Construction and Analysis of Two Genome-Scale Deletion Libraries for Bacillus subtilis.
Construction and Analysis of Two Genome-Scale Deletion Libraries for Bacillus subtilis.
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DOI：
10.1016/j.cels.2016.12.013
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发表时间：
2017-03-22
期刊：
影响因子：
9.3
通讯作者：
Gross CA
中科院分区：
文献类型：
--
作者：
Koo BM;Kritikos G;Farelli JD;Todor H;Tong K;Kimsey H;Wapinski I;Galardini M;Cabal A;Peters JM;Hachmann AB;Rudner DZ;Allen KN;Typas A;Gross CA
从环境和健康的角度来看，对革兰氏阳性菌的系统水平了解都很重要，并且当可以获得高质量、经过验证的基因组资源时，最容易获得这种了解。为此，我们构建了革兰氏阳性模型生物枯草芽孢杆菌的两个有序、条形码、红霉素抗性和卡那霉素抗性标记的单基因删除文库。这些文库分别包含 3968 个和 3970 个基因，并且除了 4 个基因外，所有基因都有重叠。使用这些文库，我们更新了该生物体已知的一组必需基因，提供了枯草芽孢杆菌营养缺陷型基因的综合纲要，并确定了利用特定碳源和氮源以及低温生长所需的基因。我们报告了催化氨基酸生物合成中几个缺失步骤的酶的鉴定。最后，我们描述了一套高通量表型分析方法，并将其应用于能力和孢子形成的全基因组分析。总而言之，我们为研究革兰氏阳性细菌的基因功能、通路和网络结构提供了多种资源。
A systems level understanding of Gram-positive bacteria is important from both an environmental and health perspective, and is most easily obtained when high-quality, validated genomic resources are available. To this end, we constructed two ordered, barcoded, erythromycin-resistance- and kanamycin-resistance-marked single-gene deletion libraries of the Gram-positive model organism, Bacillus subtilis. The libraries comprise 3968 and 3970 genes, respectively, and overlap in all but four genes. Using these libraries, we update the set of essential genes known for this organism, provide a comprehensive compendium of B. subtilis auxotrophic genes, and identify genes required for utilizing specific carbon and nitrogen sources, as well as those required for growth at low temperature. We report the identification of enzymes catalyzing several missing steps in amino acid biosynthesis. Finally, we describe a suite of high-throughput phenotyping methodologies and apply them to provide a genome-wide analysis of competence and sporulation. Altogether, we provide versatile resources for studying gene function and pathway and network architecture in Gram-positive bacteria.