Identification of the para-nitrophenol catabolic pathway, and characterization of three enzymes involved in the hydroquinone pathway, in pseudomonas sp. 1-7
© Zhang et al; licensee BioMed Central Ltd. 2012
Received: 18 July 2011
Accepted: 2 March 2012
Published: 2 March 2012
para-Nitrophenol (PNP), a priority environmental pollutant, is hazardous to humans and animals. However, the information relating to the PNP degradation pathways and their enzymes remain limited.
Pseudomonas sp.1-7 was isolated from methyl parathion (MP)-polluted activated sludge and was shown to degrade PNP. Two different intermediates, hydroquinone (HQ) and 4-nitrocatechol (4-NC) were detected in the catabolism of PNP. This indicated that Pseudomonas sp.1-7 degraded PNP by two different pathways, namely the HQ pathway, and the hydroxyquinol (BT) pathway (also referred to as the 4-NC pathway). A gene cluster (pdcEDGFCBA) was identified in a 10.6 kb DNA fragment of a fosmid library, which cluster encoded the following enzymes involved in PNP degradation: PNP 4-monooxygenase (PdcA), p-benzoquinone (BQ) reductase (PdcB), hydroxyquinol (BT) 1,2-dioxygenase (PdcC), maleylacetate (MA) reductase (PdcF), 4-hydroxymuconic semialdehyde (4-HS) dehydrogenase (PdcG), and hydroquinone (HQ) 1,2-dioxygenase (PdcDE). Four genes (pdcDEFG) were expressed in E. coli and the purified pdcDE, pdcG and pdcF gene products were shown to convert HQ to 4-HS, 4-HS to MA and MA to β-ketoadipate respectively by in vitro activity assays.
The cloning, sequencing, and characterization of these genes along with the functional PNP degradation studies identified 4-NC, HQ, 4-HS, and MA as intermediates in the degradation pathway of PNP by Pseudomonas sp.1-7. This is the first conclusive report for both 4-NC and HQ- mediated degradation of PNP by one microorganism.
Keywordspara-Nitrophenol Catabolism Hydroquinone pathway Hydroxyquinol pathway Pseudomonas
para-Nitrophenol (PNP) is a common environmental pollutant owing to its wide application in pharmaceuticals, explosives, dyes and agrochemicals. PNP also accumulates in the soil due to the hydrolysis of organophosphorus insecticides such as parathion or methyl parathion (MP) . Although PNP is less toxic than MP, it is also considered a significant potential toxic contaminant [2, 3] and belongs to one of 275 hazardous substances commonly found at Superfund sites [4, 5].
Although some studies examining PNP degradation have been reported, genetic information related to the PNP degradation pathways remains limited. In the BT pathway, two enzymes were first characterized from Rhodococcus opacus SAO101: one was the two-component PNP monooxygenase NpcAB; the other was the one-component BT 1,2-dioxygenase NpcC. However, the other enzymes involved in this pathway have not been identified . In Arthrobacter sp. strain JS443, another two-component monooxygenase gene NpdA1A2 has been identified . Recently, Chauhan A et al. identified two lower stream genes (pnpCD) encoding BT 1,2-dioxygenase and maleylacetate (MA) reductase in this pathway . It is worth mentioning that there are two clusters involved in PNP degradation in the gram-positive bacterium Rhodococcus sp. strain PN1. Within these two clusters, two kinds of two-component PNP monooxygenase genes (nphA1A2 and npsA1A2), a regulator protein gene (npcR) and a BT 1,2-dioxygenase gene (npsB) have been identified [9, 10]. For the HQ pathway, the first gene cluster was obtained from Pseudomonas sp. strain WBC-3, and three enzymes involved in PNP degradation, PNP 4-monooxygenase (PnpA), p-benzoquinone (BQ) reductase (PnpB) and BT 1,2-dioxygenase (PnpG), have been characterized [3, 11]. The rest of the genes in this PNP degradation gene cluster have not been identified. Recently, Shen W et al. identified five genes (pnpACC1C2R) in another gram-negative PNP-degrading bacterium, Pseudomonas putida DLL-E4, but the rest of the genes (pnpBDE) in this gene cluster were not identified . To date, all the studies have focused on identifying the upper stream genes in the HQ pathway, while the knowledge of the lower stream pathway genes, especially that of the 4-HS dehydrogenase , remains limited.
