- Original article
- Open Access
Branching of the p-nitrophenol (PNP) degradation pathway in burkholderia sp. Strain SJ98: Evidences from genetic characterization of PNP gene cluster
- Surendra Vikram1,
- Janmejay Pandey1, 2,
- Nidhi Bhalla1,
- Gunjan Pandey1, 3,
- Anuradha Ghosh1, 4,
- Fazlurrahman Khan1,
- Rakesh K Jain^1 and
- Gajendra PS Raghava1Email author
© Vikram et al.; licensee Springer. 2012
Received: 18 April 2012
Accepted: 24 April 2012
Published: 8 June 2012
Aerobic microbial degradation of p-nitrophenol (PNP) has been classically shown to proceed via ‘Hydroquinone (HQ) pathway’ in Gram-negative bacteria, whereas in Gram-positive PNP degraders it proceed via ‘Benzenetriol (BT) pathway’. These pathways are characterized by the ring cleavage of HQ and BT as terminal aromatic intermediates respectively. Earlier reports on PNP degradation have indicated these pathways to be mutually exclusive. We report involvement of both ‘HQ’ and ‘BT’ ring cleavage pathways in PNP degradation by Burkholderia sp. strain SJ98. Genetic characterization of an ~41 Kb DNA fragment harboring PNP degradation gene cluster cloned and sequenced from strain SJ98 showed presence of multiple orfs including pnpC and pnpD which corresponded to previously characterized ‘benzenetriol-dioxygenase (BtD)’ and ‘maleylacetate reductase (MaR)’ respectively. This gene cluster also showed presence of pnpE1 and pnpE2, which shared strong sequence identity to cognate sub-units of ‘hydroquinone dioxygenase’ (HqD). Heterologous expression and biochemical characterization ascertained the identity of PnpE1 and PnpE2. In in vitro assay reconstituted heterotetrameric complex of PnpE1 and PnpE2 catalyzed transformation of hydroquinone (HQ) into corresponding hydroxymuconic semialdehyde (HMS) in a substrate specific manner. Together, these results clearly establish branching of PNP degradation in strain SJ98. We propose that strain SJ98 presents a useful model system for future studies on evolution of microbial degradation of PNP.
As variations to the above generalization, a few studies have reported alternative PNP degradation pathways. Chauhan and co-workers reported PNP degradation in Arthrobacter protophormiae strain RKJ100 to proceed via conversion of BT into hydroxy-benzoquinone (HBQ), which is subsequently converted into benzoquinone (BQ) and HQ (Chauhan et al., 2000). In another study, Jain et al. (1994) reported accumulation of both BT and HQ with 14 C trapping studies (at ~ 88% and 8% respectively) during PNP degradation by Arthrobacter sp. strain JS443. However, HQ could not be degraded further by strain JS443 and therefore it was suggested to be a degradation byproduct and/or dead-end degradation product (Jain et al. 1994). Genetic characterization of PNP degradation gene cluster of strain JS443 ascertained the above observation (Perry and Zylstra 2007). Another study reported PNP degradation by Pseudomonas sp. strain WBC-3 to selectively proceed via HQ. Noticeably, strain WBC-3 could also degraded 4-NC, wherein degradation selectively proceeds via formation of BT (Wei et al. 2010a). Based on these reports, it could be argued that microbial degradation of PNP proceeds via either BT or HQ branch of the pathway. Previously, we reported that Burkholderia sp. strain SJ98 (previously identified as Ralstonia sp. SJ98 (Samanta et al; 2000; Paul et al. 2008)); a Gram-negative bacterium, metabolizes PNP as sole source of carbon and energy. PNP induced culture of strain SJ98 degrade PNP with formation of BT and maleylacetate (MA) as major transformation intermediate, whereas 4-nitrocatechol (4-NC) and HQ are detected as transient intermediates (Chauhan et al. 2010). A 6.8 Kb DNA fragment cloned from genome of strain SJ98 showed presence of some of the genes involved in lower part of the PNP degradation pathway. Two genes viz., pnpC and pnpD showed highest sequence similarity with genes encoding for 1,2,3-benzenetriol dioxygenase (BtD) and maleylacetate reductase (MaR) respectively (Chauhan et al. 2010). Heterologous expression and biochemical characterization of these genes ascertained their identity.
In this study we have carried out further genetic characterization of PNP catabolic cluster/genes in strain SJ98. Our observations indicate co-occurrence of functional genes, encoding for enzymes involved in both ‘HQ and BT’ ring cleavage pathways of PNP degradation, within the PNP degradation gene cluster of strain SJ98. Based on these genetic evidences together with earlier report by Chauhan et al. (2010) we propose that PNP degradation in strain SJ98 shows branching and proceed via both BT and HQ branches of catabolic pathway. Similar observation for involvement of both the branches of PNP degradation pathway has been recently reported in Pseudomonas sp. 1–7 (Zhang et al. 2012). To the best of our knowledge, Burkholedria sp. strain SJ98 along with Pseudomonas sp.1-7 is one of the first strains showing involvement of both the branches of PNP degradation pathway.
Materials and methods
Bacterial strains, plasmids, chemicals, media and culture conditions
Bacterial strains and plasmids used in this study
Source or reference
Burkholderia sp. strain SJ98
Wild type PNP degrading isolate
Lab stock and DSM = 23195
E. coli DH5α
Host strain for cosmid cloning vectorHost strain for GateWay entry clone
E. coli BL-21 AI
Host strain for expression vector (LacY1DE3, F_ompT hsdS, gal, dcm, ara-I)
Invitrogen Inc. CA- USA
Cosmid cloning vector, Dual cos sites
Agilent Technologies- Genomics. CA, USA.
