The Operon Encoding Hydrolytic Dehalogenation of 4-Chlorobenzoate Is Transcriptionally Regulated by the TetR-Type Repressor FcbR and Its Ligand 4-Chlorobenzoyl Coenzyme A

Abstract

The bacterial hydrolytic dehalogenation of 4-chlorobenzoate (4CBA) is a coenzyme 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 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 Comamonas sediminis strain CD-2. The fcbR knockout strain exhibited constitutive expression of the fcb cluster. In the host Escherichia coli, the expression of the P_fcb -fused green fluorescent protein (gfp) reporter was repressed by the introduction of the fcbR gene, and genetic studies combining various catabolic genes suggest that the ligand for FcbR may be an intermediate metabolite. Purified FcbR could bind to the P_fcb DNA probe in vitro, and the metabolite 4-chlorobenzyl-CoA (4CBA-CoA) prevented FcbR binding to the P _fcb DNA probe. Isothermal titration calorimetry (ITC) measurements showed that 4CBA-CoA could bind to FcbR at a 1:1 molar ratio. DNase I footprinting showed that FcbR protected a 42-bp DNA motif (5'-GGAAATCAATAGGTCCATAGAAAATCTATTGACTAATCGAAT-3') that consists of two sequence repeats containing four pseudopalindromic sequences (5'-TCNATNGA-3'). This binding motif overlaps with the -35 box of P_fcb and was proposed to prevent the binding of RNA polymerase. This study characterizes a transcriptional repressor of the fcb operon, together with its ligand, thus identifying halogenated benzoyl-CoA as belonging to the class of ligands of transcriptional regulators.IMPORTANCE The bacterial hydrolytic dehalogenation of 4CBA is a special CoA-activation-type catabolic pathway that plays an important role in the biodegradation of polychlorinated biphenyls and some 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.

Keywords: 4-chlorobenzoate; 4-chlorobenzoyl-CoA; FcbR; TetR-type transcriptional regulator; hydrolytic dehalogenation.

FIG 1

Organization and transcriptional analysis of the fcb cluster in strain CD-2. (A) Steps of the 4CBA dehalogenation pathway. (B) Schematic diagram of the fcb cluster. The P_fcb promoter is indicated by an arrow. The five amplification fragments (F1 to F5) for transcriptional unit evaluation are shown under the fcb cluster as lines. The fcb clusters in Comamonas sp. strain DJ12, Acidovorax sp. strain T1, Alcaligenes sp. strain AL3007, an uncultured bacterial clone, a consortium cosmid clone, Pseudomonas sp. strain CBS3, Arthrobacter globiformis strain KZT1, and Arthrobacter sp. strain TM1 are also presented, for which the GenBank accession numbers are AF0517717, MF189566, AF537222, KY207245, DQ826744, EF569604, AF304300, and AF042490, respectively. The identities of amino acids compared with those for strain CD-2 are shown for each gene. (C) TSS determination of the fcb cluster by 5′-RACE. The TSS C is indicated by an arrow. (D) TSS determination of fcbR by 5′-RACE. The TSS A is indicated by an arrow. (E) DNA elements and the location of the FcbR-binding site in the P_fcb promoter of the fcb gene cluster. The −35 box TTGACT and the −10 box TATAGT are indicated by lines, and the TSS is indicated with an arrow. The FcbR-binding site is indicated by a bold orange line above the sequence. (F) DNA elements in the P_fcbR promoter of the fcbR regulator gene itself. The −35 box GCGCCA and the −10 box TAGAAT are indicated by lines, and the TSS is indicated with an arrow. The proposed FcbR-binding site is indicated by a bold orange line above the sequence. (G) Transcription unit determination of the fcb cluster. Five fragments (F1 to F5), as shown in panel B, were PCR amplified and resolved by electrophoresis. Samples using total RNA, cDNA, and genomic DNA (gDNA) as the templates are indicated. Total RNA was extracted from strain CD-2 cells grown in MSM with 0.5 mM 4CBA as the carbon source, and cDNA was synthesized using random primers from the extracted total RNA.

FIG 2

Effects of fcbR on the dehalogenation of 4CBA, cell growth using 4CBA as the sole carbon source, and gene expression of the fcb operon. (A) 4CBA dehalogenation by the wild-type strain CD-2 (WT, blue), the fcbR knockout mutant (MT, orange), the fcbR-complemented strain (MTC, gray), and absent of bacterial cells (NCK, black). (B) Cell growth of strains WT, MT, and MTC on 4CBA. (C) Relative transcriptional expression analysis of fcbB, fcbA, fcbT1, fcbT2, fcbT3, and fcbC in the WT (blue), MT (orange), and MTC (gray) strains in the presence of 0.5 mM succinate or 0.5 mM 4CBA. (D) Relative transcriptional expression analysis of fcbR in the WT (blue) and MTC (gray) strains in the presence of 0.5 mM succinate or 0.5 mM 4CBA. Data shown are means ± standard deviation (SD) of three replicates. Asterisks indicate P values assessed by Student’s t test: ***, P < 0.001; NS, nonsignificant.