In this study, a gram-negative bacterium Pseudomonas sp. 1-7, with the ability to degrade both MP and PNP, was isolated from MP-polluted activated sludge. Microbial degradation studies showed that the intermediate products were HQ and 4-NC, which indicated that both the HQ pathway and BT pathway were utilized in Pseudomonas sp. 1-7. Additionally, a 10.6 Kb gene cluster (pdcEDGFCBA) was identified from a genomic library. Genes: pdcDE, pdcF and pdcG were chosen to be expressed in Escherichia coli for characterization.
Strains, plasmids, and chemicals
Bacterial strains and plasmids used in this study
Strains and plasmids
Relevant genotype or characteristic(s)
Reference or source
methyl parathion and p-nitrophenol utilizer, wild type
F-Φ80(lacZ) M15 lacX74hsdR(rK-mK+) recA1398endA1tonA
F- ompT hsdS (rB- mB-) gal dcm lacY1(DE3)
Kmr, Expression vector
Ampr, Expression vector
Ampr, Expression vector
Ampr, Cloning vector
BamHI-HindIII fragment containing pdcF inserted into pET30a
BamHI-XhoI fragment containing pdcG inserted into pET30a
BamHI-XhoI fragment containing pdcD inserted into pET30a
BamHI-XhoI fragment containing pdcE inserted into pET2230
Isolation of Pseudomonas degrading MP and PNP
Activated sludge (0.5 g) collected from a pesticide factory (Tianjin, China) was cultured overnight at 30°C in 100 ml liquid Burk medium, before being diluted and spread on solid Burk medium containing 0.1% (v/v) MP pesticide and incubated at 30°C. The positive strain able to degrade MP produced a visible hydrolysis halo around the colonies on the plate. Positive colonies were inoculated in liquid Burk medium containing 0.1% (v/v) MP pesticide and cultured overnight at 30°C. MP degradation to PNP was assessed by monitoring whether the medium changed color from colorless to yellow. Further incubation for 2 days was used to test whether this was followed by PNP degradation, confirmed by a subsequent color change from yellow to colorless. Finally, the ability of this bacterium to degrade MP and PNP was confirmed by a second inoculation on a Burk agar plate containing 0.1% (v/v) MP .
Extraction of the intermediates from culture
After the cultures had reached late log-phase in LB medium supplemented with 0.5 mM PNP, bacteria were harvested and washed in Burk medium by centrifugation. The bacteria were then incubated as concentrated cell suspensions (optical density of 1.5 at 600 nm) in Burk medium containing 1.5 mM PNP. Samples were collected at different time points, centrifuged, and aromatic compounds were extracted from the cell-free supernatants as described by Samanta et al .
Characterization of intermediate compounds by HPLC and MS
Identification and quantification of intermediates was performed based on their UV-visible spectra, MS spectra and by chromatographic comparison with standards. The HPLC system consisted of an Agilent 1100 model G1312A binary pump, a model G1330B autosampler and a model G1315B DAD (Agilent Technologies, Inc., Wilmington, DE) equipped with a C18 reversed phase column (5 μm; 250 × 4.6 mm; SunFire) using a column temperature of 30°C. The mobile phase was 30% methanol (pH 3.0) at a flow rate of 0.5 ml min-1. PNP, HQ and 4-NC were all detected in the range 220-400 nm. Under these conditions, authentic PNP, HQ, and 4-NC had retention times of 75, 10.5 and 45 min, respectively.
MS spectra of the intermediate compounds were obtained by the following procedure: a mass selective detector (Agilent, 6430, Ion Trap) was equipped with an ESI using a cone voltage of 25 V and a capillary voltage of 3.5 kV for negative ionization of the analytes (ESI-mode). The dry nitrogen was heated to 325°C and the drying gas flow was 8 l min-1. Data were acquired in the negative scan mode in the range 30-500 Da. The mass of each compound was calculated from its peak area.
