摘要

为了加快发现编码药物靶标或抗生素抗性的基因，我们开发了称为Plas-Seq的功能基因组策略。该技术涉及将多拷贝抑制子库与新一代测序相结合。我们产生了转化到大肠杆菌中的大肠杆菌质粒基因组文库。对于头孢曲松，庆大霉素，左氧氟沙星，四环素或甲氧苄啶，使用0.25×至2×最小抑制浓度逐步选择这些转化体。在每个选择步骤分离质粒并进行Illumina测序。通过寻找测序覆盖率随抗生素压力增加的基因组座位，我们能够检测到至少一种抗生素富集的48种不同的基因组座位。在功能上研究了这些基因座中的15个，并且我们表明13可以在过表达时降低大肠杆菌对抗生素的敏感性。这些基因编码用于药物靶标，转录因子，膜蛋白和抗性因子。 Plas-Seq技术正在加速发现与抗生素作用方式或抗性相关的基因，并导致分离出影响药物敏感性的新基因。它具有应用于新型分子和其他微生物物种的潜力。

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.

http://mgen.microbiologyresearch.org/content/journal/mgen/10.1099/mgen.0.000148