Willemin Mathilde Stéphanie, Vingerhoets Marie, Holliger Christof, Maillard Julien
Laboratory for Environmental Biotechnology, School of Architecture, Civil and Environmental Engineering, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.
Front Microbiol. 2020 Mar 3;11:310. doi: 10.3389/fmicb.2020.00310. eCollection 2020.
The bioremediation of persistent organohalide molecules under anoxic conditions mostly relies on the bacterial process called organohalide respiration (OHR). Organohalide-respiring bacteria (OHRB) are phylogenetically diverse anaerobic bacteria that share the capacity to use organohalides as terminal electron acceptors in an energy-conserving process. The reductive dehalogenase () gene clusters encode for proteins specialized in the respiration of one or a limited number of organohalides. One particular OHRB may harbor up to several dozens of gene clusters suggesting a wide potential for bioremediation. To avoid wasting energy in producing unnecessary proteins, gene clusters often include a transcriptional regulator. In organohalide-respiring , RdhK is a dedicated transcriptional regulator of OHR and represents a subfamily of proteins among the CRP/FNR superfamily of regulators. RdhK proteins are composed of an effector-binding domain (EBD) which recognizes a given organohalide and subsequently controls the interaction of its C-terminal DNA-binding domain (DBD) with a DNA motif (referred to as dehalobox, or DB) located in the promoter region of the target genes. The two binding partners (i.e. an organohalide molecule and a DB sequence) of RdhK proteins are interdependent which impairs the exploration of OHR regulatory networks. Here, we propose a strategy relying on hybrid proteins to efficiently screen the DNA target of a single RdhK protein without prior knowledge on its effector. To demonstrate the potential of the method, two hybrids with alternative fusion points were designed based on RdhK6 EBD and RdhK1 DBD from . Electrophoretic mobility shift assay was performed with purified hybrids along with the parental proteins and their binding properties were further tested through a β-galactosidase reporter assay. Along with revealing new RdhK6 features, we show that both hybrids resulted in active regulatory proteins with distinct binding patterns. While Hybrid A was less specific for the DNA motif, Hybrid B successfully mimicked the binding behavior of the parental proteins and thus represents a promising template for the design of new RdhK hybrids to screen yet uncharacterized RdhK proteins and also possibly other members of the CRP/FNR superfamily.
在缺氧条件下，持久性有机卤化物分子的生物修复主要依赖于一种名为有机卤化物呼吸作用（OHR）的细菌过程。有机卤化物呼吸细菌（OHRB）是系统发育上多样化的厌氧细菌，它们具有在能量守恒过程中利用有机卤化物作为末端电子受体的能力。还原性脱卤酶（）基因簇编码专门用于一种或有限数量有机卤化物呼吸作用的蛋白质。一种特定的OHRB可能含有多达几十种基因簇，这表明其在生物修复方面具有广阔的潜力。为避免在产生不必要的蛋白质上浪费能量，基因簇通常包含一个转录调节因子。在有机卤化物呼吸作用中，RdhK是OHR的一个专门转录调节因子，属于CRP/FNR调节因子超家族中的一个蛋白质亚家族。RdhK蛋白由一个效应物结合结构域（EBD）组成，该结构域识别特定的有机卤化物，随后控制其C端DNA结合结构域（DBD）与位于目标基因启动子区域的一个DNA基序（称为脱卤盒，或DB）的相互作用。RdhK蛋白的两个结合伙伴（即一个有机卤化物分子和一个DB序列）相互依赖，这妨碍了对OHR调节网络的探索。在此，我们提出一种基于杂交蛋白的策略，无需事先了解其效应物，就能有效地筛选单个RdhK蛋白的DNA靶点。为证明该方法的潜力，基于来自的RdhK6 EBD和RdhK1 DBD设计了两种具有不同融合点的杂交体。用纯化的杂交体以及亲本蛋白进行了电泳迁移率变动分析，并通过β-半乳糖苷酶报告基因分析进一步测试了它们的结合特性。除揭示RdhK6的新特性外，我们还表明这两种杂交体都产生了具有不同结合模式的活性调节蛋白。虽然杂交体A对DNA基序的特异性较低，但杂交体B成功地模拟了亲本蛋白的结合行为，因此代表了一种有前景的模板，可用于设计新的RdhK杂交体，以筛选尚未表征的RdhK蛋白，也可能用于筛选CRP/FNR超家族的其他成员。
