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

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, , involved in biodegradation of 4-CP was identified and cloned from sp. strain YH-5B by genome walking. The sequence analysis showed that the gene cluster encoded an AraC-type transcriptional regulator and a two-component monooxygenase enzyme, while quantitative real-time PCR analysis further revealed that was constitutively expressed and positively regulated the transcription of genes in response to 4-CP or phenol, as evidenced by gene knockout and complementation experiments. Through the transcriptional fusion of the mutated promoter with the gene, it was found that the CphR regulator binding sites had two 15-bp imperfect direct repeats (TGCA-N-GGNTA) at -35 to -69 upstream of the 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.