Transposon-insertion sequencing in a clinical isolate of Legionella pneumophila to identify essential genes and determinants of natural transformation. Tn-seq of Legionella pneumophila HL77S

Transposon-insertion sequencing (TIS) is an increasingly common component of the toolkit of microbiologists to explore the genotype-phenotype relationship in bacteria. In addition to its use in identification of the genetic determinants of selectable phenotypes, TIS can also identify fitness determinants and essential genes, possible antibiotic targets. The power of TIS to identify genetic determinants of growth and/or of specific functions is largely dependent on the complexity of the transposon insertion library that can be obtained. We here sought to obtain a high-saturation transposon insertion library for Legionella pneumophila, a human pathogen for which TIS has not yet been used to its full power. We obtained high-saturation libraries in the non-ST1 clinical isolate HL77S. We therefore identified essential genes in L. pneumophila, and also revealed that TIS could be used to identify genes controlling vertical transmission of mobile genetic elements. We then applied TIS to identified all genes required for L. pneumophila to develop competence and undergo natural transformation, defining the set of major and minor Type IV pilins that are engaged in DNA uptake. This work paves the way for the functional exploration of the L. pneumophila genome and the identification of the genetic basis other life traits of this species.

转座子插入测序（Transposon-insertion sequencing, TIS）现已日益成为微生物学家探究细菌基因型-表型关系的常用研究工具之一。除用于鉴定可选择表型的遗传决定因子外，TIS还可鉴定适合度决定因子与必需基因，后者或可成为抗生素靶点。TIS鉴定生长及/或特定功能相关遗传决定因子的能力，在很大程度上取决于所获得的转座子插入文库的复杂度。本研究旨在为嗜肺军团菌（Legionella pneumophila）构建高饱和度转座子插入文库——该人类病原体此前尚未被充分利用TIS技术开展研究。我们成功在非ST1型临床分离株HL77S中构建了高饱和度文库。借此，我们不仅鉴定出嗜肺军团菌的必需基因，还证实TIS可用于鉴定控制可移动遗传元件垂直传播的基因。随后，我们利用TIS技术筛选出嗜肺军团菌建立感受态并进行自然转化所需的全部基因，明确了参与DNA摄取的主要与次要IV型菌毛蛋白亚组。本研究为嗜肺军团菌基因组的功能探索以及该物种其他生命性状的遗传基础鉴定铺平了道路。