Cloning,Characterization And Regulation Of A Gene Cluster Involved In The Catabolism Of P-Nitrophenol From Pseudomonas Putida DLL-E4

p-Nitrophenol (PNP) is widely used in the manufacture of pesticides,dyes, explosives, and drug intermediates. PNP can also accumulate in soil as a result of the hydrolysis of several organophosphorous insecticides, such as parathion or methyl parathion. Due to its potential toxicity and persistence in the environment, PNP was listed on the "Priority Pollutants List"by US EPA.Pseudomonas putida DLL-E4has the ability to grow on PNP as the sole source of carbon, nitrogen, and energy, and hydroquinone was detected as the key intermediate in PNP degradation. In this study, we cloned a pnp gene cluster involved in the catabolism of PNP from DLL-E4. The functions of five genes in the cluster were verified by expression in E. coli and gene deletion in DLL-E4.A12,540bp DNA fragment around the conserved region was obtained after several rounds of SEFA PCR. Ten open reading frames (ORFs) were found in the fragment and they were annotated on the basis of BLAST analysis. The10ORFs can be divided into three groups by functional analysis. The pnpC1C2DE genes were proposed to be involved in hydroquinone catabolism by DLL-E4. pnpA and pnpB were determined to encode PNP4-monooxygenase and p-benzoquinone reductase, they could oxidatively remove the nitro group from PNP. pnpR was located at the beginning of the hydroquinone degradation gene cluster of DLL-E4and proposed to be encode a LysR regulatory protein. The12kb fragment named as pnp gene cluster, contained all essential genes involved in PNP degradation.PnpA was overexpressed in E. coli and purified to apparent homogeneity by Ni-NTA affinity chromatography and size exclusion Sephadex G-200column. PnpA is a flavin adenine dinucleotide-dependent single-component PNP4-monooxygenase that converts PNP to para-benzoquinone. The optimal reaction temperature of PnpA is20℃The optimal reaction pH of PnpA is8.0. Purified PnpA had a specific activity of2.57±0.04U mg-1for PNP. The Km value of PnpA for PNP was302μM. PnpA activity is NADH and FAD dependent. NADH can be replaced by NADPH with the lower efficiency. PnpA transformed4-NC with a lower specific activity (1.17±0.032U·mg-1) than that of PNP transformation. No activity was observed by PnpA on o-nitrophenol,m-nitrophenoland2,4-Dinitrophenol. Transition metal (Cu2+、Ni2+、Zn2+、Fe2+、Fe3+and Cr3+)inhibit the activity of PnpA and metal chelators(EDTA, EGTA,1,10-phenanthrolin)has no effect on PnpA activity, these results indicate that PnpA is non-metallic enzyme. To elucidate the phylogenetic relationships among the functionally identified nitroarene monooxygenases, PnpA and other flavoprotein monooxygenases were used to construct the distance neighbor-joining tree. The results indicated that PnpA belongs to a different group of flavin monooxygenases.PnpCl and PnpC2from the pnp gene cluster in DLL-E4are the oc-(PnpCl) and P-subunits (PnpC2) of hydroquinone dioxygenase. Purified PnpC1and PnpC2failed to reconstitute into active complex in vitro. Most of separately expressed PnpC2existed as inclusion bodies, and a mixture of separately expressed and purified PnpC1and PnpC2showed no activity on hydroquinone. A poly-cistronic plasmid pET-pnpC1C2containing pnpCl and pnpC2was constructed to express the dual component hydroquinone dioxygenase complex (PnpC1C2BL21)-Size-exclusion spectrometry revealed that hydroquinone dioxygenase is a α2and β2heterotetramer of112.4kDa. The reconstitution of a hydroquinone dioxygenase complex in vitro failed. Most of separately expressed PnpC2existed as inclusion bodies, and a mixture of separately expressed and purified PnpC1and PnpC2showed no activity on hydroquinone. A poly-cistronic plasmid pET-pnpC1C2containing pnpCl and pnpC2was constructed to express the dual component hydroquinone dioxygenase complex (PnpC1C2BL21).Hydroquinone is transformed to4-hydroxymuconic semialdehyde by PnpC1C2BL21under aerobic conditions. The expressed PnpC1C2BL21seemed to be unstable when acting on hydroquinone since complete conversion of hydroquinone was only possible by repeated addition of fresh PnpC1C2BL21.This phenomenon had not been observed from PnpC1C2DLL-E4of P. putida DLL-E4. Since a large number of factors have been studied, we believe that different behavior of the two complexes is caused by differences in protein structure. The activity of PnpC1C2DLL-E4is greatly inhibited by the product (4-hydroxymuconic semialdehyde). The inhibition can be overcome by increasing the substrate (hydroquinone) concentration, and the maximum velocity (Vmax) of the reaction is unchanged. These results suggest that the product inhibit the activity of PnpC1C2DLL-E4by competitive inhibition. In an attempt to identify the function of pnpR, a pnpR deletion mutant, strain DLL-ApnpR was constructed by homologous recombination in DLL-E4. Activity assay showed that DLL-△pnpR could still degradate PNP with nitrite release, but lost the ability to degrade hydroquinone. The accumulated product was determined by HPLC analysis to be hydroquinone. RT-PCR analysis showed that the pnpCl can be transcripted under the presence of PnpR. These results indicated that PnpR positively controls the transcription of hydroquinone catabolic genes but not pnpA in pnp gene cluster. The analysis of amino acid sequence shows that PnpR contains a conserved helix-turn-helix DNA binding motif, as well as a LysR substrate-binding domain.Since PnpA can transform4-nitrocatechol to1,2,4-trihydroxybenzene, and PnpC converted1,2,4-trihydroxybenzene to maleylacetate, the pnp gene cluster from DLL-E4must contain the essential genes to catalyze the degradation of4-nitrocatechol; however, DLL-E4did not degrade4-nitrocatechol when it was used as the single substrate, but degraded up to92%of4-nitrocatechol when mixed with PNP. The RT-PCR analysis showed that4-nitrocatechol cannot act as an inductor for inducible transcription of the pnp gene cluster in DLL-E4.