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Appl Environ Microbiol. Jul 2011; 77(13): 4547–4552.
PMCID: PMC3127694

Patchwork Assembly of nag-Like Nitroarene Dioxygenase Genes and the 3-Chlorocatechol Degradation Cluster for Evolution of the 2-Chloronitrobenzene Catabolism Pathway in Pseudomonas stutzeri ZWLR2-1[down-pointing small open triangle]


Pseudomonas stutzeri ZWLR2-1 utilizes 2-chloronitrobenzene (2CNB) as a sole source of carbon, nitrogen, and energy. To identify genes involved in this pathway, a 16.2-kb DNA fragment containing putative 2CNB dioxygenase genes was cloned and sequenced. Of the products from the 19 open reading frames that resulted from this fragment, CnbAc and CnbAd exhibited striking identities to the respective α and β subunits of the Nag-like ring-hydroxylating dioxygenases involved in the metabolism of nitrotoluene, nitrobenzene, and naphthalene. The encoding genes were also flanked by two copies of insertion sequence IS6100. CnbAa and CnbAb are similar to the ferredoxin reductase and ferredoxin for anthranilate 1,2-dioxygenase from Burkholderia cepacia DBO1. Escherichia coli cells expressing cnbAaAbAcAd converted 2CNB to 3-chlorocatechol with concomitant nitrite release. Cell extracts of E. coli/pCNBC exhibited chlorocatechol 1,2-dioxygenase activity. The cnbCDEF gene cluster, homologous to a 3-chlorocatechol degradation cluster in Sphingomonas sp. strain TFD44, probably contains all of the genes necessary for the conversion of 3-chlorocatechol to 3-oxoadipate. The patchwork-like structure of this catabolic cluster suggests that the cnb cluster for 2CNB degradation evolved by recruiting two catabolic clusters encoding a nitroarene dioxygenase and a chlorocatechol degradation pathway. This provides another example to help elucidate the bacterial evolution of catabolic pathways in response to xenobiotic chemicals.


Chloronitrobenzenes (CNBs) are used as intermediates in the synthesis of drugs, dyes, and pesticides. They are serious environmental pollutants, being toxic to both humans and animals. Microbial degradation of CNBs has attracted a great deal of interest, and developments in this field have recently been reviewed (8). Thus far, several bacterial strains have been reported to use CNBs as a sole carbon and energy source for growth (9, 12, 22, 24). Among them, the 4-chloronitrobenzene degraders Comamonas sp. strain CNB-1 (22) and Pseudomonas putida ZWL73 (23, 24) were reported to use nitroreductase to catalyze 4-chloronitrobenzene reduction to 1-chloro-4-hydroxylaminobenzene. This intermediate is then transformed to the ring cleavage substrate 2-amino-5-chlorophenol by a hydroxylaminobenzene mutase or Bamberger rearrangement. A 2-aminophenol 1,6-dioxygenase catalyzes a ring cleavage reaction to produce 2-amino-5-chloromuconate, which is converted to tricarboxylic acid cycle intermediates after additional enzymatic steps (22). However, the 2-chloronitrobenzene (2CNB) utilizer Pseudomonas stutzeri ZWLR2-1 was reported to metabolize 2CNB in an oxidative pathway based on nitrite release (12). This study reports the genetic determinants of a metabolic pathway for 2CNB degradation in strain ZWLR2-1 and reveals the evolution of the pathway by patchwork assembly of a Nag-like nitroarene dioxygenase gene cluster and a 3-chlorocatechol degradation cluster.


Bacterial strains, plasmids, chemicals, and culture conditions.

The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli strains were grown in lysogeny broth (LB) medium at 37°C with 100 μg/ml ampicillin. P. stutzeri ZWLR2-1 was grown at 30°C in LB medium or M9 minimal medium (12) supplemented with 0.5 mM 2CNB or 10 mM succinate. 3-Methylcatechol and 4-chlorocatechol were purchased from Sigma (St. Louis, MO); 4-methylcatechol, 2CNB, and 3-chloronitrobenzene were from Fluka Chemical Co. (Buchs, Switzerland); and 3-chlorocatechol was from TCI (Tokyo, Japan).

