Explore RdhK based regulatory network of organohalide respiration using a hybrid proteins strategy

Reductive dehalogenase (rdh) gene clusters are encoding proteins that enable organohalide respiring bacteria (OHRB) to couple the degradation of halogenated molecules to energy conservation. The transcription of rdh gene clusters is regulated by RdhK regulators belonging to the CRP/FNR-family. RdhK6 (previously called CprK1) in Desulfitobacterium hafniense was shown to activate the transcription of the chlorophenol rdh genes in presence of 3-hydroxy-4-chlorophenylacetate1,2. RdhK effector-binding domain binds to organohalides which triggers protein conformational change and allows the interaction with specific DNA motifs (dehalobox, DB) upstream of rdh gene clusters3,4. The genome of Dehalobacter restrictus PER-K23 encodes 24 rdh gene clusters, suggesting a great OHR potential. Each cluster has a rdhK paralogue in close proximity5. The elucidation of the regulation network represents an indirect way to reveal yet unknown substrates for D. restrictus. However, the challenge resides in the fact that for each new RdhK, there are a large number of potential organohalides and possible DB sequences, resulting in a high amount of combinations to be tested. This project aims to develop a strategy involving RdhK hybrid proteins to screen for DB and organohalides individually. The hybrids are composed by one domain (i.e. DNA- or effector-binding domain) of D. hafniense RdhK6 and the complementary domain from any RdhK of interest. The proof of concept as well as strategy limitations and alternatives will be discussed. References 1. Kim et al. (2012). BMC Microbiol. 12:21. 2. Gábor et al. (2006). J Bacteriol. 188:2604. 3. Joyce et al. (2006). J Biol Chem. 281:28318. 4. Levy et al. (2008). Mol Microbiol. 70:151. 5. Rupakula et al. (2013). Philos Trans R Soc Lond B Biol Sci. 368(20120325):1.