A novel 2-aminophenol 1,6-dioxygenase involved in the degradation of p-chloronitrobenzene by Comamonas strain CNB-1: purification, properties, genetic cloning and expression in Escherichia coli
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- Wu, J., Sun, C., Jiang, C. et al. Arch Microbiol (2005) 183: 1. doi:10.1007/s00203-004-0738-5
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Comamonas strain CNB-1 was isolated from a biological reactor treating wastewater from a p-chloronitrobenzene production factory. Strain CNB-1 used p-chloronitrobenzene as sole source of carbon, nitrogen, and energy. A 2-aminophenol 1,6-dioxygenase was purified from cells of strain CNB-1. The purified 2-aminophenol 1,6-dioxygenase had a native molecular mass of 130 kDa and was composed of α- and β-subunits of 33 and 38 kDa, respectively. This enzyme is different from currently known 2-aminophenol 1,6-dioxygenases in that it: (a) has a higher affinity for 2-amino-5-chlorophenol (Km=0.77 μM) than for 2-aminophenol (Km=0.89 μM) and (b) utilized protocatechuate as a substrate. These results suggested that 2-amino-5-chlorophenol, an intermediate during p-chloronitrobenzene degradation, is the natural substrate for this enzyme. N-terminal amino acids of the α- and β-subunits were determined to be T-V-V-S-A-F-L-V and M-Q-G-E-I-I-A-E, respectively. A cosmid library was constructed from the total DNA of strain CNB-1 and three clones (BG-1, BG-2, and CG-13) with 2-aminophenol 1,6-dioxygenase activities were obtained. DNA sequencing of clone BG-2 revealed a 15-kb fragment that contained two ORFs, ORF9 and ORF10, with N-terminal amino acid sequences identical to those of the β- and α-subunits, respectively, from the purified 2-aminophenol 1,6-dioxygenase. The enzyme was actively synthesized when the genes coding for the ORF9 and ORF10 were cloned into Escherichia coli.
KeywordsComamonas2-Aminophenol 16-DioxygenaseChloronitrobenzene degradation
Choloronitrobenzenes are serious environmental pollutants that are discharged in certain industrial wastes (e.g., wastewater from chloronitrobenzene production factories) and also from the microbial conversion of certain aromatic compounds (Häggblom 1992). Steinwandter reported the occurrence of p-chloronitrobenzene in fish from the River Main (Germany) (as cited in Katsivela et al. 1999), and chloronitrobenzenes were present in municipal water systems of Shanghai, China. p-Chloronitrobenzene causes methemoglobinemia, is harmful to the liver and kidney (Linch 1974) and is weakly mutagenic (Shimizu et al. 1983) and carcinogenic (Weisburger et al. 1978). Thus, p-chloronitrobenzene is a registered priority pollutant in China and the European Economic Community (EEC) (1982) .
Microbial degradation and mineralization of chloronitrobenzenes are important processes during the treatment of wastewater (Livingston and Brookes 1994). However, information on the microbial degradation of p-chloronitrobenzene is very limited. The two known p-chloronitrobenzene degraders are bacterial strain LW1 (Katsivela et al. 1999) and a coculture of Pseudomonas putida and Rhodococcus sp. (Park et al. 1999). Based on the intermediates formed during conversion of p-chloronitrobenzene by a cellular lysate of strain LW1, Katsivela et al. (1999) proposed that p-chloronitrobenzene was degraded through meta-cleavage of 2-amino-5-chlorophenol. However, the enzymes and genes involved in this process have not been identified or characterized. The main objectives of the present study were to purify and characterize the enzyme and to identify the genes that are responsible for this meta-cleavage reaction in a new strain of Comamonas that utilizes p-chloronitrobenzene as sole source of carbon, nitrogen, and energy.
