CAS Key Laboratory of Urban Pollutant Conversion, Department of Applied Chemistry, University of Science & Technology of China, Hefei, 230026, P. R. China.
School of Life Sciences, University of Science & Technology of China, Hefei, 230026, P. R. China.
Environ Sci Technol. 2020 Mar 17;54(6):3306-3315. doi: 10.1021/acs.est.9b07191. Epub 2020 Mar 5.
species are indigenous in diverse aquatic environments and play important roles in environmental remediation. However, the pollutant transformation mechanisms of these bacteria remain elusive, and their potential in pollution control is largely unexploited so far. In this work, we report an efficient and simple genome regulation tool to edit and identify its biomolecular pathways for pollutant transformation. The genome regulation system, which is based on the type II clustered regularly interspaced short palindromic repeat interference (CRISPRi) system from , can serve as a reversible and multiplexible platform for gene knockdown in . A single-plasmid CRISPRi system harboring both dCas9 and the sgRNA was constructed in and used to silence diverse genes with varied sizes and expression levels. With this system, up to 467-fold repression of expression was achieved, and the function of the essential gene- was identified quickly and accurately. Furthermore, simultaneous transcriptional repression of multiple targeted genes was realized. We discovered that the operon played an essential role in arsenic detoxification, and the extracellular electron transfer (EET) pathway was involved in methyl orange reduction, but not in vanadium reduction by . Our method allows better insights and effective genetic manipulation of the pollutant transformation processes in , which might facilitate more efficient utilization of the species and other microbial species for environmental remediation applications.
这些细菌在环境修复中扮演着重要的角色。然而，它们的污染物转化机制仍然难以捉摸，其在污染控制中的潜力尚未得到充分利用。在这项工作中，我们报告了一种高效、简单的基因组调控工具，用于编辑和鉴定其污染物转化的生物分子途径。该基因组调控系统基于 型 II 类簇状规则间隔短回文重复干扰（CRISPRi）系统，可以作为 中基因敲低的可逆和多路复用平台。我们构建了一个单质粒 CRISPRi 系统，其中包含 dCas9 和 sgRNA，用于沉默具有不同大小和表达水平的多种基因。利用该系统，可实现高达 467 倍的 表达抑制，并且能够快速准确地鉴定必需基因 的功能。此外，还可以实现多个靶向基因的同时转录抑制。我们发现 操纵子在砷解毒中起着至关重要的作用，而细胞外电子转移（EET）途径参与了甲基橙的还原，但不参与 对钒的还原。我们的方法可以更好地了解和有效地对 中的污染物转化过程进行遗传操作，这可能有助于更有效地利用 种和其他微生物种进行环境修复应用。
