The TroR transcription factor regulates iron homeostasis in the halophilic archaeon Haloferax volcanii [RNA-Seq]

Iron is essential for almost all organisms, but can be toxic in excess. Cells use regulatory mechanisms to control the iron uptake, iron utilization and iron release mechanisms to ensure the availability of enough iron to be used for biological processess but minimizing the oxidative stress caused by the oxidation of free molecules of iron. In this paper we show that TroR is a transcription factor from the conserved family of proteins DtxR that works as the major regulator of iron homeostasis in the halophilic archaeon Haloferax volcanii. Using RNA seq, we compared the trancriptome of cells from parental strain versus the troR knockout strain, growing in minimal media containing iron in excess to understand if the TF TroR was involved in those cellular mechanims that determine iron homeostasis. RNA was checked for quality, and 300-500 micrograms of total RNA were depleted using RIBOZERO to remove rRNA.Libraries were constructed using ~50 ng of mRNA using the Kapa Stranded RNA-Seq Library Preparation Kit (KAPA Biosystems, Illumina).

铁几乎是所有生物体不可或缺的营养物质，但过量存在时会产生毒性。细胞通过调控机制管控铁摄取、铁利用与铁释放过程，以保障生物过程所需铁元素的充足供应，同时最大限度降低因游离铁分子氧化所引发的氧化应激。本研究证实，TroR是一类属于保守蛋白家族DtxR的转录因子（transcription factor），在嗜盐古生菌沃氏嗜盐盒菌（*Haloferax volcanii*）中作为铁稳态（iron homeostasis）的主要调控因子。本研究借助RNA测序（RNA-seq）技术，以过量含铁的基础培养基培养亲本菌株与troR敲除菌株，对二者的转录组（transcriptome）进行比较分析，以探究转录因子TroR是否参与调控铁稳态相关的细胞机制。对提取的总RNA进行质量检测后，使用RIBOZERO试剂去除300~500微克总RNA中的核糖体RNA（rRNA）。随后使用约50纳克的信使RNA（mRNA），借助Kapa链特异性RNA-seq文库制备试剂盒（KAPA Biosystems, Illumina）构建测序文库。