Burger Brian T, Imam Saheed, Scarborough Matthew J, Noguera Daniel R, Donohue Timothy J
Great Lakes Bioenergy Research Center, Wisconsin Energy Institute, University of Wisconsin, Madison, Wisconsin, USA.
Department of Bacteriology, University of Wisconsin, Madison, Wisconsin, USA.
mSystems. 2017 Jun 6;2(3). doi: 10.1128/mSystems.00015-17. eCollection 2017 May-Jun.
is one of the best-studied alphaproteobacteria from biochemical, genetic, and genomic perspectives. To gain a better systems-level understanding of this organism, we generated a large transposon mutant library and used transposon sequencing (Tn-seq) to identify genes that are essential under several growth conditions. Using newly developed Tn-seq analysis software (TSAS), we identified 493 genes as essential for aerobic growth on a rich medium. We then used the mutant library to identify conditionally essential genes under two laboratory growth conditions, identifying 85 additional genes required for aerobic growth in a minimal medium and 31 additional genes required for photosynthetic growth. In all instances, our analyses confirmed essentiality for many known genes and identified genes not previously considered to be essential. We used the resulting Tn-seq data to refine and improve a genome-scale metabolic network model (GEM) for . Together, we demonstrate how genetic, genomic, and computational approaches can be combined to obtain a systems-level understanding of the genetic framework underlying metabolic diversity in bacterial species. Knowledge about the role of genes under a particular growth condition is required for a holistic understanding of a bacterial cell and has implications for health, agriculture, and biotechnology. We developed the Tn-seq analysis software (TSAS) package to provide a flexible and statistically rigorous workflow for the high-throughput analysis of insertion mutant libraries, advanced the knowledge of gene essentiality in , and illustrated how Tn-seq data can be used to more accurately identify genes that play important roles in metabolism and other processes that are essential for cellular survival.
从生化、遗传和基因组学角度来看，它是研究最深入的α-变形菌之一。为了从系统层面更好地了解这种生物体，我们构建了一个大型转座子突变体文库，并使用转座子测序（Tn-seq）来鉴定在多种生长条件下必需的基因。利用新开发的Tn-seq分析软件（TSAS），我们鉴定出493个基因对于在丰富培养基上有氧生长是必需的。然后，我们利用该突变体文库鉴定在两种实验室生长条件下的条件必需基因，又鉴定出85个在基本培养基中进行有氧生长所需的额外基因以及31个光合生长所需的额外基因。在所有情况下，我们的分析都证实了许多已知基因的必需性，并鉴定出了以前未被认为是必需的基因。我们利用所得的Tn-seq数据来完善和改进一个针对[具体物种]的基因组规模代谢网络模型（GEM）。我们共同展示了如何将遗传、基因组和计算方法结合起来，以从系统层面理解细菌物种代谢多样性背后的遗传框架。对于全面理解细菌细胞而言，了解特定生长条件下基因的作用是必要的，这对健康、农业和生物技术都有影响。我们开发了Tn-seq分析软件（TSAS）包，为插入突变体文库的高通量分析提供了一个灵活且统计严谨的工作流程，推进了对[具体物种]中基因必需性的认识，并说明了如何利用Tn-seq数据更准确地鉴定在代谢及其他对细胞存活至关重要的过程中发挥重要作用的基因。
