The MarR-Type Regulator Rdh2R Regulates rdh Gene Transcription in Dehalococcoides mccartyi Strain CBDB1
The MarR-Type Regulator Rdh2R Regulates rdh Gene Transcription in Dehalococcoides mccartyi Strain CBDB1
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DOI：
10.1128/jb.00419-16
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发表时间：
2016-12-01
影响因子：
3.2
通讯作者：
Lechner, Ute
中科院分区：
文献类型：
--
作者：
Krasper, Lydia;Lilie, Hauke;Lechner, Ute
还原性脱卤酶是有机卤化物呼吸中的必需酶，由催化亚基A和膜蛋白B组成，由rdhAB基因编码。Mccartyi脱卤球菌CBDB 1菌株基因组中存在32个rdhAB基因。为了获得对rdh操纵子的调控的第一个了解，使用异源表达和体外研究研究了直接上游编码的MarR型调节因子Rdh 2 R（cbdbA 1456）对两个rdhAB基因（cbdbA 1453/cbdbA 1452和cbdbA 1455/cbdbA 1454）的基因表达的控制。产生启动子-lacZ报告基因融合体并整合到大肠杆菌宿主的基因组中。两个rdhA启动子的lacZ报告活性降低后，转化的细胞与携带的rdh 2 R基因的质粒，表明Rdh 2 R作为阻遏物，而rdh 2 R启动子的lacZ报告活性不受影响。菌株CBDB 1和/或异源宿主中的两个rdhA基因的转录起始位点映射到具有11- 13-bp半位点的保守的直接重复。DNA酶I足迹显示Rdh 2 R与一个类似于30 bp的序列结合，该序列覆盖了两个启动子中的完整直接重复序列，包括转录起始位点。平衡沉降超电泳显示，Rdh 2 R结合作为四聚体的直接重复基序的rdhA（cbdbA 1455）启动子。使用电泳迁移率变动分析，发现两个rdhA启动子的类似的结合亲和力。在只有一个半站点的直接重复的存在下，相互作用强烈减少，这表明一个积极的协同性的结合，其中不寻常的短palinertium内的直接重复半sites可能发挥重要作用。IMPORTANCEDehalococcoides mccartyi菌株是专性厌氧菌生长的有机卤化物呼吸。它们具有重要的生物修复潜力，因为它们能够将大量卤代化合物还原为毒性较低的产品。我们现在开始了解这些生物体如何利用这种巨大的分解代谢潜力。McCartyi以化合物特异性方式表达脱卤酶。MarR型调节子通常在还原脱卤酶基因附近编码。在这项研究中，我们利用异源表达和体外研究，以证明马尔型转录因子Rdh 2 R作为一个负调节。我们确定其结合位点的DNA，这表明了一种机制，它控制两个相邻的还原脱卤酶操纵子的表达。
Reductive dehalogenases are essential enzymes in organohalide respiration and consist of a catalytic subunit A and a membrane protein B, encoded by rdhAB genes. Thirty-two rdhAB genes exist in the genome of Dehalococcoides mccartyi strain CBDB1. To gain a first insight into the regulation of rdh operons, the control of gene expression of two rdhAB genes (cbdbA1453/cbdbA1452 and cbdbA1455/cbdbA1454) by the MarR-type regulator Rdh2R (cbdbA1456) encoded directly upstream was studied using heterologous expression and in vitro studies. Promoter-lacZ reporter fusions were generated and integrated into the genome of the Escherichia coli host. The lacZ reporter activities of both rdhA promoters decreased upon transformation of the cells with a plasmid carrying the rdh2R gene, suggesting that Rdh2R acts as repressor, whereas the lacZ reporter activity of the rdh2R promoter was not affected. The transcriptional start sites of both rdhA genes in strain CBDB1 and/or the heterologous host mapped to a conserved direct repeat with 11- to 13-bp half-sites. DNase I footprinting revealed binding of Rdh2R to a similar to 30-bp sequence covering the complete direct repeat in both promoters, including the transcriptional start sites. Equilibrium sedimentation ultracentrifugation revealed that Rdh2R binds as tetramer to the direct-repeat motif of the rdhA (cbdbA1455) promoter. Using electrophoretic mobility shift assays, a similar binding affinity was found for both rdhA promoters. In the presence of only one half-site of the direct repeat, the interaction was strongly reduced, suggesting a positive cooperativity of binding, for which unusual short palindromes within the direct-repeat half-sites might play an important role.IMPORTANCEDehalococcoides mccartyi strains are obligate anaerobes that grow by organohalide respiration. They have an important bioremediation potential because they are capable of reducing a multitude of halogenated compounds to less toxic products. We are now beginning to understand how these organisms make use of this large catabolic potential, whereby D. mccartyi expresses dehalogenases in a compound-specific fashion. MarR-type regulators are often encoded in the vicinity of reductive dehalogenase genes. In this study, we made use of heterologous expression and in vitro studies to demonstrate that the MarR- type transcription factor Rdh2R acts as a negative regulator. We identify its binding site on the DNA, which suggests a mechanism by which it controls the expression of two adjacent reductive dehalogenase operons.