Materials and methods
Bacterial strains, plasmids, media and culture conditions
Bacterial strains, plasmids, and primers used in this study. The EcoRI site is underlined. The start and stop codons are in bold
Characteristics or sequences
Comamonas strain CNB-1
Isolated from activated sludge, assimilating p-chloronitrobenzene
Escherichia coli strain BL21(D3)
E. coli strain XL1-blue MR
Cloning host for cosmid library
Plasmid carrying 2-aminophenol 1,6-dioxygenase genes
Constructed for expression of 2-aminophenol 1,6-dioxygenase
Unless otherwise indicated, purification steps were carried out at 4°C. Cellular lysate was prepared from 6 g of cells (wet weight) of Comamonas strain CNB-1 grown in LB broth. Cells that had been stored at −20°C were thawed and suspended in 40 ml buffer A (pH 8.0) composed of 20 mM Tris–HCl, 10% (v/v) ethanol, 1 mM dithiothreitol and 0.5 mM L-ascorbic acid. The suspended cells were disrupted by sonification. Cellular debris was removed by centrifugation at 17,000 g for 10 min and at 4°C. The supernatant was subjected to (NH4)2SO4 fractionation. Proteins that precipitated between 60 and 70% (NH4)2SO4 saturation were dissolved in 8 ml of buffer A and further concentrated to 2 ml by ultrafiltration. The concentrate was applied to a Superdex 200 column (Pharmacia, Sweden) equilibrated with 280 ml of buffer A. The proteins were fractionated with buffer A at a flow rate of 0.6 ml min−1. Eluant was collected (1 ml per tube) and subjected to assays of protein concentration and enzyme activity. Fractions (tubes 60–68) with 2-aminophenol 1,6-dioxygenase activities were pooled and loaded onto a Mono Q HR 5/5 ion-exchange column (Pharmacia, Sweden). The column was washed with buffer A, and proteins were eluted at a flow rate of 0.4 ml min−1 with a linear gradient (50–500 mM) of NaCl in 200 ml buffer A. Eluants of 100 μl/tube were collected. The 2-aminophenol 1,6-dioxygenase was recovered in tubes 46–49.
Determinations of enzymatic activities and protein concentrations
The activity of 2-aminophenol 1,6-dioxygenase was determined at room temperature by measuring the increase in absorbance (due to the formation of 2-aminomuconic acid semialdehyde) at 380 nm. The reaction mixture (2.95 ml) contained 10 mM sodium phosphate buffer (pH 8.0) and 0.16 mM 2-aminophenol. The reaction was started by adding 50 μl of cellular lysate or enzyme preparation.
The use of various substituted aromatic compounds by purified 2-aminophenol 1,6-dioxygenase was determined as described above, except that 2-aminophenol was replaced by other substrates and the reactions were monitored at the maximal absorption wavelengths of the products. The Km values of 2-aminophenol and 2-amino-5-chlorophenol were calculated with Lineweaver-Burk plots. The following molar coefficients were used for calculations of the enzymatic activities (M−1 cm−1): ɛ380nm=15,100 for 2-aminophenol (Lendenmann et al. 1996), ɛ395nm=21,000 for 2-amino-5-chlorophenol (Katsivela et al. 1999), ɛ290nm=2300 for protocatechuate and ɛ375nm=33,000 for catechol (Iwagami et al. 2000; Sala-Trepat et al. 1971). Protein concentrations were determined according to the method of Bradford (1976).
Identification of products from 2-amino-5-chlorophenol cleavage
2-Amino-5-chlorophenol was oxidized for 10 min in an enzyme assay system. The reaction mixture was adjusted to 2.0 with HCl solution and extracted with ethyl acetate. After centrifugation at 12,000 g for 10 min, the organic phases were collected, concentrated by evaporation of the organic solvent under vacuum and then analyzed by GC-MS (Micromass, UK). The mass spectrometer was operated in the electron impact mode at 70 eV. Gas chromatography (GC) was run with helium as the carrier gas and at a flow rate of 0.5 ml min−1. The oven temperature was maintained at 80°C for 2 min, and then increased to 250°C at a rate of 20°C min−1. Samples (1.0 μl) were injected into the gas chromatograph operating in the splitless mode with an injector temperature of 250°C.
