Toward functional genomics in bacteria:: Analysis of gene expression in Escherichia coli from a bacterial artificial chromosome library of Bacillus cereus
Toward functional genomics in bacteria:: Analysis of gene expression in Escherichia coli from a bacterial artificial chromosome library of Bacillus cereus
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DOI：
10.1073/pnas.96.11.6451
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发表时间：
1999-05-25
影响因子：
11.1
通讯作者：
Handelsman, J
中科院分区：
文献类型：
--
作者：
Rondon, MR;Raffel, SJ;Handelsman, J
随着微生物研究进入功能基因组学时代，越来越需要用于分析各种微生物的分子工具。目前，农业，医学和基础科学的兴趣，许多原核生物的生物学研究是有限的缺乏足够的遗传工具。我们报告了细菌人工染色体（BAC）载体在原核生物学中的应用，作为满足这一需求的有力方法。我们从革兰氏阳性细菌蜡样芽孢杆菌的基因组DNA中构建了大肠杆菌BAC文库。该文库提供了B的5.75倍覆盖。cereus基因组，平均插入片段大小为98kb。确定B的异源表达程度。在文库中的蜡样芽胞杆菌基因，我们筛选了它的几个B的表达。cereus在E. coli宿主。在文库中鉴定了表达10种测试活性中的6种的克隆，即氨苄青霉素抗性、两性霉素A抗性、七叶苷水解、溶血、橙子色素产生和卵磷脂酶活性。我们对所选BAC克隆进行遗传分析以快速鉴定特异性B。蜡状体这些结果表明BAC文库将为研究不同原核生物的基因表达提供一种强有力的方法。
As the study of microbes moves into the era of functional genomics, there is an increasing need for molecular tools for analysis of a wide diversity of microorganisms. Currently, biological study of many prokaryotes of agricultural, medical, and fundamental scientific interest is limited by the lack of adequate genetic tools. We report the application of the bacterial artificial chromosome (BAC) vector to prokaryotic biology as a powerful approach to address this need. We constructed a BAC library in Escherichia coli from genomic DNA of the Gram-positive bacterium Bacillus cereus. This library provides 5.75-fold coverage of the B. cereus genome, with an average insert size of 98 kb. To determine the extent of heterologous expression of B. cereus genes in the library, we screened it for expression of several B. cereus activities in the E. coli host. Clones expressing 6 of 10 activities tested were identified in the library, namely, ampicillin resistance, zwittermicin A resistance, esculin hydrolysis, hemolysis, orange pigment production, and lecithinase activity. We analyzed selected BAC clones genetically to identify rapidly specific B. cereus loci. These results suggest that BAC libraries will provide a powerful approach for studying gene expression from diverse prokaryotes.