Hebdon Skyler D, Gerritsen Alida T, Chen Yi-Pei, Marcano Joan G, Chou Katherine J
Biosciences Center, National Renewable Energy Laboratory, Golden, CO, United States.
Computational Sciences Center, National Renewable Energy Laboratory, Golden, CO, United States.
Front Microbiol. 2021 Sep 7;12:695517. doi: 10.3389/fmicb.2021.695517. eCollection 2021.
is a thermophilic bacterium recognized for its natural ability to effectively deconstruct cellulosic biomass. While there is a large body of studies on the genetic engineering of this bacterium and its physiology to-date, there is limited knowledge in the transcriptional regulation in this organism and thermophilic bacteria in general. The study herein is the first report of a large-scale application of DNA-affinity purification sequencing (DAP-seq) to transcription factors (TFs) from a bacterium. We applied DAP-seq to > 90 TFs in and detected genome-wide binding sites for 11 of them. We then compiled and aligned DNA binding sequences from these TFs to deduce the primary DNA-binding sequence motifs for each TF. These binding motifs are further validated with electrophoretic mobility shift assay (EMSA) and are used to identify individual TFs' regulatory targets in . Our results led to the discovery of novel, uncharacterized TFs as well as homologues of previously studied TFs including RexA-, LexA-, and LacI-type TFs. We then used these data to reconstruct gene regulatory networks for the 11 TFs individually, which resulted in a global network encompassing the TFs with some interconnections. As gene regulation governs and constrains how bacteria behave, our findings shed light on the roles of TFs delineated by their regulons, and potentially provides a means to enable rational, advanced genetic engineering of and other organisms alike toward a desired phenotype.
是一种嗜热细菌，因其具有有效解构纤维素生物质的天然能力而闻名。尽管迄今为止已有大量关于该细菌的基因工程及其生理学的研究，但对于该生物体以及一般嗜热细菌中的转录调控，人们了解有限。本文的研究是首次大规模应用DNA亲和纯化测序（DAP-seq）来研究细菌转录因子（TFs）的报告。我们对该细菌中的90多个TFs应用了DAP-seq，并检测到其中11个TFs的全基因组结合位点。然后，我们整理并比对了这些TFs的DNA结合序列，以推断每个TF的主要DNA结合序列基序。这些结合基序通过电泳迁移率变动分析（EMSA）进一步验证，并用于识别该细菌中各个TFs的调控靶点。我们的结果导致发现了新的、未表征的TFs以及先前研究过的TFs的同源物，包括RexA -、LexA -和LacI型TFs。然后，我们利用这些数据分别重建了11个TFs的基因调控网络，结果形成了一个包含这些TFs且有一些相互连接的全局网络。由于基因调控决定并限制了细菌的行为方式，我们的发现揭示了由其调控子描绘的TFs的作用，并有可能提供一种手段，使该细菌和其他生物体能够朝着期望的表型进行合理、先进的基因工程改造。