Native and subunit molecular mass determinations
The molecular mass of the native 2-aminophenol 1,6-dioxygenase was estimated by gel filtration with a Superdex 200 column (Pharmacia) at a flow rate of 0.5 ml min−1 and Tris–HCl (50 mM, pH 8.0)-NaCl (0.10 M) as the buffer. Carbonic anhydrase (29 kDa), albumin (66 kDa), alcohol dehydrogenase (150 kDa), β-amylase (200 kDa), apoferritin (443 kDa) and thyroglobulin (669 kDa) (all from Sigma) served as standards. The molecular masses of the subunits were determined by SDS-PAGE with a 15% resolving gel and a 5% stacking gel. Protein molecular mass standards for SDS-PAGE were purchased from the Institute of Biochemistry and Cell Biology, CAS (Shanghai, China).
Determination of N-terminal amino acid sequences
Purified 2-aminophenol 1,6-dioxygenase was subject to SDS-PAGE (12% acrylamide). The proteins were transferred to PVDF membrane by electro-blotting and visualized by Coomasie brilliant blue staining. The protein bands of the α- and β-subunits were cut out and sequenced by the Edman degradation method using Applied Biosystems (USA) Procise 491 Protein Sequencer.
Effects of chemicals on 2-aminophenol 1,6-dioxygenase activity
In order to test the inhibition or stimulation of various metal ions, EDTA, and oxidizing reagents (all at concentrations of 2 mM), the purified 2-aminophenol 1,6-dioxygenase was incubated with the chemicals for 1 h in the enzymatic assay buffer without substrates, and determination of activity was started by addition of 2-aminophenol as described above. The recovery of activity by 2-aminophenol 1,6-dioxygenase after treatment with reductant or oxidant, was monitored by exposing the enzyme to ascorbate (5 mM) and then further incuabtion for 1 h before activity measurement.
Construction of cosmid library of Comamonas strain CNB-1
Total DNA of Comamonas strain CNB-1 was prepared according to the protocol described by Sambrook et al. (2001). Total DNA partially digested with MboI was ligated to arms-separated SuperCos 1 vector. The cosmid library of strain CNB-1 was constructed by following the protocol provided by the supplier (Gigapack III XL Packaging Extract, #200208, Stratagene, La Jolla, Calif., USA). E. coli XL 1- Blue MR was used as host.
Screening for 2-aminophenol 1,6-dioxygense-positive clones
Ampicillin-resistant transformants were picked and transferred to 96-well microtiter plates containing 0.2 ml of one-fourth-strength LB broth with 100 μg ampicillin per ml and 50 μM 2-aminophenol. 2-Aminophenol is unstable in aqueous solution and gradually forms a yellow product under the assay conditions, whereas cleavage of 2-aminophenol by 2-aminophenol 1,6-dioxygenase prevents formation of the yellow product. Therefore, after 2 days of cultivation at 37°C, wells that were not yellow were selected and presumably expressed 2-aminophenol 1, 6-dioxygenase activity.
DNA sequencing, sequence assembly and analysis
The 2-aminophenol-1,6-dioxygenase-positive clone, E. coli/pBG-2, containing a 30-kb DNA fragment from strain CNB-1 was partially sequenced using the shotgun method by Beijing Genome Institute (Huada, Beijing, China). Sequences were assembled by Phred/Phrap/Consed System (University of Washington, USA).
ORF finding and sequence BLAST
The ORFs, amino acid sequence similarity and sequence alignment were analyzed respectively with ORF finder program, AlignX and BLASTx (translated query vs. protein database) from the NCBI website (http://www.ncbi.nlm.nih.gov). The DNA sequences reported here are available under the GenBank accession number AY605054.
Cloning and expression of 2-aminophenol 1,6-dioxygenase in Escherichia coli
PCR primers (Table 1) were designed according to the DNA sequence obtained as described above, and the entire 2-aminophenol 1,6-dioxygenase gene (1,770 bp) was amplified from the strain CNB-1 genome. The PCR product was purified, treated with EcoRI, and then ligated into pET-21a(+) which had been digested previously with NdeI plus EcoRI. The resulting plasmid, designated pETA16D, was used to transform E. coli BL21(DE3) for expression of the 2-aminophenol 1,6-dioxygenase gene. Synthesis of 2-aminophenol 1,6-dioxygenase in cells of E. coli strains carrying plasmid pETA16D was induced with 1 mM IPTG.