Table 1.
Strains and plasmids used in this study

Gene cloning and sequence analysis.

An EcoRI-SacI-digested genomic DNA library of strain ZWLR2-1 was constructed using pBluescript II SK (Stratagene, La Jolla, CA). The library was screened for positive clones by PCR using primers nitro-dioF (5′-ACCCACCTTCAAGCACTCTG-3′) and nitro-dioR (5′-CGAWGGCATACGTCCAAWCC-3′), which were derived from a fragment encoding a portion of the ring-hydroxylating dioxygenase α subunit in strain ZWLR2-1 (12). The insert from positive clone pZWL01 was sequenced, and the upstream sequence was obtained by a genome-walking strategy (16). Nucleotide sequences were determined by Invitrogen Technologies Co. (Shanghai, China). Open reading frames (ORFs) were identified using the ORF Finder program at the National Center for Biotechnology Information website. A promoter search was performed using the Neural Network Promoter Prediction software (http://www.fruitfly.org/seq_tools/promoter.html).

Expression of cnbAaAbAcAd and cnbC in E. coli.

A 1,725-bp SacI-SmaI-digested PCR fragment containing cnbAaAb amplified from strain ZWLR2-1 genomic DNA and a 2,365-bp SmaI-SphI-digested DNA fragment containing cnbAcAd from pZWL01 were ligated into SacI-SphI-digested pUC18 in a three-fragment ligation. The resultant plasmid, pCNBA containing cnbAaAbAcAd, was transformed into E. coli DH5α for whole-cell biotransformation. cnbC was PCR amplified from genomic DNA and cloned into the NdeI-BamHI sites of pET5α to produce pCNBC. Subsequently, the resultant plasmid was introduced into E. coli BL21(DE3) Rosetta for expression. Cloned sequences were determined to ensure that mutations were not incorporated during PCR. Transformed E. coli strains were grown in LB medium at 37°C to an optical density at 600 nm (OD600) of 0.6 and then induced with 0.4 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 4 h at 30°C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was then performed using a discontinuous gel.

Whole-cell biotransformation.

E. coli DH5α/pCNBA cells were harvested by centrifugation, washed, and resuspended in 20 ml phosphate buffer (50 mM, pH 7.4) to a final OD600 of 2.0. Succinate-grown strain ZWLR2-1 was prepared similarly. For qualitative analysis, each nitroaromatic substrate was added to recombinant E. coli cell suspensions at a final concentration of ~0.5 mM. Cell suspensions were incubated with shaking (180 rpm, 26°C). Samples were collected at appropriate intervals to monitor reaction progress by nitrite analysis. After 1 h, the supernatant of the reaction mixture was extracted with ethyl acetate for high-performance liquid chromatography (HPLC) or liquid chromatography (LC)-mass spectrometry (MS) analysis. For a time course analysis, 2CNB was added to recombinant E. coli and strain ZWLR2-1 suspensions at a final concentration of ~0.3 mM. Collected samples were stored at −20°C until use. 2CNB biotransformation activity was determined by measuring the nitrite released from the substrate at 26°C. Nitrite was assayed as described elsewhere (11).

HPLC and LC-MS analyses.

HPLC analysis of nitroaromatic substrates and their oxidation products was performed on an Agilent series 1200 system (Agilent Technologies, Palo Alto, CA) equipped with a C18 reversed-phase column (5 μm, 4.6 by 250 mm; Agilent Technologies) maintained at 30°C. The monitoring wavelength was set to 280 nm. The mobile phase consisted of water containing 0.1% (vol/vol) acetic acid (A) and methanol (B) at a 1.0-ml/min flow rate. A stepped solvent gradient was used as follows: 0 to 7 min, 5 to 15% B; 7 to 15 min, 15 to 37% B; 15 to 40 min, 37 to 73% B; 40 to 45 min, 73 to 80% B; 45 to 50 min, 80% B. Under these conditions, authentic catechol, 4-methylcatechol, 3-methylcatechol, 3-chlorocatechol, and 4-chlorocatechol had retention times of 11.0 min, 18.4 min, 19.7 min, 20.8 min, and 23.0 min, respectively.

