Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi
Functional genomics of the rapidly replicating bacterium Vibrio natriegens by CRISPRi
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DOI：
10.1038/s41564-019-0423-8
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发表时间：
2019-07-01
影响因子：
28.3
通讯作者：
Church, George M.
中科院分区：
文献类型：
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作者：
Lee, Henry H.;Ostrov, Nili;Church, George M.
快速生长的革兰氏阴性细菌产钠弧菌是一种有吸引力的微生物系统，由于其显着短的世代时间（1，2）和代谢能力（3，4）的分子生物学和生物技术。然而，努力发现和利用其快速增长的机制受到功能基因组数据稀缺的阻碍。在这里，我们开发了一个汇集的全基因组聚类规则间隔短回文重复序列（CRISPR）干扰（CRISPRi）筛选，以确定快速野生型生长所需的最小基因集。针对4，565（99.7%）预测的蛋白质编码基因，我们的筛选发现了核心基因，包括推定的必需和生长支持基因，这些基因富含呼吸途径。我们发现，96%的核心基因位于较大的1号染色体上，而核心基因的生长中性重复主要位于2号染色体上。我们的筛选还通过区分功能性生物合成酶与基于比较基因组学预测的酶来细化代谢途径注释。总之，这项工作为高通量功能基因组学提供了一个广泛适用的平台，以加速产钠弧菌的生物学研究和工程。
The fast-growing Gram-negative bacterium Vibrio natriegens is an attractive microbial system for molecular biology and biotechnology due to its remarkably short generation time(1,2) and metabolic prowess(3,4). However, efforts to uncover and utilize the mechanisms underlying its rapid growth are hampered by the scarcity of functional genomic data. Here, we develop a pooled genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) interference (CRISPRi) screen to identify a minimal set of genes required for rapid wild-type growth. Targeting 4,565 (99.7%) of predicted protein-coding genes, our screen uncovered core genes comprising putative essential and growth-supporting genes that are enriched for respiratory pathways. We found that 96% of core genes were located on the larger chromosome 1, with growth-neutral duplicates of core genes located primarily on chromosome 2. Our screen also refines metabolic pathway annotations by distinguishing functional biosynthetic enzymes from those predicted on the basis of comparative genomics. Taken together, this work provides a broadly applicable platform for high-throughput functional genomics to accelerate biological studies and engineering of V. natriegens.