Intracellular Screen To Identify Metagenomic Clones That Induce or Inhibit a Quorum-Sensing Biosensor
Intracellular Screen To Identify Metagenomic Clones That Induce or Inhibit a Quorum-Sensing Biosensor
复制标题
细胞内筛选识别诱导或抑制群体感应生物传感器的宏基因组克隆
DOI：
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发表时间：
2005
影响因子：
4.4
通讯作者：
J. Handelsman
中科院分区：
文献类型：
--
作者：
L. Williamson;B. Borlee;P. Schloss;C. Guan;Heather K. Allen;J. Handelsman
摘要 这项研究的目的是设计和评估快速筛选，以识别产生生物活性小分子的宏基因组克隆。我们利用阿拉斯加塔纳纳河漫滩土壤中的 DNA 构建了宏基因组文库。我们直接从土壤中提取 DNA，并将其克隆到 fosmid 和细菌人工染色体载体中，构建了 8 个宏基因组文库，其中包含 53,000 个克隆，插入片段范围为 1 至 190 kb。为了识别感兴趣的克隆，我们设计了一种高通量“细胞内”筛选，命名为 METREX，其中宏基因组 DNA 位于宿主细胞中，该宿主细胞含有诱导细菌群体感应的化合物的生物传感器。如果宏基因组克隆产生群体感应诱导剂，则细胞会产生绿色荧光蛋白（GFP），并且可以通过荧光显微镜识别或通过荧光激活细胞分选捕获。我们的初步筛选确定了 11 个诱导 GFP 表达的克隆和两个抑制 GFP 表达的克隆。细胞内筛选在宏基因组克隆中检测到了群体感应诱导剂，而传统的重叠筛选则无法检测到。一个诱导克隆携带 LuxI 同源物，指导 N-酰基高丝氨酸内酯群体感应信号分子的合成。 LuxI 同源物与 GenBank 中最接近的匹配荧光假单胞菌 AmfI 具有 62% 的氨基酸序列同一性，并且位于包含 67 个开放阅读框的 78 kb 插入片段上。另一个诱导克隆携带与高柠檬酸合酶同源的基因。我们的结果证明了细胞内筛选在识别宏基因组文库中具有功能活性的克隆和生物活性小分子方面的能力。
ABSTRACT The goal of this study was to design and evaluate a rapid screen to identify metagenomic clones that produce biologically active small molecules. We built metagenomic libraries with DNA from soil on the floodplain of the Tanana River in Alaska. We extracted DNA directly from the soil and cloned it into fosmid and bacterial artificial chromosome vectors, constructing eight metagenomic libraries that contain 53,000 clones with inserts ranging from 1 to 190 kb. To identify clones of interest, we designed a high throughput “intracellular” screen, designated METREX, in which metagenomic DNA is in a host cell containing a biosensor for compounds that induce bacterial quorum sensing. If the metagenomic clone produces a quorum-sensing inducer, the cell produces green fluorescent protein (GFP) and can be identified by fluorescence microscopy or captured by fluorescence-activated cell sorting. Our initial screen identified 11 clones that induce and two that inhibit expression of GFP. The intracellular screen detected quorum-sensing inducers among metagenomic clones that a traditional overlay screen would not. One inducing clone carries a LuxI homologue that directs the synthesis of an N-acyl homoserine lactone quorum-sensing signal molecule. The LuxI homologue has 62% amino acid sequence identity to its closest match in GenBank, AmfI from Pseudomonas fluorescens, and is on a 78-kb insert that contains 67 open reading frames. Another inducing clone carries a gene with homology to homocitrate synthase. Our results demonstrate the power of an intracellular screen to identify functionally active clones and biologically active small molecules in metagenomic libraries.
影响因子：
56.9
作者：
PASSADOR, L;COOK, JM;IGLEWSKI, BH
通讯作者：
IGLEWSKI, BH
影响因子：
3.5
作者：
Dunn, AK;Handelsman, J
通讯作者：
Handelsman, J
影响因子：
2.9
作者：
EBERHARD, A;BURLINGAME, AL;OPPENHEIMER, NJ
通讯作者：
OPPENHEIMER, NJ