FIG 3

FcbR represses the expression of the P_fcb-gfp fusion in host E. coli cells. (A) GFP expression phenotype of E. coli DH5α colonies harboring different plasmids. (B) Quantitative measurement of GFP expression in E. coli DH5α colonies harboring different plasmids. pSRGFP-18, a pUC18-based gfp reporter plasmid containing two multiple cloning sites (60); pSR-R, pSRGFP-18 harboring fcbR; pSR-P, pSRGFP-18 harboring P_fcb upstream of gfp; pSR-PR, pSRGFP-18 harboring P_fcb upstream of gfp and the transcriptional regulator gene fcbR; pBAC, a BAC plasmid (61); pBAC-fcbT, pBAC harboring fcbT1T2T3; pBAC-fcbAT, pBAC harboring fcbAT1T2T3; pBAC-fcbBAT, pBAC harboring fcbBAT1T2T3; and pBAC-hbaA, pBAC harboring the predicted 4-hydroxybenzoate-CoA ligase gene hbaA. BLANK, cells cultured in LB; 4FBA, cells cultured in 0.5 mM 4FBA; 4CBA, cells cultured in 0.5 mM 4CBA; 4BBA, cells cultured in 0.5 mM 4BBA; 4IBA, cells cultured in 0.5 mM 4IBA; 4HBA, cells cultured in 0.5 mM 4HBA.

FIG 4

EMSA of FcbR and the P_fcb or P_fcbR probe and effects of 4CBA-CoA on their binding. (A) FcbR binds to the promoter DNA probe of the fcb operon and the effect of diverse molecules on FcbR-specific binding. Each lane contains 60 ng of DNA probe. The first 5 lanes show samples incubated with increasing amounts of FcbR (0 to 3.32 μM), and the next 6 lanes show samples incubated with 3.32 μM FcbR and different small molecules (5 mM), including 4CBA, 4CBA-CoA, 4HBA-CoA, 4HBA, CoA, and BA-CoA. (B) Effects of increasing 4CBA-CoA (0 to 32 μM) on the binding between 3.32 μM FcbR and the P_fcb DNA probe. S0 indicates the 198-bp negative-control DNA fragment that was amplified from the fcbA gene; S1 indicates the 241-bp P_fcb DNA probe; S2 indicates the complex of FcbR and the P_fcb DNA probe. (C) FcbR binds to the DNA probe of its own promoter. Each lane contains 60 ng of DNA probe and samples were incubated with increasing amounts of FcbR (0 to 5.81 μM). SR1 indicates the 249-bp P_fcbR DNA probe; SR2 indicates the complex of FcbR and the P_fcbR DNA probe. (D) ITC measurement of 4CBA-CoA binding to FcbR. The ITC raw data are shown on the top, and the fitted curve is shown on the bottom. A 500 μM 4CBA-CoA solution was titrated into the reaction cell containing 32 μM FcbR. The first drop was one-fourth of the volume of the subsequent drops, and it was not used in the curve fit.

FIG 5

The FcbR-binding site in P_fcb and ITC measurement of FcbR binding to its own DNA-binding site. (A) DNase I footprinting analysis of FcbR and P_fcb. 6-Carboxyfluorescein-labeled DNA probe (500 ng) was incubated with 0 or 5 μg FcbR (red and blue lines, respectively). The FcbR-protected region is indicated in a box, and the protected sequence is shown at the bottom. (B) Sequence alignment of FcbR-binding sites. The 42-bp FcbR-binding motif among strains CD-2, DJ12, T1, AL3007, an uncultured clone, a cosmid clone, and strain CBS-3 were selected for sequence alignment. The proposed FcbR-binding site in P_fcbR was also aligned and is shown under the figure; sequence exhibiting high similarity to repeat 1 is indicated by a black box. (C) The Logo of the FcbR-binding motif in seven samples was generated using WebLogo 2.8.2. Two sequence repeats in the motif are indicated by lines, and four palindromic sequences (PS1, PS2, PS3, and PS4) are indicated by arrow-headed lines. (D) ITC measurement of FcbR binding to the binding site-containing DNA probe. The 42-bp FcbR binding site-containing DNA (40 μM) was titrated into a reaction cell containing 23 μM FcbR. The first drop was one-fourth of the volume of the subsequent points, and it was not used in the curve fit.

FIG 6

Proposed transcriptional regulation model for FcbR. (I) FcbR represses the transcription of the fcb cluster in the absence of 4CBA in the medium. (II) 4CBA is transported into cells and converted to the ligand 4CBA-CoA by basal expression of FcbT and FcbA, respectively. (III) 4CBA-CoA binds to FcbR and releases FcbR from the promoter region of the fcb operon. The fcb gene cluster is then highly transcribed to produce more 4CBA transporters and dehalogenation enzymes.