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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 ( rdh ) 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  rdh  gene clusters suggesting a wide potential for bioremediation. To avoid wasting energy in producing unnecessary proteins,  rdh  gene clusters often include a transcriptional regulator. In organohalide-respiring  Firmicutes , 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  rdh  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  Desulfitobacterium hafniense . Electrophoretic mobility shift assay was performed with purified hybrids along with the parental proteins and their binding properties were further tested  in vivo  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.

Hybrid Transcriptional Regulators for the Screening of Target DNA Motifs in Organohalide-Respiring Bacteria