Functional Analysis Of A Regulatory Gene, Mphr, Involved In Phenol Degradation Of Acinetobacter Calcoaceticus PHEA-2

Phenol is a harmful aromatic compound that widely contaminates the environment due to industrial activities. Thus, there is research worldwide concerning the biodegradation and regulation mechanisms of phenol.In this study, the regulatory gene of phenol hydroxylase in Acinetobacter calcoaceticus PHEA-2 was cloned. Sequence analysis shows that the deduced amino acid sequence predict a XylR/DmpR-type regulator involved in the transcription of the operon mphKLMNOP, which is 69.11% identical to the MopR in A. calcoaceticus NCIB8250. The mphR is 1671bp in length and encodes a protein consisting of 556 amino acids with a predicted molecular mass of 63 kDa. Transcriptional assays for mphK revealed that mphR activated mphKLMNOP transcription in the presence of phenol. After purification of MphR, the protein was crystallized using the sitting-drop vapour-diffusion technique.Aσ70-type promoter sequence for mphR (PmphR) and a -24/-12 promoter (σ54-type promoter) sequence for mphK (PmphK) are found in the intergenic region between mphR and mphK. The region contains a putative upstream activating site (UAS) consisting of three inverted repeat regions (IR1, IR2, and IR3) and a putative integration host factor (IHF) binding site just upstream of PmphK. In addition, the transcriptional start site of mphK was determined at 40 bp upstream of the start codon of mphK by primer extension. An Escherichia coli bioreporter incorporating the truncated mphK promoter (PmphK) of A. calcoaceticus PHEA-2 fused to aβ-galactosidase gene (lacZ) and the regulatory gene, mphR, was constructed. Deletion analysis of PmphK revealed that IR2 and IR3 were essential for the promoter's activity while IR1 was involved in transcriptional repression. The sensitivity of the bioreporter for the detection of phenol (0.1-5μM) was increased by approximately 100% with the deletion of IR1 in PmphK. Transcriptional analysis using PmphK::lacZ fusions revealed that, of the 21 tested compounds, phenol,ο-cresol, m-cresol and 2-chlorophenol were amongst the 9 kinds of phenol derivates which stimulated the transcription of mphK when mphR was expressed. MphR is constitutively expressed from aσ70-type promoter and partially enhances its own expression. Gel mobility shift assay confirmed the interaction between His-MphR and the promoter of the mphK gene. MphR can bind separately to all three IR sequence of PmphK with IR2 showing the strongest binding. Our results demonstrate that MphR is necessary for the transcriptional activation of the phenol degradation mphKLMNOP gene cluster in A. calcoaceticus PHEA-2.