基于 T6SS毒素 Txe1的负筛选系统在杀鱼爱德华氏菌基因组编辑中的应用

Application of a T6SS effector Txe1-based counterselection system for genome editing in Edwardsiella piscicida

摘要:

以果聚糖蔗糖酶(sucrose-6-fructosyltransferase, SacB)为基础的双交换同源重组技术虽常用于革兰氏阴性菌基因组编辑，但其负筛选效率在不同菌株间常存在显著差异，部分菌株因代谢特性或基因组组成差异导致效率低下。 目的 构建基于VI型分泌系统(T6SS)效应蛋白Txe1的新型负筛选体系，以提升无痕基因组编辑效率。 方法 改造传统自杀质粒pDM4，引入卡那霉素抗性基因，获得衍生质粒pDM4K；再以阿拉伯糖诱导型Txe1毒素C端结构域表达盒( araC-P _BAD:: txe1 ^CTD )替换质粒中的 sacB基因，构建新型负筛选质粒pTL1010。以杀鱼爱德华氏菌FC2株毒力相关基因 tssB为靶点，系统比较Txe1与SacB系统的负筛选效率。 结果 阿拉伯糖诱导下，Txe1系统的负筛选效率达91.1% (假阳性率8.9%)，显著优于传统SacB系统(假阳性率100%， P<0.01)。 结论 新型Txe1负筛选质粒pTL1010显著提高了杀鱼爱德华氏菌的无痕基因编辑效率，为革兰氏阴性菌精准遗传操作提供了高效新工具。

Abstract:

Double-crossover homologous recombination using the SacB negative selection system is commonly employed for genome editing in Gram-negative bacteria. However, its negative selection efficiency varies significantly across strains, being frequently compromised by differences in metabolic characteristics or genomic composition. Objective To develop a novel counterselection system based on the type VI secretion system (T6SS) effector Txe1 to enhance the efficiency of seamless genome editing. Methods We first modified the conventional suicide plasmid pDM4 by introducing a kanamycin resistance gene, generating the derivative plasmid pDM4K. Subsequently, we replaced sacB with an l-arabinose-inducible expression cassette encoding the C-terminal domain of Txe1 ( araC-P _BAD:: txe1 ^CTD ), constructing a novel counterselection plasmid pTL1010. Using the virulence gene tssB of Edwardsiella piscicida FC2 as the target, we systematically evaluated and compared the counterselection efficiency of the Txe1 system with that of the conventional SacB system. Results Under induction with l-arabinose, the Txe1-based counterselection system achieved the efficiency of 91.1% (false-positive rate of 8.9%), outperforming the SacB system which had a false-positive rate of 100% ( P<0.01). Conclusion The newly developed Txe1-based counterselection plasmid pTL1010 significantly enhances the efficiency of seamless genome editing in E. piscicida and provides a highly effective tool for precise genetic manipulation in Gram-negative bacteria.