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The bacterial hydrolytic dehalogenation of 4-chlorobenzoate (4CBA) is a CoA-activation type catabolic pathway that is usually a common part of the microbial mineralization of chlorinated aromatic compounds. Previous studies have shown that the transport and dehalogenation genes for 4CBA are typically clustered as an fcbBAT1T2T3C operon and are inducibly expressed in response to 4CBA. However, the associated molecular mechanism remains unknown. In this study, a gene (fcbR) adjacent to the fcb operon was predicted to encode a TetR-type transcriptional regulator in strain Comamonas sediminis CD-2. The fcbR knockout strain exhibited constitutive expression of the fcb cluster. In the host E. coli, the expression of the Pfcb-fused gfp reporter was repressed by the introduction of the fcbR gene, and genetic studies combining various catabolic genes suggest that the FcbR ligand may be an intermediate metabolite. Purified FcbR could bind to the Pfcb DNA probe in vitro, and the metabolite 4-chlorobenzyl-CoA (4CBA-CoA) prevented FcbR binding to the Pfcb DNA probe. Isothermal titration calorimetry (ITC) measurements showed that 4CBA-CoA could bind to FcbR at a 1:1 mole ratio. DNase I footprinting showed that FcbR protected a 42-bp DNA motif (5′-GGAAATCAATAGGTCCATAGAAAATCTATTGACTAATCGAAT-3′) that consists of two sequence repeats containing four pseudo-palindromic sequences (5’-TCNATNGA-3’). This binding motif overlaps with the -35 box of Pfcb and was proposed to prevent the binding of RNA polymerase. This study identified a transcriptional repressor and its ligand of the fcb operon, extending halogenated benzoyl-CoA as a member of known ligands of transcriptional regulators. Importance The bacterial hydrolytic dehalogenation of 4CBA is a special CoA-activation type catabolic pathway, which plays an important role in the biodegradation of polychlorinated biphenyls and many certain herbicides. With genetic and biochemical approaches, the present study identified the transcriptional repressor and its cognate effector of a 4CBA hydrolytic dehalogenation operon. This work extends halogenated benzoyl-CoA as a new member of CoA-derived effector compounds that mediate allosteric regulation of transcriptional regulators.

Due to their key role in methane cycling and their industrial potential, methanotrophs have drawn increasing attention. Genome-wide experimental approaches for gene-phenotype mapping accelerate our understanding and engineering of a bacterium.