Preliminary Study On The Molecular Mechanism Of Para-nitrophenol Metabolism And PnpA Crystallography In Pseudomonas Putida DLL-E4

Para-nitrophenol (PNP) is an important class of nitrophenolic pollutant, having strong toxic effect on human or animal blood, liver and central nervous system. It has been listed on the "Priority Pollutants List" of the US ATSDR and US EPA. Nowadays many PNP-degrading bacteria have been isolated,and the PNP-degrading pathway and PNP-degrading related genes have been studied extensively. In this study, We use Pseudomonas putida DLL-E4,a PNP- and hydroquinone- (HQ, PNP’s metabolic intermediate) degrading microorganism, as raw material to study the regulatory mechanism of PNP degradation and crystal structure and catalytic mechanism of PnpA by means of reverse transcription PCR,5’-RACE,RNA-Seq,gene knockout, quantitative PCR and protein expression,purification and crystallization technology.There are two PNP degradation gene clusters in strain DLL-E4 genome. By Rockhopper software and reverse transcription PCR analysis, we divided the two PNP degradation gene clusters into six operons pnpAb,pnpA,pnpB, pnpR,pnpC1C2DECX1X2 and pnpC1bC2bDbEbCbX1bX2b. The transcription start sites (TSSs) of pnpR, pnpCl were determined through 5’-RACE method. Relative to the base A of the start codon, the base G of the TSS of pnpR and the base A of the TSS of pnpCl were located at positions 31, 30 in the upstream of the coding sequence, respectively.Semi-quantitative PCR method was applied to analyze the expression profiles of the two PNP degradation gene clusters during growth on PNP, PNP plus glucose, HQ and HQ plus glucose. PCR results revealed that pnpA and pnpR could be transcribed at basal levels without any inducers, and the transcription levels of pnpA and pnpB remained stable when pnpR was deleted. pnpC1C2DECX1X2 was positively regulated by PnpR and its expression could be induced by HQ or PNP. However, the deletion of pnpR might have no obvious effect on the transcription of pnpC1bC2bDbEbCbX1bX2b.Comparative analysis of cell growth between strain DLL-E4 and DLL-△pnpR revealed that the deletion of pnpR had no effect on cell growth and cell morphology. However, the degradation characteritics of PNP and HQ were greatly changed when pnpR was deleted.pnpR-deleted mutant strain lost the ability to degrade HQ and could only catalize the denitration reaction of PNP. The ability to use carbon source also changed when pnpR was deleted. The pnpR deletion mutant strain strengthened the ability to assimilate L-serine, L-pyroglutamic acid and D-glucaric acid.Based on the results of semi-quantitative PCR, PNP degradation and BIOLOG experiments, we used PNP and glucose as the carbon source to cultivate RNA-Seq samples.Through RNA-Seq technology, we could understand the expression profiles of mRNA, the regulatory mechanism of PNP degradation and the relationship between PNP degradation and central carbon metabolism. RNA-Seq results showed that most of genes and ncRNAs significantly changed when wild-type strain DLL-E4 and pnpR-deleted mutant strain DLL-△pnpR were grown on PNP plus glucose, indicating that PNP was a global effector.However, only few of genes and ncRNAs altered when pnpR was deleted.RNA-Seq data and qPCR results showed that the presence of PNP could efficiently induce the transcription of the two PNP degradation gene clusters. However, PNP could not directly induce the transcription of operons pnpC1C2 DECX1X2,pnpC1bC2bDbEbCbX1bX2b and pnpR by means of qPCR method to determine the expression profiles of the two HQ degradation gene clusters when pnpA was inactivated.RNA-Seq results also revealed that ncRNAs ins1 and ins2 prominently upregulated in the presence of PNP. However, ins1 could not be deleted, and the deletion of ins2 only slightly delay PNP degradation and had no significant effect on cell growth. Combined with the previous studies of our laboratory, we concluded that (i) pnpA, pnpR, pnpC1C2DECX1X2 and pnpR1 were the key genes in PNP degradation, and pnpAb and pnpC1bC2bDbEbCbX1bX2b lost function in PNP and HQ metabolism, respectively. (ii)pnpA was regulated by multiple transcriptional factors and its effector was PNP. (iii) operons