Zhao Youfu, Sundin George W, Wang Dongping
Department of Crop Sciences, University of Illinois at Urbana-Champaign, 1201 W. Gregory Dr, Urbana, IL 61801, USA.
Can J Microbiol. 2009 Apr;55(4):457-64. doi: 10.1139/w08-147.
An easy gene-knockout technique, PCR-based one-step inactivation of chromosomal genes, is widely used in Escherichia coli and related enterobacteria to construct mutants. In this study, we adapted this technique to construct genomic island and large operon deletion mutants of Erwinia amylovora, including the 33.4 kb hrp-type III secretion (T3SS) pathogenicity island (PAI1) and the 15.8 kb amylovoran biosynthesis (AMS) operon. Deletion of 2 novel T3SS pathogenicity islands (PAI2 and PAI3) and an operon encoding a type II secretion system (T2SS) demonstrated that these determinants are not involved in virulence in plants. Co-inoculation experiments demonstrated that the hrp-T3SS and AMS deletion mutants could complement each other. These results further confirmed that the one-step inactivation technique can be used to generate large deletions in E. amylovora.
一种简单的基因敲除技术，即基于PCR的染色体基因一步失活技术，被广泛应用于大肠杆菌及相关肠道细菌中以构建突变体。在本研究中，我们采用该技术构建了梨火疫病菌的基因组岛和大操纵子缺失突变体，包括33.4 kb的hrp III型分泌（T3SS）致病岛（PAI1）和15.8 kb的支链淀粉生物合成（AMS）操纵子。对2个新的T3SS致病岛（PAI2和PAI3）以及一个编码II型分泌系统（T2SS）的操纵子的缺失表明，这些决定因素不参与植物中的毒力作用。共接种实验表明，hrp-T3SS和AMS缺失突变体可以相互补充。这些结果进一步证实，一步失活技术可用于在梨火疫病菌中产生大片段缺失。