LC-diode array detector (DAD)-MS measurements of the oxidation product from 2CNB were performed with an Agilent 1200 series system (Agilent Technologies, Waldbronn, Germany) coupled with a Bruker microQTOF mass spectrometer (Bruker Daltonics, Bremen, Germany) with an electrospray ionization interface. The LC system included a quaternary solvent delivery system, an online degasser, an autosampler, a column temperature controller, and a DAD. An ACE C18-HL column (5 μm, 250 by 4.6 mm) was used with a C18 guard column with a column temperature of 30°C and a sample injection volume of 20 μl (ethyl acetate extract). The monitoring wavelength was 280 nm, and the DAD acquisition wavelength was 210 to 900 nm. The mobile phase consisted of water (A) and acetonitrile (B), both containing 0.1% (vol/vol) formic acid. A stepped solvent gradient was used as follows: 0 to 7 min, 5 to 15% B, 7 to 15 min, 15 to 37% B; 15 to 40 min, 37 to 73% B; 40 to 45 min, 73 to 80% B; 45 to 50 min, 80% B. The flow rate was 1.0 ml/min with 5% of the eluant directed to a mass spectrometer using a Bruker nuclear magnetic resonance-MS interface unit (Bruker BioSpin, Rheinstetten, Germany). The optimized mass spectrometric parameters were as follows: capillary voltage, 4,500 V; nebulizer gas pressure, 0.8 × 105 Pa; drying gas flow rate, 8 liters/min; gas temperature, 180°C; negative-ion mode. Spectra were recorded within m/z 50 to 1,000.

Cell extract preparation and chlorocatechol 1,2-dioxygenase activity assay.

Cell extracts were prepared by resuspending bacteria pellets in ice-cold phosphate buffer (40 mM, pH 7.4) and sonicating them in an ice-water bath at 5.0-s on and 9.0-s off intervals for 10 min. Cell debris was removed by centrifugation at 17,000 × g for 30 min at 4°C. Chlorocatechol 1,2-dioxygenase activity was determined by spectrophotometric assay using the method of Spain and Nishino (17) with a Lambda 25 spectrophotometer (Perkin-Elmer Cetus, Norwalk, CT). Reaction mixtures contained 50 mM phosphate buffer (pH 7.4), 50 μM catechol or substituted catechols, and cell extracts, and reactions were initiated by substrate addition. The activity against substrate catechol or substituted catechols was assayed by measuring the increase in absorbance at 260 nm due to the formation of their corresponding products, cis,cis-muconate or substituted cis,cis-muconates. Enzyme activities were calculated using molar extinction coefficients determined as described by Dorn and Knackmuss (4), i.e., 16,800 M−1·cm−1 for cis,cis-muconate, 17,100 M−1·cm−1for 2-chloro-cis,cis-muconate, 12,400 M−1·cm−1 for 3-chloro-cis,cis-muconate, 18,000 M−1·cm−1 for 2-methyl-cis,cis-muconate, and 13,900 M−1·cm−1 for 3-methyl-cis,cis-muconate. One unit of enzyme activity is defined as the amount of activity required for the production of 1 μmol of product per minute. Specific activities were expressed as units per milligram of protein. Protein concentrations were determined by the Bradford method (2) with bovine serum albumin used as the standard.

Nucleotide sequence accession number.

The 16.2-kb 2CNB degradation gene sequence has been deposited in GenBank under accession number GU181397.


