Quadram Institute Bioscience, Norwich Research Park, Norwich, NR4 7UQ, United Kingdom.
School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia 4072, Queensland, Australia.
Genome Res. 2020 Feb;30(2):239-249. doi: 10.1101/gr.254391.119. Epub 2020 Feb 12.
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.
理解表型的遗传基础是生物学研究的一个核心目标。通过创建大型突变文库并寻找条件重要基因，人们对细菌基因组有了很多了解。然而，当前的全基因组方法在很大程度上无法检测到不适于破坏的必需基因。为了克服这一限制，我们开发了一种新版本的“TraDIS”（转座子定向插入位点测序），我们称之为“TraDIS-Xpress”，它将诱导型启动子结合到转座子盒中。这允许通过饱和所有开放阅读框附近的插入物以及常规失活来控制所有基因的过表达和抑制。我们应用 TraDIS-Xpress 来确定对生物杀灭剂三氯生的一系列浓度的反应。三氯生在现代生活中普遍存在，但对其作用模式存在不确定性，其浓度依赖性从抑菌作用到杀菌作用的转变尚未得到解释。我们的结果显示，三氯生的浓度依赖性反应与静态和杀菌暴露之间生存相关的不同基因有关。这些基因包括先前报道的与三氯生抗性有关的基因以及一组新的基因，包括必需基因。被鉴定为对三氯生暴露敏感的新基因包括参与屏障功能、小分子摄取以及转录和翻译完整性的基因。我们预计，我们在这里展示的方法，通过允许在 TraDIS 数据的多个实验条件下进行比较，并包括必需基因，将成为未来研究不同药物条件如何影响细菌生存机制的起点。
