Infection and Immunity Division, The Roslin Institute and The Royal (Dick) School of Veterinary Studies, University of Edinburgh Edinburgh, UK.
Front Microbiol. 2011 Aug 17;2:168. doi: 10.3389/fmicb.2011.00168. eCollection 2011.
Enterohemorrhagic E. coli (EHEC) O157:H7 can cause serious gastrointestinal and systemic disease in humans following direct or indirect exposure to ruminant feces containing the bacterium. The main colonization site of EHEC O157:H7 in cattle is the terminal rectum where the bacteria intimately attach to the epithelium and multiply in the intestinal mucus. This study aimed to identify genomic regions of EHEC O157:H7 that contribute to colonization and multiplication at this site. A bacterial artificial chromosome (BAC) library was generated from a derivative of the sequenced E. coli O157:H7 Sakai strain. The library contains 1152 clones averaging 150 kbp. To verify the library, clones containing a complete locus of enterocyte effacement (LEE) were identified by DNA hybridization. In line with a previous report, these did not confer a type III secretion (T3S) capacity to the K-12 host strain. However, conjugation of one of the BAC clones into a strain containing a partial LEE deletion restored T3S. Three hundred eighty-four clones from the library were subjected to two different selective screens; one involved three rounds of adherence assays to bovine primary rectal epithelial cells while the other competed the clones over three rounds of growth in bovine rectal mucus. The input strain DNA was then compared with the selected strains using comparative genomic hybridization (CGH) on an E. coli microarray. The adherence assay enriched for pO157 DNA indicating the importance of this plasmid for colonization of rectal epithelial cells. The mucus assay enriched for multiple regions involved in carbohydrate utilization, including hexuronate uptake, indicating that these regions provide a competitive growth advantage in bovine mucus. This BAC-CGH approach provides a positive selection screen that complements negative selection transposon-based screens. As demonstrated, this may be of particular use for identifying genes with redundant functions such as adhesion and carbon metabolism.
肠出血性大肠杆菌（EHEC）O157:H7 可以通过直接或间接接触含有该细菌的反刍动物粪便引起人类严重的胃肠道和全身疾病。EHEC O157:H7 在牛中的主要定植部位是直肠末端，细菌在那里紧密附着在上皮细胞并在肠道黏液中繁殖。本研究旨在鉴定有助于该部位定植和繁殖的 EHEC O157:H7 基因组区域。从已测序的大肠杆菌 O157:H7 坂井衍生株中生成了细菌人工染色体（BAC）文库。该文库包含 1152 个克隆，平均大小为 150kbp。为了验证文库，通过 DNA 杂交鉴定了含有完整肠上皮细胞脱落（LEE）位点的克隆。与之前的报告一致，这些克隆不能赋予 K-12 宿主菌株 III 型分泌（T3S）能力。然而，将一个 BAC 克隆与含有部分 LEE 缺失的菌株进行接合恢复了 T3S。从文库中挑选了 384 个克隆进行两种不同的选择性筛选；一种涉及三轮对牛直肠原代上皮细胞的粘附测定，另一种在三轮牛直肠黏液中生长竞争克隆。然后使用大肠杆菌微阵列上的比较基因组杂交（CGH）比较输入菌株 DNA 与选择菌株。粘附测定富集了 pO157 DNA，表明该质粒对直肠上皮细胞定植的重要性。黏液测定富集了多个参与碳水化合物利用的区域，包括己糖酸盐摄取，表明这些区域在牛黏液中提供了竞争生长优势。这种 BAC-CGH 方法提供了一种正选择筛选，补充了基于负选择转座子的筛选。如所证明的，这对于鉴定具有冗余功能（如粘附和碳代谢）的基因可能特别有用。