pnpC1C2DECX1X2 and pnpC1bC2bDbEbCbX1bX2b responded differently to HQ. (iv)LysR-type transcriptional regulator PnpR positively regulated the pnpA and pnpC1C2DECX1X2 operons, and regulator PnpRl positively regulated pnpA but had no impact on operon pnpC1C2DECX1X2. It seemed that the regulation of PNP degradation was complex.With the comparative analysis, we further discovered that transcripts of genes encoding enzymes for PNP metabolism and the TCA cycle significantly increased, whereas those of genes involved in the synthesis of ribosomal proteins, rRNA and RNA polymerase sigma factors conspicuously decreased in both strains grown on glucose and PNP. Meanwhile,genes encoding the outer membrane porin OprB and glucose dehydrogenase Gcd were upregulated. However, genes in the Embden-Meyerhof pathway and the pentose phosphate pathway were unaffected or downregulated under the same conditions. Furthermore, genes required for central carbon metabolism remained unaffected in strain DLL-△pnpR grown on glucose only. The results of glucose utilization assays validated the comparative analysis results and showed that the presence of PNP led to accelerated glucose assimilation at the early degradation stage. However, glucose assimilation ceased when PNP was exhausted.The accelerated glucose assimilation at the early stage might be due to the increased expression of genes encoding the outer membrane porin OprB and the glucose dehydrogenase Gcd. The termination of glucose utilization at later stages seemed to be attributable to (i) the downregulated expression of RNA polymerase sigma factors and genes involved in ribosomal proteins and rRNA synthesis as revealed by RNA-Seq, (ii) the repression of the glucose transport system by the products of PNP catabolism.All results of RNA-Seq, PNP degradation, HQ degradation and qPCR experiments showed that there was a complex link between HQ metabolism and glucose utilization. The accelerated PNP degradation and retarded HQ degradation in the presence of glucose could be attributed to the relief and reinforce of carbon catabolite repression, respectively. The variation in substrate utilization and cell growth in the presence of PNP was a consequence of the interaction of multiple factors.Native-PnpA protein with high purity and suitability for crystallization was obtained through sonication, Ni-NTA affinity chromatography, dialysis, and ultrafiltration. After several rounds of preliminary screening and optimized screening, an appropriate condition for high-quality protein crystal growth was established. The growth condition of native-PnpA protein crystal was 10% isopropanol, 0.1 M Tris-HCl pH 8.5, 13.5% PEG4000, 5% or 7.5%glycerol as well as native-PnpA protein with 5.2 mg/ml. The condition of PnpA-PNP protein crystal was 10% isopropanol, 0.1 M Tris-HCl pH 8.5, 12% PEG4000 and 0.5 mM PNP,meanwhile, the desired protein concentration is 5 mg/ml. The condition of PnpA-FAD/PNP protein crystal was 10% isopropanol, 0.1 M Tris-HCl pH 8.5, 12% PEG4000, 0.5 mM PNP,0.01 mM FAD and native-PnpA with 5 mg/ml. The crystal with high-quality to X-ray diffraction experiments would be obtained using sitting drop vapor diffusion method and cultivating at 293 K for about 2 days under the above-mentioned condition.The purification of Se-PnpA was identical to that of native-PnpA. Since the unstable property of Se-PnpA, β-mercaptoethanol should be added during the whole purification and crystallization process. A suitable Se-PnpA protein crystal growth condition consisted of 10% isopropanol,0.1M Tris-HCl pH 8.5,8.0～12.0% PEG4000,10 mM β- mercaptoethanol,and Se-PnpA protein with 3 to 5 mg/ml.The diffraction resolution of native-PnpA, PnpA-PNP, PnpA- FAD/PNP and Se -PnpA could be up to 1.7 A, 2.0 A, 2.0 A and 1.5 A, respectively. Their space group were P21212,and the unit cell parameters of the four crystals were listed as follows: (i) native-PnpA: a=54.2, b = 76.5, c = 208.8, α= 90.00°,β= 90.00°,γ=90.00°.(ii) PnpA-PNP: a = 53.9, b=76.4, c = 208.3,α= 90.00°,β= 90.00 °,γ = 90.00 °. (iii) PnpA- FAD/PNP: a = 54.4, b=77.6, c = 211.0,α= 90.00 °,β =90.00 °,y= 90.00 °. (iv) Se -PnpA: a = 54.3, b = 77.0, c =208.7, α= 90.00。,β= 90.00 °,γ=90.00 °.