Cloning and sequence analysis of genes involved in 2CNB degradation.

A positive clone, pZWL01, was identified from the constructed genomic library, and DNA sequencing revealed a 9.3-kb insert. Subsequently, a farther upstream ~7-kb DNA fragment was obtained by genome walking. The assembled sequence formed a 16.2-kb DNA contig with 19 complete ORFs (Fig. 1 B) annotated by BLAST analysis (Table 2). CnbAc and CnbAd exhibited significant homology to the respective α (90 to 92% identity) and β (91 to 95% identity) subunits of Nag-like ring-hydroxylating dioxygenases (type III Rieske nonheme iron aromatic oxygenase [10]). These enzymes are involved in the catabolism of nitrotoluene in Pseudomonas sp. strain JS42 (14), nitrobenzene in Comamonas sp. strain JS765 (11), 2,4-dinitrotoluene in Burkholderia cepacia R34 (6, 8) and Burkholderia sp. strain DNT (6, 18), and naphthalene in Ralstonia sp. strain U2 (25). A comparison of the cluster organizations containing the above dioxygenase genes and cnbAcAd is shown in Fig. 1D. Noticeably, cnbAcAd were located in a 3,085-bp region between two IS6100 insertion sequences consisting of a typical class I transposon (Fig. 1B). The transposon was identical to those in a transposable catabolic transposon, Tnmph, from the methyl parathion utilizer Pseudomonas sp. strain WBC-3 (20). The nagR-like regulator-encoding gene, found to be transcribed divergently from nitroarene dioxygenase genes in all identified clusters, was absent in the 2CNB degradation cluster (Fig. 1D). However, a putative promoter (nucleotides 9787 to 9832) was located within the IS6100 insertion sequence, ~900 nucleotides upstream of the predicted cnbAc start codon. Upstream of cnbAcAd was a cluster (cnbCEFAbAa) sharing significant homology (82 to 94% of the encoded products) with the same organization as the chlorocatechol gene cluster tfdC2E2F2orf5orf6 of Sphingomonas sp. strain TFD44 (19) (Fig. 1C). Among them, CnbAa and CnbAb shared moderate homology with ferredoxin reductase (40% identity) and ferredoxin (55% identity) of a three-component anthranilate 1,2-dioxygenase (type IV oxygenase) identified in B. cepacia DBO1 (3). The DNA sequences of ORFs 4 to 7, located downstream of cnbAcAd, were almost identical to those on plasmid Rms149 from Pseudomonas aeruginosa Ps142 (5).

Fig. 1.
Catabolic pathway of 2CNB degradation and comparative analysis of catabolic genes encoding 2CNB degradation in P. stutzeri ZWLR2-1 with homologs in other nitroarene utilizers. (A) Proposed pathway of 2CNB catabolism. (B) Organization of the cnb gene cluster. ...
Table 2.
Sequence comparisons of 2CNB gene clusters of P. stutzeri ZWLR2-1 with database entries

CnbAaAbAcAd catalyzes 2CNB conversion to 3-chlorocatechol.

