晶体蛋白 Cry19A（ORF1；75 kDa）和一个未知功能的 ORF2（60 kDa）。在苏云金芽孢杆菌无晶体形成菌株（4Q7）中表达 cry19A 操纵子，每个细胞产生一个小晶体，而单独表达 cry19A 或 orf2 时则不产生晶体。为了确定 ORF2 蛋白的功能，在 4Q7 菌株中合成了不同组合的 Cry19A、ORF2 和 Cry1C 的 N 或 C 末端半部分。在产孢细胞中观察到这些融合蛋白的稳定晶体内含物，其形状与携带野生型操纵子的菌株中的晶体相似。比较分析表明，ORF2 与大 Cry 蛋白的 C 末端区域具有相当的氨基酸序列同一性。综上所述，这些结果表明，ORF2 通过类似于 130 kDa 质量范围内的 Cry 蛋白的 C 末端结构域的功能，协助 Cry19A 的合成和结晶。此外，为了确定 cry19A 操纵子的过表达是否稳定其形状并增加 Cry19A 的产量，它在强嵌合 cyt1A-p/STAB-SD 启动子的控制下表达。有趣的是，与天然启动子的表达相比，操纵子的过表达产生了均匀的双锥形晶体，其平均比野生型晶体大 4 倍。在使用 Culex quinquefasciatus 第 4 龄幼虫的生物测定中，产生更大 Cry19A 晶体的菌株表现出适度的杀幼虫活性，比携带野生型操纵子的菌株（LC（95）= 8.2 μg/ml）的活性高 4 倍（95%致死浓度 [LC（95）]= 1.9 μg/ml）。
