苏云金芽孢杆菌亚种 kurstaki HD1 作为一个工厂，用于合成碱不稳定 ChiA74∆sp 几丁质酶内含体、Cry 晶体和孢子，用于实际应用。
Bacillus thuringiensis subsp. kurstaki HD1 as a factory to synthesize alkali-labile ChiA74∆sp chitinase inclusions, Cry crystals and spores for applied use.
机构信息
Universidad de Guanajuato Campus Irapuato-Salamanca, División Ciencias de la Vida, Posgrado en Biociencias, Irapuato, Guanajuato 36500, México.
出版信息
Microb Cell Fact. 2014 Jan 24;13:15. doi: 10.1186/1475-2859-13-15.
BACKGROUND
The endochitinase ChiA74 is a soluble secreted enzyme produced by Bacillus thuringiensis that synergizes the entomotoxigenecity of Cry proteins that accumulate as intracellular crystalline inclusion during sporulation. The purpose of this study was to produce alkaline-soluble ChiA74∆sp inclusions in B. thuringiensis, and to determine its effect on Cry crystal production, sporulation and toxicity to an important agronomical insect, Manduca sexta. To this end we deleted the secretion signal peptide-coding sequence of chiA74 (i.e. chiA74∆sp) and expressed it under its native promoter (pEHchiA74∆sp) or strong chimeric sporulation-dependent cytA-p/STAB-SD promoter (pEBchiA74∆sp) in Escherichia coli, acrystalliferous B. thuringiensis (4Q7) and B. thuringiensis HD1.
RESULTS
Based on mRNA analyses, up to 9-fold increase in expression of chiA74∆sp was observed using the cytA-p/STAB-SD promoter. ChiA74∆sp (70 kDa) formed intracellular inclusions that frequently accumulated at the poles of cells. ChiA74∆sp inclusions were dissolved in alkali and reducing conditions, similar to Cry crystals, and retained its activity in a wide range of pH (5 to 9), but showed a drastic reduction (~70%) at pH 10. Chitinase activity of E. coli-pEHchiA74∆sp was ~150 mU/mL, and in E. coli-pEBchiA74∆sp, 250 mU/mL. 4Q7-pEBchiA74∆sp and 4Q7-pEHchiA74∆sp had activities of ~127 mU/mL and ~41 mU/mL, respectively. The endochitinase activity in HD1-pEBchiA74∆sp increased 42x when compared to parental HD1 strain. HD1-pEBchiA74∆sp and HD1 harbored typical bipyramidal Cry inclusions, but crystals in the recombinant were ~30% smaller. Additionally, a 3x increase in the number of viable spores was observed in cultures of the recombinant strain when compared to HD1. Bioassays against first instar larvae of M. sexta with spore-crystals of HD1 or spore-crystal-ChiA74∆sp inclusions of HD1-pEBchiA74∆sp showed LC₅₀s of 67.30 ng/cm² and 41.45 ng/cm², respectively.
CONCLUSIONS
Alkali-labile ChiA74∆sp inclusion bodies can be synthesized in E. coli and B. thuringiensis strains. We demonstrated for the first time the applied utility of synthesis of ChiA74∆sp inclusions, Cry crystals and spores in the same sporangium of HD1, a strain used successfully worldwide to control economically significant lepidopteran pests of agriculture. Our findings will allow to us develop strategies to modify expression of ChiA74∆sp while maximizing Cry crystal synthesis in commercial strains of B. thuringiensis.
背景
内几丁质酶 ChiA74 是一种由苏云金芽孢杆菌产生的可溶性分泌酶，它与 Cry 蛋白协同作用，Cry 蛋白在孢子形成过程中积累为细胞内的结晶内含物。本研究的目的是在苏云金芽孢杆菌中产生碱性可溶的 ChiA74∆sp 内含物，并确定其对 Cry 晶体产生、孢子形成和对重要农业昆虫烟夜蛾的毒性的影响。为此，我们删除了 chiA74 的分泌信号肽编码序列（即 chiA74∆sp），并在大肠杆菌、无晶体的苏云金芽孢杆菌（4Q7）和苏云金芽孢杆菌 HD1 中分别使用其天然启动子（pEHchiA74∆sp）或强嵌合的孢子依赖性 cytA-p/STAB-SD 启动子（pEBchiA74∆sp）进行表达。
结果
基于 mRNA 分析，使用 cytA-p/STAB-SD 启动子可使 chiA74∆sp 的表达增加约 9 倍。ChiA74∆sp（70 kDa）形成细胞内内含物，通常在细胞的两极积累。ChiA74∆sp 内含物可在碱性和还原条件下溶解，类似于 Cry 晶体，并且在广泛的 pH 值（5 至 9）范围内保持其活性，但在 pH 值为 10 时活性急剧下降（70%）。大肠杆菌-pEHchiA74∆sp 的几丁质酶活性约为 150 mU/mL，而大肠杆菌-pEBchiA74∆sp 为 250 mU/mL。4Q7-pEBchiA74∆sp 和 4Q7-pEHchiA74∆sp 的活性分别约为 127 mU/mL 和 41 mU/mL。与亲本 HD1 菌株相比，HD1-pEBchiA74∆sp 中的内几丁质酶活性增加了 42 倍。HD1-pEBchiA74∆sp 和 HD1 含有典型的双金字塔形 Cry 内含物，但重组体中的晶体小 30%。此外，与 HD1 相比，重组菌株培养物中的活孢子数量增加了 3 倍。用 HD1 的孢子晶体或 HD1-pEBchiA74∆sp 的孢子晶体-ChiA74∆sp 内含物对烟夜蛾初孵幼虫进行的生物测定显示 LC₅₀ 分别为 67.30 ng/cm²和 41.45 ng/cm²。
结论
可在大肠杆菌和苏云金芽孢杆菌菌株中合成碱性不稳定的 ChiA74∆sp 内含物。我们首次证明了在 HD1 中合成 ChiA74∆sp 内含物、Cry 晶体和孢子的应用实用性，HD1 是一种在全球范围内成功用于控制具有经济重要性的鳞翅目农业害虫的菌株。我们的发现将使我们能够开发在商业苏云金芽孢杆菌菌株中修改 ChiA74∆sp 表达的策略，同时最大限度地提高 Cry 晶体的合成。
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