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首页> 外文期刊>Infection and immunity >Two pathogenicity islands in uropathogenic Escherichia coli J96: cosmid cloning and sample sequencing.
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Two pathogenicity islands in uropathogenic Escherichia coli J96: cosmid cloning and sample sequencing.

机译：尿路致病性大肠杆菌J96中的两个致病岛：粘粒克隆和样品测序。

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Many of the virulence genes of pathogenic strains of Escherichia coli are carried in large multigene chromosomal segments called pathogenicity islands (PAIs) that are absent from normal fecal and laboratory K-12 strains of this bacterium. We are studying PAIs in order to better understand factors that govern virulence and to assess how such DNA segments are gained or lost during evolution. The isolation and sample sequencing of a set of 11 cosmid clones that cover all of one and much of a second large PAI in the uropathogenic E. coli J96 are described. These PAIs were mapped to the 64- and 94-min regions of the E. coli K-12 chromosome, which differ from the locations of three PAIs identified in other pathogenic E. coli strains. Analysis of the junction sequences with E. coli K-12-like DNAs showed that the insert at 94 min is within the 3' end of a phenylalanine tRNA gene, pheR, and is flanked by a 135-bp imperfect direct repeat. Analysis of the one junction recovered from the insert at 64 min indicated that it lies near another tRNA gene, pheV. To identify possible genes unique to these PAIs, 100 independent subclones of the cosmids were made by PstI digestion and ligation into a pBS+ plasmid and used in one-pass sample DNA sequencing from primer binding sites at the cloning site in the vector DNA. Database searches of the J96 PAI-specific sequences identified numerous instances in which the cloned DNAs shared significant sequence similarities to adhesins, toxins, and other virulence determinants of diverse pathogens. Several likely insertion sequence elements (IS100, IS630, and IS911) and conjugative R1 plasmid and P4 phage genes were also found. We propose that such mobile genetic elements may have facilitated the spread of virulence determinants within PAIs among bacteria.
机译：大肠杆菌的致病菌株的许多毒力基因都携带在称为致病岛（PAI）的大型多基因染色体片段中，而该细菌的正常粪便和实验室K-12菌株却没有这种片段。我们正在研究PAI，以便更好地理解控制毒力的因素，并评估在进化过程中此类DNA片段如何获得或丢失。描述了一组11个粘粒克隆的分离和样品测序，这些粘粒克隆覆盖了尿路致病性大肠杆菌J96中的一个或多个第二大PAI的全部。这些PAI被定位到大肠杆菌K-12染色体的64分钟和94分钟区域，这与在其他致病性大肠杆菌菌株中鉴定出的三个PAI的位置不同。对与大肠杆菌K-12样DNA的连接序列的分析表明，在94分钟处的插入片段位于苯丙氨酸tRNA基因pheR的3'末端，并且侧翼为135 bp不完美的直接重复序列。分析在64分钟时从插入片段中回收的一个连接点，表明它位于另一个tRNA基因pheV附近。为了鉴定这些PAI特有的可能基因，通过PstI消化并连接到pBS +质粒中，制成了100个独立的粘粒亚克隆，并用于从载体DNA克隆位点的引物结合位点进行单次样品DNA测序。对J96 PAI特定序列的数据库搜索确定了许多实例，其中克隆的DNA与粘附素，毒素和其他多种病原体的毒力决定因素共享显着的序列相似性。还发现了几种可能的插入序列元件（IS100，IS630和IS911）以及结合型R1质粒和P4噬菌体基因。我们建议这种移动的遗传因素可能已经促进了细菌中PAIs内的毒力决定因素的传播。

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