Construction of a genomic DNA library
All DNA isolation and cloning procedures were carried out essentially as described by Sambrook et al. . Construction of the fosmid library strictly followed the protocol of the CopyControl™ HTP Fosmid Library Production Kit of EPI (Epicentre Biotechnologies, Madison, WI, USA).
Cloning of the genes involved in PNP degradation
The fosmid library was screened for the positive strains that contained the genes involved in PNP degradation using a PCR-based library screening method. The primers (Ps-F and Ps-R) (Additional file 1: Table S1) were designed based on a conserved region which was identified by comparing the amino acid sequences of available BT dioxygenase gene sequences.
The nucleotide sequence of the positive clone was determined by the National Key Facility Open Laboratory of the Chinese Academy of Agricultural Sciences (Beijing, China). The sequences were assembled using the Contig Express program of the Vector NTI suite 7.0 (InforMax, Frederick, MD, USA). Open reading frames (ORFs) in the assembled sequence were analyzed by the ORF finder tool , and deduced amino acid sequences were examined by BLASTP in NCBI . The potential signal peptides and hydrolytic domains of the identified genes were predicted using SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP). Multiple alignments between protein sequences were performed using ClustalW1.83.
Expression in E. coli of genes involved in PNP degradation
Four genes were selected for expression in E. coli. Genes (pdcDEFG) were amplified by PCR from the positive clones, inserted into expression vectors pET30a (Novagen) or pET2230, and transformed into the expression host E. coli BL21 (DE3), respectively. The primers with their restriction sites are shown in Additional file 1: Table S1. The backbone and the multiple cloning sites of pET2230 originated from pET22b and pET30a, respectively. All positive colonies harboring the corresponding gene were confirmed by DNA sequencing. All host cells harboring the recombinant vectors were grown in LB at 37°C to an OD600 of 0.6 and then induced by the addition of IPTG (0.4 mM final concentration) and incubation at 16°C for 16 h to yield the recombined proteins with fused His6 tags.
Purification of recombinant proteins
E. coli BL21 (DE3) cells harboring the expression plasmid of interest were harvested by centrifugation and resuspended in 20 mM Tris-HCl buffer (pH 8.0). The crude cell extracts were prepared by sonication . All His-tagged recombined proteins (His6-PdcF, His6-PdcG and His6-PdcDE) were purified from the corresponding E. coli crude cell extract using Ni-nitrilotriacetic acid agarose (Ni2+-NTA) (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. The purified proteins were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
The enzyme assays are described in the Additional file 1 (Methods of Enzyme Assays). All assays, where applicable, were performed using cell extracts prepared from non-induced BL21 (DE3) cells that harbored the corresponding recombinant vector and from BL21 (DE3) cells that harbored the non-recombinant expression vector as the negative controls.
GenBank accession number
The nucleotide sequences of the Pseudomonas sp. 1-7 16S rDNA and the PNP degradation gene cluster were deposited in the GenBank database [GenBank FJ821774 and GenBank FJ821777, respectively].
Isolation of Pseudomonas sp. 1-7
Strain 1-7, capable of degrading both MP and PNP and collected from a pesticide factory in Tianjin, China, was identified as a Pseudomonas sp. by 16S rDNA analysis, which sequence has been deposited in the Agricultural Culture Collection of China (ACCC), with collection number [ACCC 05510] . When Pseudomonas sp. 1-7 was cultured in Burk medium containing 0.1% (v/v) MP pesticide, the color of the culture changed to yellow from colorless, indicating that the MP had been hydrolyzed to PNP. After incubation for a further 2 days, the color reverted to colorless, indicating PNP degradation. Moreover, this strain exhibited the same phenomenon on a culture plate containing 0.1% (v/v) MP pesticide: generation of a distinct hydrolysis halo, the color of which first turned yellow and then became colorless. Pseudomonas sp. 1-7 was thus able to degrade both MP and PNP.