To determine the function of the putative cnbAaAbAcAd-encoded 2CNB dioxygenase, IPTG-induced E. coli DH5α/pCNBA cells were analyzed for the ability to transform 2CNB. Enzyme activity was confirmed by the formation of a pink complex in a nitrite assay. LC-MS analysis indicated that the product from whole-cell biotransformation of 2CNB appeared at a retention time of 18.91 min, which is consistent with that of the authentic 3-chlorocatechol. Analysis gave an [M-H] ion at m/z 142.9910 corresponding to the parent ion with the same major fragments (at m/z 122.0376, 107.0180) as those of the 3-chlorocatechol standard. In contrast, authentic 4-chlorocatechol gave an [M-H] ion at m/z 142.9910 with a single major fragment at m/z 107.0180 and a 19.80-min retention time. The product formed from 2CNB oxidation by CnbAaAbAcAd was therefore assigned to 3-chlorocatechol, and 4-chlorocatechol was not detected. 3-Chlorocatechol was not detected in the control containing identically treated E. coli DH5α/pUC18. In the time course assay, stoichiometric amounts of 3-chlorocatechol accumulated with concomitant nitrite release when 2CNB was transformed with E. coli DH5α/pCNBA. The assays were performed on three separate occasions, and similar results were obtained; a representative example is shown in Fig. 2. These data indicated that CnbAaAbAcAd is a 2CNB dioxygenase capable of oxidizing 2CNB to 3-chlorocatechol with concomitant nitrite release, as shown in Fig. 1A. E. coli cells with functional CnbAaAbAcAd were also analyzed for the ability to transform other nitroaromatic substrates. Besides nitrite being formed in all cases, catechol from nitrobenzene transformation was detected, both 3-chlorocatechol and 4-chlorocatechol from 3-chloronitrobenzene transformation were detected, 3-methylcatechol from 2-nitrotoluene transformation was detected, and both 3-methylcatechol and 4-methylcatechol from 3-nitrotoluene transformation were detected.

Fig. 2.
Time course of 2CNB conversion to 3-chlorocatechol with nitrite release by IPTG-induced E. coli DH5α/pCNBA expressing CnbAaAbAcAd. Cells were washed and resuspended in 20 ml phosphate buffer (50 mM, pH 7.4) to a final OD600 of 2.0. Assays were ...

2CNB dioxygenase activity was also detected by nitrite analysis in the assay of 2CNB biotransformation by succinate-grown or 2CNB-grown strain ZWLR2-1 cells. The 2CNB biotransformation activity in succinate-grown strain ZWLR2-1 cells was 5.17 nmol min−1 mg cell dry weight−1, and it was 13.7 nmol min−1 mg cell dry weight−1 in 2CNB-grown strain ZWLR2-1 cells. This indicated that the enzyme-catalyzed 2CNB conversion was constitutively expressed in the wild-type strain. Intermediate 3-chlorocatechol was detected at a low concentration during the transformation of 2CNB by succinate- or LB-grown ZWLR2-1 cells.

CnbC is a chlorocatechol 1,2-dioxygenase.

A PCR product containing the entire cnbC gene was ligated into the expression vector pET5a, and the result was designated plasmid pCNBC and transformed into E. coli BL21(DE3) Rosetta. Following IPTG induction, SDS-PAGE of cell extracts showed elevated levels of a polypeptide at ~30 kDa, which correlated with the amino acid composition of CnbC. Chlorocatechol 1,2-dioxygenase activity was detected in the same extracts with a specific activity of 0.021 U/mg. CnbC exhibited extended substrate specificity for catechol and available methylated or halogenated catechols (Table 3). Furthermore, cell extracts from E. coli/pCNBC containing cnbC and succinate-grown and 2CNB-grown ZWLR2-1 cells exhibited similar relative activities of intradiol cleavage dioxygenase against catechol or substituted catechols, as shown in Table 3. This result suggested that CnbC may be the native form of chlorocatechol 1,2-dioxygenase in strain ZWLR2-1.

Table 3.
Intradiol cleavage dioxygenase relative activities with different substrates in cell extracts from 2CNB-grown strain ZWLR2-1, succinate-grown strain ZWLR2-1, and E. coli BL21(DE3) Rosetta/pCNBC containing cnbC induced by IPTG

Genetic stability of the cnb catabolic cluster.

Intriguingly, a spontaneous mutant of strain ZWLR2-1, designated Pseudomonas sp. strain ZWLR2-1D, was obtained during repetitive culturing in LB medium. Despite possessing the same morphological characteristics as strain ZWLR2-1, this mutant was not able to degrade 2CNB. PCR diagnostic assays indicated that this mutant had lost several 2CNB catabolic genes, including cnbAc, cnbAd, and cnbC. This further confirmed the involvement of the cnb catabolic cluster in 2CNB degradation.


