Zhang Hui, Yu Ting, Wang Yiran, Li Jie, Wang Guangli, Ma Yingqun, Liu Yu
College of Life Sciences, Huaibei Normal University, Huaibei, China.
Advanced Environmental Biotechnology Centre, Nanyang Environment & Water Research Institute, Nanyang Technological University, Singapore, Singapore.
Front Microbiol. 2018 Oct 23;9:2481. doi: 10.3389/fmicb.2018.02481. eCollection 2018.
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.
4-氯苯酚（4-CP）氧化在4-CP解毒过程中起着至关重要的作用。然而，尚未对4-CP在基因和生化水平上的氧化调控进行研究。为了探究4-CP氧化的调控机制，通过基因组步移从sp. 菌株YH-5B中鉴定并克隆了一个参与4-CP生物降解的新型基因簇。序列分析表明，该基因簇编码一个AraC型转录调节因子和一种双组分单加氧酶，而定量实时PCR分析进一步揭示，该基因簇组成型表达，并响应4-CP或苯酚正向调控基因的转录，基因敲除和互补实验证明了这一点。通过将突变的启动子与基因进行转录融合，发现在转录起始位点上游-35至-69处，CphR调节因子结合位点有两个15 bp的不完全直接重复序列（TGCA-N-GGNTA）。值得注意的是，-46至-49位置的亚基序在与CphR二聚体的适当相互作用中起关键作用。此外，经His标签亲和层析纯化的单加氧酶亚基CphA1和CphA2能够催化4-CP转化为4-氯邻苯二酚，这表明菌株YH-5B可通过4-氯邻苯二酚途径降解4-CP。本研究增进了我们对革兰氏阳性菌中4-CP氧化转录调控的遗传和生化多样性的理解。
