Molecular Characterization Of Catabolism Of Para-Nitrophenol In Pseudomonas Sp.1-7

A former identified para-Nitrophenol (PNP) degradation bacterial strain, Pseudomonas sp.1-7, was carried out to study in this work. The morphology, physiological and biochemical properties studies revealed that this strain was a Gram negative bacterium. Its optimal culture temperature was30℃, had resistance to Ampicillin and Chloromycetin, and could use PNP as the sole carbon, nitrogen and energy resources. Two different intermediates, hydroquinone (HQ) and4-nitrocatechol (4-NC) were detected in the catabolism of PNP in the strain1-7using the High Performance Liquid Chromatography (HPLC) and LC-MS analysis. It revealed that Pseudomonas sp.1-7degraded PNP by two different pathways, the HQ pathway, and the hydroxyquinol (BT) pathway. Through the construction of strain1-7genome fosmid library and the detection of the positive fosmid library clone (4-2M) nucleotide sequence, a12.3kb DNA fragment was cloned and named Clusterl. BLAST analysis revealed that there are7genes (pdcEDGFCBA) involved in PNP degradation in Clusterl. These genes were expressed in E. coli and followed the enzyme activity assays in vitro. The results revealed that the gene pdcA encodes PNP4-monooxygenase, which takes part in the initial reaction in PNP degradation, catalyzes PNP into p-benzoquinone (BQ); pdcB encodes BQ reductase which catalyzes BQ into HQ; pdcD and pdcE encode a two component HQ1,2-dioxygenase which catalyzes HQ to4-hydroxymuconic semialdehyde (4-HS); the gene pdcG encodes4-HS dehydrogenase which dehydrogenates4-HS forming maleylacetate (MA) and pdcF encodes MA reductase which catalyzes MA to β-ketoadipate. Additionally, the gene pdcC encoded a BT1,2-dioxygenase which catalyzes the BT into MA involved in the BT pathway. RT-PCR and quantitative PCR results indicated that the pdcEDGFCBA expression is induced by PNP. To clone the PNP degradation regulator genes, through the method of homology-based cloning, another PNP degradation gene cluster was cloned from strain1-7, and named Cluster2. Cluster2is5.06kb long and inclueds three PNP related genes:pdcR1A1R2. The gene pdcRl and pdcR2encode two PNP degradation regulators, and pdcA1encoded another PNP4-monooxygenase, which takes part in the PNP degradation. RT-PCR results revealed that there are6promoters (P1, P2m, P2f, P5m, P5r and P6) related in PNP degradation in Clusterl and Cluster2. The interaction studies between two regulators (PdcRl and PdcR2) and6promoters were carried out by Bacterial one-hybird system and Electrophoretic Mobility Shift Assay. The results revealed that PNP was important for the interaction between regulators and promoters, and when PNP existed in the systems, PdcRl only could interact with P1and P2m, but PdcR2could interact with5of6promoters (P1, P2m, P2f, P5m andP5r). These results indicated that PdcR2is a more important regulator than PdcRl in PNP degradation in strain1-7. But the exact regulate mechanism needs further study.