Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli.
Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli.
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DOI：
10.1186/1471-2199-4-11
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发表时间：
2003-12-13
影响因子：
--
通讯作者：
Campellone KG
中科院分区：
文献类型：
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作者：
Murphy KC;Campellone KG
λ Red重组技术已在大肠杆菌和鼠伤寒沙门氏菌中广泛应用，以便于通过PCR介导产生缺失突变体，但在致病性大肠杆菌种类如肠出血性大肠杆菌（EHEC）和肠致病性大肠杆菌（EPEC）中应用较少。我们早期在EHEC和EPEC中使用λ Red的实验得到了零星的结果，这促使了当前的研究去确定可能提高在这些致病性大肠杆菌菌株中Red重组效率的因素。
在本报告中，我们确定了在EHEC和EPEC中优化使用λ Red进行重组的条件。利用含有一个Ptac - red - gam操纵子和一个温度敏感型复制起点的质粒，我们在已知的毒力基因中产生了多种突变（有标记的和无标记的）。此外，我们轻松地删除了EHEC中五个疑似含有毒力因子的O157特异性岛（O岛）。我们研究了使用PCR产生的底物（两侧有40bp同源序列）和质粒衍生的底物（两侧约1kb同源序列）的情况；两者效果都很好，且各有优势。高重组表型的建立仅需要对red和gam进行20分钟的IPTG诱导。这个重组窗口很重要，因为red和gam的组成型表达会使对利福平的自发抗性增加10倍。其他因素，如重组底物中药物标记的方向和热激效应，在EHEC和EPEC中Red介导的重组成功中也起作用。
λ Red重组技术已针对致病性大肠杆菌种类，即EHEC和EPEC进行了优化。作为这项技术的演示，通过电穿孔将含有两侧带有40bp靶DNA的药物标记的PCR产生的底物导入，轻松且精确地从染色体上删除了EHEC的五个O岛。这些结果应该会鼓励在这些以及其他致病性细菌菌株中使用λ Red重组技术，以便更快地鉴定毒力因子，并快速产生用于疫苗开发的细菌突变体。
The λ Red recombineering technology has been used extensively in Escherichia coli and Salmonella typhimurium for easy PCR-mediated generation of deletion mutants, but less so in pathogenic species of E. coli such as EHEC and EPEC. Our early experiments with the use of λ Red in EHEC and EPEC have led to sporadic results, leading to the present study to identify factors that might improve the efficiency of Red recombineering in these pathogenic strains of E. coli. In this report, we have identified conditions that optimize the use of λ Red for recombineering in EHEC and EPEC. Using plasmids that contain a Ptac-red-gam operon and a temperature-sensitive origin of replication, we have generated multiple mutations (both marked and unmarked) in known virulence genes. In addition, we have easily deleted five O157-specific islands (O-islands) of EHEC suspected of containing virulence factors. We have examined the use of both PCR-generated substrates (40 bp of flanking homology) and plasmid-derived substrates (~1 kb of flanking homology); both work well and each have their own advantages. The establishment of the hyper-rec phenotype requires only a 20 minute IPTG induction period of red and gam. This recombinogenic window is important as constitutive expression of red and gam induces a 10-fold increase in spontaneous resistance to rifampicin. Other factors such as the orientation of the drug marker in recombination substrates and heat shock effects also play roles in the success of Red-mediated recombination in EHEC and EPEC. The λ Red recombineering technology has been optimized for use in pathogenic species of E. coli, namely EHEC and EPEC. As demonstration of this technology, five O-islands of EHEC were easily and precisely deleted from the chromosome by electroporation with PCR-generated substrates containing drug markers flanked with 40 bp of target DNA. These results should encourage the use of λ Red recombineering in these and other strains of pathogenic bacteria for faster identification of virulence factors and the speedy generation of bacterial mutants for vaccine development.