In former studies, the full-length of methyl parathion hydrolase gene ophc3 from this bacterium was cloned by constructing genomic library. The gene ophc3 was expressed in E. coli and recombinant methyl parathion hydrolase OPHC3 was purified and the enzymatic properties were studied .
Strain 1-7 degraded PNP utilizing both HQ and BT pathways
Additionally, culture supernatants collected at various time intervals showed a sharp depletion of PNP within 3.5 h, and clearly demonstrated the accumulation of HQ and 4-NC from 3.5 h onward. The maximum amount of 4-NC was detected at 3.5 h, and the maximum amount of HQ at 30 min (Additional file 1: Figure S2).
These results identified both HQ and 4-NC as intermediates in the degradation of PNP by strain 1-7. Being intermediates in the HQ and BT pathways, respectively, both of the two PNP degradation pathways are therefore utilized in PNP mineralization by strain 1-7.
Cloning of genes involved in PNP degradation
Expression and purification of PdcF, PdcG and PdcDE
Enzymatic assays of HQ 1,2-dioxygenase activity
Biochemical properties of the three enzymes
Effect of various metal ions and chemical agent on the activity of the three enzymes
Metal ion or chemical agent (5 mM)
Relative activity (%)
K + (KCl)
113.04 ± 10.80
95.79 ± 16.49
129.00 ± 27.32
Na + (NaCl)
113.42 ± 2.27
88.22 ± 17.76
123.91 ± 25.82
Ba 2+ (BaCl 2 )
99.19 ± 6.29
123.34 ± 7.79
129.02 ± 6.46
Mg 2+ (MgCl 2 )
95.41 ± 5.96
138.06 ± 8.46
129.79 ± 18.11
Zn 2+ (ZnCl 2 )
87.44 ± 8.68
145.95 ± 5.13
21.44 ± 3.71
Cu 2+ (CuCl 2 )
22.46 ± 6.83
110.18 ± 11.17
59.23 ± 12.57
Ni 2+ (NiCl 2 )
111.05 ± 2.61
183.93 ± 30.68
35.25 ± 16.67
Co 2+ (CoCl 2 )
104.15 ± 6.79
147.08 ± 17.51
79.14 ± 13.21
Mn 2+ (MnCl 2 )
77.45 ± 2.93
186.12 ± 9.99
136.59 ± 3.65
Cd 2 + (CdSO 4 )
63.24 ± 3.61
58.93 ± 3.88
39.52 ± 7.01
Fe 2+ (FeCl 2 )
82.13 ± 13.46
39.47 ± 9.49
118.90 ± 21.53
Fe 3+ (FeCl 3 )
78.33 ± 10.74
187.37 ± 15.37
134.89 ± 28.19
62.44 ± 3.90
83.17 ± 8.32
112.93 ± 40.43
97.47 ± 1.65
81.58 ± 24.05
136.59 ± 3.66
Enzymatic assays of 4-HS dehydrogenase activity
The specific activity of PdcG was calculated to be 0.44 Umg-1 (Table 2). PdcG was active over a temperature range of 20-70°C with an optimal activity at 50°C, and over a pH range of 5.0-10.0 with an optimum activity at pH 8.0 (Additional file 1: Figure S4a, S4c). Further, the purified enzyme retained 65% activity after 20 min at 60°C, 18% activity after 30 min at pH 3.0, and 75% activity after 30 min at pH 10.0 (Additional file 1: Figure S4b, S4d). The influence of different metal ions, EDTA and SDS is shown in Table 3.
Co-action of PdcDE and PdcG
Because PdcG was able to metabolize the product of PdcDE, the activities of both His6-PdcDE and His6-PdcG were assayed in one reaction mixture with HQ as the substrate. This was done spectrophotometrically by following the change of absorbance at 320 nm. At the beginning of the reaction, the absorbance at 320 nm rose continuously (Figure 7c), while no rising curve was observed in the negative control (data not shown). This indicated that 4-HS was generated in the reaction mixture containing both enzymes. After about 180 seconds, the absorbance plateaued, suggesting that the generation of 4-HS had reached a limit. NAD+ (the cofactor of PdcG) was then added to the reaction mixture to a final concentration of 0.05 mM to activate His6-PdcG. Upon addition of NAD+, the absorbance at 320 nm immediately decreased rapidly, and then leveled off. However, no such results were observed when His6-PdcG was omitted from the reaction or when His6-PdcDE was incubated with a crude cell extract of the non-induced BL21 strain that harbored pdcF instead of His6-PdcG (data not shown). This confirmed that 4-HS was the product of His6-PdcDE acting on HQ, and that 4-HS was the substrate of the enzyme His6-PdcG.