Characterization of the p-chloronitrobenzene-degrading Comamonas strain CNB-1
Strain CNB-1 was isolated from a biological reactor used to treat the wastewater of a p-chloronitrobenzene production factory. The strain CNB-1 is obligately aerobic and heterotrophicrod-shaped with polar flagella, and gram-negative. Catalase was negative but oxidase was positive. Sodium citrate, sodium gluconate, L-valine, L-alanine, L-proline, L-phenylalanine, and histidine were utilized. Sugars were rarely assimilated. Poly(3-hydroxybutyrate) accumulated in cells. These phenotypic properties, in addition to the 16S rRNA gene sequence (GenBank accession no. AY291591) analysis identified strain CNB-1 as a member of the genus Comamonas. The closest phylogenetic relative of CNB-1 is C. testosteroni (16S rRNA gene sequence identity was 99%).
Comamonas strain CNB-1 utilized p-chloronitrobenzene, nitrobenzene, catechol, and protocatechuate as sole sources of carbon and energy. When strain CNB-1 was cultivated in mineral salts with p-chloronitrobenzene, the optimal temperature and pH were 28°C and 9.0, respectively, for cell growth and p-chloronitrobenzene degradation.
Purification of 2-aminophenol 1,6-dioxygenase from Comamonas strain CNB-1
Purification of 2-aminophenol 1,6-dioxygenase from Comamonas strain CNB-1
Total protein (mg)
Total activity (units)
Specific activity (units mg−1)
Biochemical properties of 2-aminophenol 1,6-dioxygenase
Effects of metal ions and chemicals on 2-aminophenol 1,6-dioxygenase
Activity of untreated enzyme (%)
Metal ions (2 mM)
Chelator (2 mM)
Oxidizing agents (2 mM)
In addition to utilizing 2-aminophenol (100% relative activity), 2-aminophenol dioxygenase of strain CNB-1 also utilized 2-amino-5-chlorophenol (68% activity), protocatechuate (33% activity), catechol (5% activity) as substrates. The action of this 2-aminophenol 1,6-dioxygenase on protocatechuate differentiated it from other 2-aminophenol 1,6-dioxygenases of Pseudomonas spp., since none of these catalyzed the cleavage of protocatechuate. 4-Methylcatechol, 4-chlorocatechol, 2,4-dihydroxybenzoic acid, o-nitrophenol, p-nitrophenol, and 4-nitrocatechol did not serve as substrates for the 2-aminophenol 1,6-dioxygenase from strain CNB-1. The Km and Vmax values of the purified 2-aminophenol 1,6-dioxygenase for 2-aminophenol were 0.89 μM and 44.6 μmol min−1 mg−1, respectively. The Km and Vmax values for 2-amino-5-chlorophenol were 0.77 μM and 19.3 μmol min−1 mg−1, respectively.
The product of 2-amino-5-chlorophenol oxidation catalyzed by this dioxygenase was identified by GC-MS. The product had a molecular ion at m/z=157 (M+), indicative of C6H4ClNO2, and the major fragments of this compound appeared at m/z=113 (M+, loss of CO2) and m/z=78 (M+, loss of CO2Cl). A GC-MS database search revealed that this spectrum completely matched that of 4-chloropicolinic acid. The latter is a spontaneous conversion product of 2-amino-5-chloromuconic semialdehyde (Katsivela et al. 1999; Davison et al. 1996); thus the original product of 2-amino-5-chlorophenol cleavage was 2-amino-5-chloromuconic semialdehyde.
