TraDIS-Xpress: a high-resolution whole-genome assay identifies novel mechanisms of triclosan action and resistance
TraDIS-Xpress: a high-resolution whole-genome assay identifies novel mechanisms of triclosan action and resistance
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DOI：
10.1101/gr.254391.119
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发表时间：
2020-02-01
期刊：
影响因子：
7
通讯作者：
Charles, Ian G.
中科院分区：
文献类型：
--
作者：
Yasir, Muhammad;Turner, A. Keith;Charles, Ian G.
了解表型的遗传基础是生物学研究的中心目标。通过建立大型突变文库和寻找有条件的重要基因，人们对细菌基因组有了很多了解。然而，目前的全基因组方法在很大程度上无法测定不可破坏的基本基因。为了克服这一限制，我们开发了一个新版本的“TraDIS”（转座子定向插入位点测序），我们称之为“TraDIS- xpress”，它将一个诱导启动子结合到转座子盒中。由于邻近所有开放阅读框的插入物饱和以及常规失活，这允许所有基因的可控过表达和抑制。我们应用TraDIS-Xpress来确定在不同浓度下对杀菌剂三氯生的反应。三氯生在现代生活中是一种地方性疾病，但其作用模式尚不确定，从抑菌作用到杀菌作用的浓度依赖性转换无法解释。我们的研究结果显示，在静态和杀虫暴露中，三氯生对不同基因的浓度依赖性反应对生存有重要影响。这些基因包括先前报道的在三氯生耐药性中起作用的基因，以及一组新的基因，包括必需基因。新发现的对三氯生暴露敏感的基因包括那些参与屏障功能、小分子摄取、转录和翻译完整性的基因。我们期待我们在这里展示的方法，通过允许对TraDIS数据的多种实验条件进行比较，包括必要的基因，将成为未来研究不同药物条件如何影响细菌生存机制的工作的起点。
Understanding the genetic basis for a phenotype is a central goal in biological research. Much has been learnt about bacterial genomes by creating large mutant libraries and looking for conditionally important genes. However, current genome-wide methods are largely unable to assay essential genes which are not amenable to disruption. To overcome this limitation, we developed a new version of "TraDIS" (transposon directed insertion-site sequencing) that we term "TraDIS-Xpress" that combines an inducible promoter into the transposon cassette. This allows controlled overexpression and repression of all genes owing to saturation of inserts adjacent to all open reading frames as well as conventional inactivation. We applied TraDIS-Xpress to identify responses to the biocide triclosan across a range of concentrations. Triclosan is endemic in modern life, but there is uncertainty about its mode of action with a concentration-dependent switch from bacteriostatic to bactericidal action unexplained. Our results show a concentration-dependent response to triclosan with different genes important in survival between static and cidal exposures. These genes include those previously reported to have a role in triclosan resistance as well as a new set of genes, including essential genes. Novel genes identified as being sensitive to triclosan exposure include those involved in barrier function, small molecule uptake, and integrity of transcription and translation. We anticipate the approach we show here, by allowing comparisons across multiple experimental conditions of TraDIS data, and including essential genes, will be a starting point for future work examining how different drug conditions impact bacterial survival mechanisms.