The PucR-type activator IppR regulates 2-isopropylphenol hydroxylation in strain Rhodococcus sp. D-6

Although genes involved in isoprocarb (IPC) degradation, including the IPC hydrolase gene ipcH and the 2-isopropylphenol (IPP) hydroxylation gene cluster ippA1A2 , have been identified in Rhodococcus sp. D-6, the transcriptional regulatory mechanism remains unclear. In this study, we characterized a PucR-type transcriptional regulator, IppR, which controls the inducible expression of the ippA1A2 cluster. Gene disruption and complementation analyses revealed that ippR was essential for activation of ippA1A2 expression in response to IPP in strain D-6. Quantitative reverse transcription-PCR (qRT-PCR) and promoter activity assays demonstrated that IPP was the effector molecule of IppR. Electrophoretic mobility shift assay (EMSA) showed that IppR bound to a 24-bp motif in the promoter region, with the CC/CG box within this motif identified as the critical site for IppR binding. Moreover, IppR residues likely involved in IPP binding include Val188, Leu192, Val218, Trp257, Trp259, His297, and Ala300, along with Lys382 and Thr384, which are predicted to be essential for DNA recognition. This study elucidates the regulatory mechanism of IPC degradation in strain D-6, and the identification of IppR expands the diversity of regulatory models within the PucR-type regulator.