Enzymatic assays of MA reductase activity
His6-PdcF was active over a temperature range of 20-70°C with an optimal activity at 40°C, and over a pH range of 5.0-9.0 with an optimum activity at pH 7.0 (Table 2, Additional file 1: Figure S5a, S5c). Its specific activity was calculated to be 446.97 Umg-1. Further, the purified enzyme retained 10% activity after 20 min at 60°C, 20% activity after 30 min at pH 3.0, and 58% activity after 30 min at pH 10.0 (Additional file 1: Figure S5b, S5d). The influence of different metal ions, EDTA and SDS is shown in Table 3.
Pseudomonas sp.1-7, a gram-negative bacterium isolated from MP-polluted activated sludge, was able to catabolize both MP and its initial hydrolysis product PNP. Using HPLC and LC-MS, we demonstrated that strain 1-7 degraded PNP through two different pathways, the HQ pathway and the BT pathway. A gene cluster pdcABCDEFG involved in PNP degradation was identified in Pseudomonas sp.1-7. Genes pdcABDEFG were involved in the HQ pathway, and genes pdcCG were involved in the BT pathway. The BT pathway also needs a two-component PNP monooxygenase (Figure 1) to catalyze PNP to 4-NC and BT ; however, we did not find the relevant PNP monooxygenase in the gene cluster. We speculate that the monooxygenase PdcA in the HQ pathway may have two functions, catalyzing PNP to both BQ and 4-NC. This is supported by recent reports indicating that the HQ pathway monooxygenase has the ability to catalyze 4-NC to BT, normally thought to be the work of the BT pathway monooxygenase . This suggests that the HQ pathway monooxygenase could be substituted for the BT pathway monooxygenase in the process of PNP degradation. In future studies, we will identify whether there are BT pathway-specific PNP monooxygenase genes, or whether the HQ pathway monooxygenase is a bi-functional enzyme in strain 1-7.
We also identified three enzymes (PdcDE, PdcF and PdcG) in the HQ pathway. PdcDE was a two-component dioxygenase and catalyzed HQ to 4-HS. PdcG was a 4-HS dehydrogenase that catalyzed 4-HS to MA. PdcF was a MA reductase which transformed MA to β-ketoadipate. All three enzymes performed optimally at temperatures of 40-50°C, and at nearly neutral pH (pH 6.0-8.0). Regarding stability, only PdcG has a better thermal stability at 60°C (65% retention of activity after 20 min exposure) than the other two enzymes (10% to 35% retention). All of the enzymes had better alkali stability at pH 10.0 (58% to 75% retention of activity after 30 min exposure) than acid stability at pH 3.0 (18% to 20% retention).
The HQ dioxygenase gene has been identified in other bacteria [12, 21], but little is known about the properties of its corresponding enzyme. Our research on the enzyme (PdcDE) will hopefully contribute to our understanding. Of the two, the MA reductase PdcF was the more active enzyme, with a specific activity of 446.97 Umg-1 as opposed to 13.33 Umg-1. It is also the first time that a 4-HS dehydrogenase (PdcG) has been extensively characterized.
Pseudomonas sp.1-7, with the capability of degrading MP and PNP, was isolated from MP-polluted activated sludge. The bacterium utilized two pathways for PNP degradation, the HQ pathway and the BT pathway. Three enzymes (PdcDE, PdcF and PdcG) in the HQ pathway were expressed, purified, and characterized. Our research will pave the way for a better understanding of the PNP degradation pathway in gram-negative bacteria.
The work was supported by the National Natural Science Foundation of China (Grant No.31170036).
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