Genetic approaches to uncover gene products involved in iron-sulfur protein maturation.
Genetic approaches to uncover gene products involved in iron-sulfur protein maturation.
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揭示与铁硫蛋白成熟有关的基因产物的遗传学方法。
DOI：
10.1007/978-1-0716-1605-5_3
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发表时间：
2021
影响因子：
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通讯作者：
Valerie Carabetta, Karla Esquilin-Lebron
中科院分区：
文献类型：
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作者：
Valerie Carabetta, Karla Esquilin-Lebron
铁硫（Fe-S）簇合物是自然界中最普遍和最通用的辅基之一。Fe-S簇有助于进行氧化还原反应、碳活化和环境传感。本章介绍了一个概述的遗传方法，已被用于识别和表征参与铁-S蛋白组装的细菌因子。传统的遗传筛选，评估生存力或条件营养缺陷型和生物信息学方法已经确定了大多数所述的基因用于Fe-S蛋白组装。在本文中，我们通过详细说明转座子测序（TnSeq）的使用来确定需要Fe-S酶的代谢途径的正常功能所必需的基因产物，从而扩展了这一遗传方法列表。TnSeq利用基因组学和大规模并行DNA测序的力量，使研究人员能够量化特定生长条件下单个基因产物的必要性。这允许鉴定在给定代谢过程中具有作用但对于该过程不是必需的基因产物或基因网络。这种方法的一个优点是，它允许研究人员鉴定具有部分表型的突变体，这些表型通常使用传统的基于平板的选择而被遗漏。应用TnSeq来解决Fe-S蛋白成熟的问题将导致对Fe-S生物发生和Fe-S蛋白组装中使用的遗传相互作用和因素的更全面的理解。
Iron-sulfur (Fe-S) clusters are one of the most ubiquitous and versatile prosthetic groups exploited by nature. Fe-S clusters aid in conducting redox reactions, carbon activation, and environmental sensing. This chapter presents an overview of the genetic approaches that have been useful for identifying and characterizing bacterial factors involved in Fe-S protein assembly. Traditional genetic screens that assess viability or conditional auxotrophies and bioinformatic approaches have identified the majority of the described genes utilized for Fe-S protein assembly. Herein, we expand upon this list of genetic methods by detailing the use of transposon sequencing (TnSeq) to identify gene products that are necessary for the proper function of metabolic pathways that require Fe-S enzymes. TnSeq utilizes the power of genomics and massively parallel DNA sequencing to allow researchers to quantify the necessity of individual gene products for a specific growth condition. This allows for the identification of gene products or gene networks that have a role in a given metabolic process but are not essential for the process. An advantage of this approach is that it allows researchers to identify mutants that have partial phenotypes that are often missed using traditional plate-based selections. Applying TnSeq to address questions of Fe-S protein maturation will result in a more comprehensive understanding of genetic interactions and factors utilized in Fe-S biogenesis and Fe-S protein assembly.