Cosmid library construction and screening for 2-aminophenol 1,6-dioxygenase
Sequence analysis of the 2-aminophenol 1,6-dioxygenase and discovery of other ORFs
The seqenced 15 -kb DNA fragment contains the following additional genes that are putatively related to aromatic compound degradation (Fig. 2). A putative aminophenol operon repressor (ORF7) and a putative ferredoxin (ORF8) precede ORF9. A putative 2-aminomuconic semialdehyde dehydrogenase (ORF12) occurred after ORF10. This gene (orf12) was cloned and actively expressed in E. coli BL21 (data not shown) and results indicated that this enzyme is active towards 2-aminomuconic semialdehyde and 2-amino-5-chloromuconic semialdehyde. Several genes (orf4–6) involved in the catechol intradiol cleavage pathway were also located in this 15-kb DNA fragment, indicating that a second pathway for aromatic compound degradation might exist in strain CNB-1. These genes include the transcriptional regulator (ORF4), muconate cycloisomerase (ORF5), and catechol 1,2-dioxygenase (ORF6).
Cloning and expression of 2-aminophenol 1,6-dioxygenase genes in E. coli
The genes encoding the 2-aminophenol 1,6-dioxygense α- and β-subunits were amplified by PCR and cloned into the expressional plasmid pETA16D at its NdeI and EcoRI sites. Upon the induction by IPTG, prominent synthesis of proteins was observed (Fig. 4). 2-Aminophenol 1,6-dioxygenase activities were detected in the cellular lysates of induced recombinant E. coli cells.
In this study, we have demonstrated that a novel 2-aminophenol 1,6-dioxygenase catalyzes the cleavage of 2-amino-5-chlorophenol and also promotes the formation of 5-chloropicolinic acid, a spontaneous conversion product of 2-amino-5-chloromuconic semialdehyde. The gene encoding this enzyme was cloned and actively expressed in E. coli. The higher affinity of the enzyme for 2-amino-5-chlorophenol (as indicated by the low Km) suggests that this dioxygenase prefers 2-amino-5-chlorophenol as substrate. The lower reaction rate with 2-amino-5-chlorophenol (the Vmax was only 68% of that obtained with 2-aminophenol) is probably due to the greater chemical stability of 2-amino-5-chlorophenol than of 2-aminophenol. Since the newly isolated Comamonas strain CNB-1 assimilates both p-chloronitrobenzenes and nitrobenzene and the same 2-aminophenol dioxygenase activity was observed during growth, we propose that both 2-amino-5-cholorophenol and 2-aminophenol are native substrates for this dioxygenase and it is involved in the degradation of both p-chloronitrobenzene and nitrobenzene.
Previous studies have shown the involvement of 2-aminophenol 1,6-dioxygenases in the degradation of nitrobenzene and 2-aminophenol by P. pseudoalcaligenes JS45 (Lendenmann et al. 1996; Davis et al. 1999), P. putida HS12 (Park et al. 2000) and Pseudomonas species AP-3 (Takenaka et al. 1997, 2000), but the importance of these dioxygenases in chloronitrobenzene degradation has not been reported. It remains unknown whether these Pseudomonas strains assimilate chloronitrobenzenes and whether their 2-aminophenol 1,6-dioxygenases can utilize 2-amino-5-chlorophenol as a substrate. Interestingly, 2-aminophenol 1,6-dioxygenase was constitutively synthesized in strain CNB-1 and in a previously reported p-chloronitrobenzene degrader, strain LW1 (Katsivella et al. 1999). A gene encoding an aminophenol operon repressor-like protein (ORF7) preceded the 2-aminophenol 1,6-dixoygenase (ORF9 and ORF10) genes. Although the function of this ORF7 and how 2-aminophenol 1,6-dioxygenase is regulated are unresolved, the loose control of the synthesis of 2-aminophenol 1,6-dioxygenase in Comamonas strain CNB-1, and the occurrence of a putative transposase (ORF2) in its vicinity (Fig. 2), suggest that the chloronitrobenzene degradative pathway in strain CNB-1 has evolved more recently than the nitrobenzene degradative pathway in Pseudomonas spp. (Johnson and Spain 2003; Lendenmann et al. 1996; Davis et al. 1999).
This work was supported by grants (NSFC30230010 and KSCX2-SW-113) from Chinese Academy of Sciences and National Natural Science Foundation of China. We are grateful to Harold L. Drake at the University of Bayreuth, Germany, for his assistance during the revision of this paper.