Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI 53706, USA.
Appl Environ Microbiol. 2011 Sep;77(18):6630-5. doi: 10.1128/AEM.05307-11. Epub 2011 Jul 22.
The traditional genetic procedure for random or site-specific mutagenesis in Escherichia coli K-12 involves mutagenesis, isolation of mutants, and transduction of the mutation into a clean genetic background. The transduction step reduces the likelihood of complications due to secondary mutations. Though well established, this protocol is not tenable for many pathogenic E. coli strains, such as uropathogenic strain CFT073, because it is resistant to known K-12 transducing bacteriophages, such as P1. CFT073 mutants generated via a technique such as lambda Red mutagenesis may contain unknown secondary mutations. Here we describe the isolation and characterization of transducing bacteriophages for CFT073. Seventy-seven phage isolates were acquired from effluent water samples collected from a wastewater treatment plant in Madison, WI. The phages were differentiated by a host sensitivity-typing scheme with a panel of E. coli strains from the ECOR collection and clinical uropathogenic isolates. We found 49 unique phage isolates. These were then examined for their ability to transduce antibiotic resistance gene insertions at multiple loci between different mutant strains of CFT073. We identified 4 different phages capable of CFT073 generalized transduction. These phages also plaque on the model uropathogenic E. coli strains 536, UTI89, and NU14. The highest-efficiency transducing phage, ΦEB49, was further characterized by DNA sequence analysis, revealing a double-stranded genome 47,180 bp in length and showing similarity to other sequenced phages. When combined with a technique like lambda Red mutagenesis, the newly characterized transducing phages provide a significant development in the genetic tools available for the study of uropathogenic E. coli.
传统的大肠杆菌 K-12 随机或定点诱变的遗传程序包括诱变、突变体分离和突变体转导到干净的遗传背景中。转导步骤降低了由于二次突变引起的并发症的可能性。尽管该方法已经建立，但对于许多致病性大肠杆菌菌株，如尿路致病性菌株 CFT073，并不可行，因为它对已知的 K-12 转导噬菌体，如 P1，具有抗性。通过 lambda Red 诱变等技术产生的 CFT073 突变体可能含有未知的二次突变。本文描述了用于 CFT073 的转导噬菌体的分离和特性。从威斯康星州麦迪逊市的一家废水处理厂收集的污水水样中获得了 77 个噬菌体分离物。噬菌体通过与 ECOR 收藏和临床尿路致病性分离株的大肠杆菌菌株进行宿主敏感性分型方案进行区分。我们发现了 49 个独特的噬菌体分离物。然后，检查它们在不同 CFT073 突变株之间的多个基因座上转导抗生素抗性基因插入的能力。我们鉴定了 4 种不同的能够进行 CFT073 普遍转导的噬菌体。这些噬菌体还在模型尿路致病性大肠杆菌菌株 536、UTI89 和 NU14 上形成噬菌斑。效率最高的转导噬菌体 ΦEB49 通过 DNA 序列分析进一步进行了表征，揭示了一个长度为 47180bp 的双链基因组，与其他测序的噬菌体具有相似性。当与 lambda Red 诱变等技术结合使用时，新鉴定的转导噬菌体为研究尿路致病性大肠杆菌提供了重要的遗传工具。
