Screening of an E. coli O157:H7 Bacterial Artificial Chromosome Library by Comparative Genomic Hybridization to Identify Genomic Regions Contributing to Growth in Bovine Gastrointestinal Mucus and Epithelial Cell Colonization.
Screening of an E. coli O157:H7 Bacterial Artificial Chromosome Library by Comparative Genomic Hybridization to Identify Genomic Regions Contributing to Growth in Bovine Gastrointestinal Mucus and Epithelial Cell Colonization.
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DOI：
10.3389/fmicb.2011.00168
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发表时间：
2011
影响因子：
5.2
通讯作者：
Tree JJ
中科院分区：
文献类型：
--
作者：
Bai J;McAteer SP;Paxton E;Mahajan A;Gally DL;Tree JJ
肠出血性大肠大肠杆菌（EHEC）O 157：H7可在人类直接或间接接触含有该细菌的反刍动物粪便后引起严重的胃肠道和全身性疾病。肠出血性大肠杆菌O 157：H7在牛体内的主要定植部位是末端直肠，在那里细菌紧密附着在上皮细胞上，并在肠粘液中繁殖。本研究旨在确定EHEC O 157：H7在该位点定植和增殖的基因组区域。从测序的E. coli O 157：H7 Sakai菌株。该文库包含1152个克隆，平均150 kbp。为了验证文库，通过DNA杂交鉴定含有肠上皮细胞消失（LEE）的完整基因座的克隆。与之前的报告一致，这些未赋予K-12宿主菌株III型分泌（T3 S）能力。然而，将BAC克隆之一缀合到含有部分LEE缺失的菌株中恢复了T3 S。三百八十四个克隆从库进行了两个不同的选择性筛选，一个涉及三轮的粘附试验，以牛初级直肠上皮细胞，而其他竞争的克隆在三轮的增长在牛直肠粘液。然后在E. coli微阵列。粘附试验富集了pO 157 DNA，表明该质粒对直肠上皮细胞定植的重要性。粘液测定富集了涉及碳水化合物利用（包括己糖醛酸摄取）的多个区域，表明这些区域在牛粘液中提供了竞争性生长优势。这种BAC-CGH方法提供了阳性选择筛选，补充了基于转座子的阴性选择筛选。正如所证明的，这可能是特别有用的识别基因的冗余功能，如粘附和碳代谢。
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.
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DOI：
10.1073/pnas.77.12.7347
发表时间：
1980-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
DITTA, G;STANFIELD, S;HELINSKI, DR
通讯作者：
HELINSKI, DR
影响因子：
3.1
作者：
Roe, AJ;Yull, H;Gally, DL
通讯作者：
Gally, DL
影响因子：
2.8
作者：
Roe, AJ;Currie, C;Gally, DL
通讯作者：
Gally, DL
DOI：
10.1073/pnas.262441699
发表时间：
2002-12-24
影响因子：
11.1
作者：
Ohnishi, M;Terajima, J;Hayashi, T
通讯作者：
Hayashi, T
影响因子：
64.8
作者：
Perna, NT;Plunkett, G;Blattner, FR
通讯作者：
Blattner, FR