Comprehensive transposon mutant library of Pseudomonas aeruginosa
Comprehensive transposon mutant library of Pseudomonas aeruginosa
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DOI：
10.1073/pnas.2036282100
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发表时间：
2003-11-25
影响因子：
11.1
通讯作者：
Manoil, C
中科院分区：
文献类型：
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作者：
Jacobs, MA;Alwood, A;Manoil, C
我们已经开发了用于创建序列定义的转座子插入突变体的饱和文库的技术，其中每个菌株都被保持。这种文库的表型分析应该提供一个几乎完整的鉴定非必需基因所需的任何过程中，一个合适的屏幕可以设计。该方法应用于铜绿假单胞菌，一种具有6.3 Mbp基因组的机会致病菌。产生的文库由30，100个序列定义的突变体组成，相当于每个基因平均有5个插入。大约12%的预测基因缺乏插入，这些基因中的许多可能是在丰富的媒体上生长所必需的。通过对E.大肠杆菌中，我们估计必需基因的实际数目为300-400个。筛选收集的菌株缺陷在两个定义的多基因过程（抽搐运动和原养型生长）确定的突变体对应于几乎所有的基因预期从早期的研究。因此，集合的表型分析可以产生不同生物活性所需的基因的基本上完整的列表。用于产生突变体集合的转座子具有应促进基因表达、蛋白质定位、上位性和染色体工程的下游研究的附加特征。
We have developed technologies for creating saturating libraries of sequence-defined transposon insertion mutants in which each strain is maintained. Phenotypic analysis of such libraries should provide a virtually complete identification of nonessential genes required for any process for which a suitable screen can be devised. The approach was applied to Pseudomonas aeruginosa, an opportunistic pathogen with a 6.3-Mbp genome. The library that was generated consists of 30,100 sequence-defined mutants, corresponding to an average of five insertions per gene. About 12% of the predicted genes of this organism lacked insertions; many of these genes are likely to be essential for growth on rich media. Based on statistical analyses and bioinformatic comparison to known essential genes in E. coli, we estimate that the actual number of essential genes is 300-400. Screening the collection for strains defective in two defined multigenic processes (twitching motility and prototrophic growth) identified mutants corresponding to nearly all genes expected from earlier studies. Thus, phenotypic analysis of the collection may produce essentially complete lists of genes required for diverse biological activities. The transposons used to generate the mutant collection have added features that should facilitate downstream studies of gene expression, protein localization, epistasis, and chromosome engineering.