Centre de Recherche en Infectiologie, Université Laval, Québec, Canada.
Microb Genom. 2018 Feb;4(2). doi: 10.1099/mgen.0.000148. Epub 2018 Jan 10.
In order to expedite the discovery of genes coding for either drug targets or antibiotic resistance, we have developed a functional genomic strategy termed Plas-Seq. This technique involves coupling a multicopy suppressor library to next-generation sequencing. We generated an Escherichia coli plasmid genomic library that was transformed into E. coli. These transformants were selected step by step using 0.25× to 2× minimum inhibitory concentrations for ceftriaxone, gentamicin, levofloxacin, tetracycline or trimethoprim. Plasmids were isolated at each selection step and subjected to Illumina sequencing. By searching for genomic loci whose sequencing coverage increased with antibiotic pressure we were able to detect 48 different genomic loci that were enriched by at least one antibiotic. Fifteen of these loci were studied functionally, and we showed that 13 can decrease the susceptibility of E. coli to antibiotics when overexpressed. These genes coded for drug targets, transcription factors, membrane proteins and resistance factors. The technique of Plas-Seq is expediting the discovery of genes associated with the mode of action or resistance to antibiotics and led to the isolation of a novel gene influencing drug susceptibility. It has the potential for being applied to novel molecules and to other microbial species.
为了加速发现编码药物靶点或抗生素耐药性的基因，我们开发了一种称为 Plas-Seq 的功能基因组策略。该技术涉及将多拷贝抑制子文库与下一代测序相结合。我们生成了一个转化到大肠杆菌中的大肠杆菌质粒基因组文库。这些转化体使用头孢曲松、庆大霉素、左氧氟沙星、四环素或甲氧苄啶的 0.25×至 2×最小抑菌浓度逐步选择。在每个选择步骤中分离质粒，并进行 Illumina 测序。通过搜索随着抗生素压力测序覆盖度增加的基因组基因座，我们能够检测到至少被一种抗生素富集的 48 个不同的基因组基因座。其中 15 个基因座进行了功能研究，我们表明当过度表达时，其中 13 个基因座可以降低大肠杆菌对抗生素的敏感性。这些基因编码药物靶点、转录因子、膜蛋白和耐药因子。Plas-Seq 技术正在加速发现与抗生素作用模式或耐药性相关的基因，并导致了一个影响药物敏感性的新基因的分离。它有可能应用于新的分子和其他微生物物种。