Based on our results, we suggest that 2CNB is subject to an initial attack by a dioxygenase to form 3-chlorocatechol with concomitant nitrite release, possibly via a nitrohydrodiol intermediate, as indicated in Fig. 1A. This reaction is analogous to nitrobenzene dioxygenation in Comamonas sp. strain JS765 (11, 13) and 2-nitrotoluene dioxygenation in Pseudomonas sp. strain JS42 (1, 14). These reactions are catalyzed by homologous enzymes of 2CNB dioxygenase without involving a dehydrogenase, and corresponding nitrohydrodiols are proposed as unstable intermediates (1, 13). The resulting 3-chlorocatechol from 2CNB oxidation is subsequently metabolized via the chlorocatechol ortho ring cleavage pathway, as shown in Fig. 1A. It is therefore reasonable to suggest that the 2CNB degradation pathway in this strain is due to patchwork assembly of nitroarene dioxygenase and 3-cholorcatechol degradation clusters in a natural evolution event (Fig. 1B to D). This theory is supported by a recently reported artificial evolution event in which a nitroarene dioxygenase introduced into Ralstonia sp. strain JS705, with a modified ortho pathway for chlorocatechol metabolism, resulted in the ability to grow on all three chloronitrobenzene isomers (7).

The Nag operon in strain U2 (25) is generally accepted as the progenitor of both the archetypal naphthalene dioxygenases and nitroarene dioxygenases (type III oxygenase) described thus far (6, 8). The nitroarene dioxygenase involved in 2CNB degradation in this study is no exception, and this is the first example of a chlorinated nitroarene oxidized by this class of dioxygenase in a natural catabolic pathway. Unlike the other four nitroarene dioxygenase clusters shown in Fig. 1D, the cnbAcAd cluster does not have an upstream nagR-like regulator-encoding gene and nagAaGHAb-like genes (encoding ferredoxin reductase and large and small oxygenase components of salicylate-5-hydroxylase and ferredoxin, respectively, in strain U2). CnbAaAb (the electron transport chain for 2CNB dioxygenase) shares the highest identities with AndAaAb for anthranilate dioxygenase, a model of type IV oxygenase (3). It appears that the α and β subunits of 2CNB dioxygenase have origins different from those of ferredoxin and ferredoxin reductase. Therefore, the cnb cluster for 2CNB degradation appears to have evolved the furthest among the identified nitroarene degradation clusters; however, enough evidence remains to trace its evolutionary origin.

Based on a comparison with the homologous cluster in Sphingomonas sp. strain TFD44 (19), the cnbCDEF cluster may contain all of the genes necessary for 3-chlorocatechol conversion to 3-oxoadipate via an ortho ring cleavage pathway. IS6100 (tnpA) upstream of the 3-chlorocatechol degradation cluster also suggests that horizontal gene transfer and movement of transposable elements have contributed to this evolution. Of the clusters recruited by strain ZWLR2-1 for a novel 2CNB catabolic pathway, the dioxygenase for the 2CNB upper pathway appears to originate from a nitroarene degradation pathway. However, the lower pathway probably arose from a chloroaromatic compound degradation pathway. Furthermore, there may be a progressive process toward such a compact region encoding the entire pathway. This observation is analogous to other excellent models for studying the molecular mechanisms of metabolic pathway evolution that have revealed the evolutionary origins of ring-hydroxylating dioxygenases in the 2,4-dinitrotoluene degrader B. cepacia R34 (6) and the diphenylamine degrader Burkholderia sp. strain JS667 (15). These are examples to increase our understanding of how bacteria have evolved to achieve efficient catabolic pathways in response to synthetic compounds.


This work was supported by grants from the National Natural Science Foundation of China (30770025 and 30970031) and the National High Technology Research and Development Program of China (2007AA10Z402).


[down-pointing small open triangle]Published ahead of print on 20 May 2011.


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