4-Chlorophenol Oxidation Depends on the Activation of an AraC-Type Transcriptional Regulator, CphR, in Rhodococcus sp. Strain YH-5B

Abstract

4-Chlorophenol (4-CP) oxidation plays an essential role in the detoxification of 4-CP. However, oxidative regulation of 4-CP at the genetic and biochemical levels has not yet been studied. To explore the regulation mechanism of 4-CP oxidation, a novel gene cluster, cphRA2A1, involved in biodegradation of 4-CP was identified and cloned from Rhodococcus sp. strain YH-5B by genome walking. The sequence analysis showed that the cphRA2A1 gene cluster encoded an AraC-type transcriptional regulator and a two-component monooxygenase enzyme, while quantitative real-time PCR analysis further revealed that cphR was constitutively expressed and positively regulated the transcription of cphA2A1 genes in response to 4-CP or phenol, as evidenced by gene knockout and complementation experiments. Through the transcriptional fusion of the mutated cphA2A1 promoter with the lacZ gene, it was found that the CphR regulator binding sites had two 15-bp imperfect direct repeats (TGCA-N₆-GGNTA) at -35 to -69 upstream of the cphA2A1 transcriptional start site. Notably, the sub-motifs at the -46 to -49 positions played a critical role in the appropriate interaction with the CphR dimer. In addition, it was confirmed that the monooxygenase subunits CphA1 and CphA2, which were purified by His-tag affinity chromatography, were able to catalyze the conversion of 4-CP to 4-chlorocatechol, suggesting that strain YH-5B could degrade 4-CP via the 4-chlorocatechol pathway. This study enhances our understanding of the genetic and biochemical diversity in the transcriptional regulation of 4-CP oxidation in Gram-positive bacteria.

Keywords: 4-chlorophenol degradation; 4-chlorophenol monooxygenase; AraC-type transcriptional regulator; Rhodococcus sp. strain YH-5B; gene cluster.

FIGURE 1

Scheme of the cphRA2A1 gene cluster and the intergenic region between cphR and cphA2 in Rhodococcus sp. strain YH-5B. The putative –10 box, –35 box, Shine-Dalgarno (SD) sequence, the proposed translational start codon ATG, and the determined transcriptional start sites (TSSs) of cphR (orange) and cphA2 (green) are in bold. The possible AraC-type CphR regulator binding sites (RBSs) (TGCA-N6-GGNTA) are shown by the two arrows and tandemly imperfect direct repeats are shaded in yellow.

FIGURE 2

Transcriptional analyses of cphA2A1 genes in the wild-type strain YH-5B (A) and the cphR knockout strain YH-5BΔcphR (B) in the presence (black) or absence (LT gray) of various phenolic compounds. Transcriptional analyses of cphA2A1 (C) and cphR (D) genes in the wild-type strain YH-5B and the cphR-complemented strain YH-5BΔcphRC in the presence (black) or absence (LT gray) of 4-CP. The transcription activities of gene tested in each sample were calculated as the fold ratio following normalization to that of 16S rRNA gene.

FIGURE 3

SDS-PAGE analyses of the purified H₆-CphA1 (lane 1), H₆-CphA2 (lane 2), and H₆-CphR (lane3). Protein molecular mass standards are indicated in lane M.

FIGURE 4

Multiple alignment of the putative regulation sequences in the intergenic region between cphR and cphA2 of Rhodococcus sp. strain YH-5B and other related strains. The identified transcriptional start sites (TSSs) are indicated by +1 in box. The proposed two tandemly imperfect direct repeats for the AraC-type regulators binding are represented by the two arrows, in which each sub-motif is boxed.

FIGURE 5

Determination of the promoter activities of the cphA2A1 genes. The β-galactosidase activities were measured in the strain JT-3 carrying each pER-lacZ derived plasmid bearing mutated promoter and pKcphRC expressing CphR in the presence (black) or absence (LT gray) of 4-CP.

FIGURE 6

HPLC and LC-MS spectrum of the 4-CP conversion catalyzed by the two-component 4-CP monooxygenase consisting of H₆-CphA1 and H₆-CphA2. (A,B) HPLC of the 4-CP conversion and the standard 4-CC, respectively; (C,D) First and second-order mass spectrum of the 4-CP conversion, respectively.