Cosmid clone with ~41 Kb insert harboring PNP degrading gene cluster of strain SJ98
Gateway entry cloning vector
Invitrogen Inc. CA, USA
Gateway expression vector
Invitrogen Inc. CA, USA
Expression clone with orf pnpE1 of strain SJ98
Expression clone with orf pnpE2 of strain SJ98
Nucleic acid isolation and genomic library construction
Total genomic DNA of strain SJ98 was isolated and purified using Qiagen DNA Isolation Kit (Qiagen, Germany). Plasmid DNA was isolated with Qiagen Miniprep plasmid DNA Purification Kit (Qiagen, Germany) according to manufacturer’s instructions. Total cellular RNA was isolated using Promega RNeasy Miniprep Kit (Promega, USA) following manufacturers protocol. For genomic library construction, restriction digestion fragments of Sau 3AI-digested genomic DNA of strain SJ98 were ligated to Bam HI-digested linearized cosmid vector SuperCos-1 (Stratagene, USA). Subsequent packaging was carried out using Gigapack III gold packaging extract (Stratagene, USA). The packaged DNA was amplified up to 10 folds and transformed into electro-competent E. coli DH5α cells, using GenePulser XcellTM Microbial Electroporation System (Bio-Rad Laboratories, USA). Transformed cells were spread plated onto LB- agar with ampicillin (100 μg/ml) and incubated at 37°C overnight.
Genomic library screening and DNA sequence analyses
In order to screen the genomic library for PNP degrading gene cluster, a PCR amplicon (~540 bp) corresponding to partial BtD gene amplified from genomic DNA of strain SJ98 (Chauhan et al., 2010), was used as radio-labeled DNA probe in colony hybridization analysis. Radio labeling of the amplicon was carried out using Megaprime DNA Labeling System (GE- Healthcare, USA). Positive clones obtained in colony hybridization assay were further confirmed with colony PCR and subsequently subjected to restriction digestion analysis for determining the size of the insert in each of these. One of these clones, designated as pSJC88, with ~41 Kb insert was selected for further studies. Nucleotide sequence of the insert was determined using an ABI Model 377 DNA Sequencer and the Auto-Assembler program (Applied Biosystems, USA). Annotation of the above DNA sequence for ORF search and homology analyses of the translated nucleotide sequence was carried out using ORF finder and BLAST programs (http://www.ncbi.nlm.nih.gov/). Pair-wise alignments and identity calculations were performed using Needlman-Wunsch global alignment algorithm (http://www.ebi.ac.uk/emboss/align). Vector NTI 10.0 (Invitrogen Technologies, USA) was used to generate the graphical output report of the annotated DNA fragment.
Cloning and expression of pnpE1 and pnpE2
Oligonucleotide primers designed and used in this study
Primer sequence (5’ → 3’)
HqD Large subunit amplification
HqD Small subunit amplification
HqD large subunit RT-PCR primer
HqD Small subunit RT-PCR primer
Partial BtD gene amplification
Preparation of cell extracts and purification of recombinant PnpE1 and PnpE2
500 ml culture of induced recombinant clones of pDEST-pnpE1 and pDEST-pnpE2 were harvested by centrifugation, washed twice with ice-cold 1X Phosphate buffered saline (PBS) and then re-suspended in 25 ml of cell lysis buffer (50 mM NaHPO4, 300 mM NaCl, 10% v/v glycerol and 10 mM Imidazole; pH 8.0) and lysed by ultrasonication. Soluble and insoluble fractions were separated through centrifugation at 20,000 g for 15 min. The separated fractions were tested for presence of over-expressed recombinant PnpE1 and PnpE2. Supernatant obtained from lysate of pDEST-pnpE2 showed presence of over-expressed protein, therefore it was purified with affinity chromatography using Ni-NTA Agarose-superflow columns (Qiagen, Germany), pre-equilibrated with lysis buffer. His-tagged PnpE2 was eluted with a linear gradient of imidazole ranging from 10 mM - 200 mM in the buffer above. Thereafter, the eluted fractions were dialyzed two times with 1 l of buffer containing 50 mM NaHPO4, 300 mM NaCl, 10% glycerol (pH 8.0). Purified protein fractions were stored with 20% glycerol at −20°C for further use. Purity of the eluted protein samples were tested by SDS–PAGE analysis. Since, majority of the over-expressed PnpE1 was found to be present in inclusion bodies, a denaturation, dialysis, and refolding procedure was followed in order to purify PnpE1.
Refolding and purification of PnpE1
Insoluble fractions collected from the cell lysate of E. coli recombinant with pDEST-pnpE1 was resuspended in denaturing buffer (50 mM NaHPO4, 6 M urea, 10 mM imidazole, 300 mM NaCl, 5 mM β-mercaptoethanol (pH 8.0)) and incubated overnight at room temperature. Afterwards, the resuspended sample was centrifuged at 14,000 g for 30 min to separate the soluble and insoluble fractions. The soluble fraction was separated and then subjected to affinity chromatography based purification using Ni-NTA Superflow column (Qiagen, Germany) under denaturing condition according to the manufacturer’s instruction. After elution with linear gradient of imidazole in denaturing buffer, PnpE1 was diluted 100 fold in the refolding buffer (50 mM NaHPO4 7.0, 10 mM imidazole, 300 mM NaCl, 5 mM BME and 10% glycerol (pH 8.0)) and allowed to refold at 4°C for 12 h. Refolded PnpE1 was again eluted through Ni-NTA Superflow beads (Qiagen, Germany) in the native purification buffer following the manufacturer’s instructions. The refolded PnpE1 was centrifuged at 14,000 × g for 30 minute to remove residual aggregate. PnpE1 was dialyzed twice with 1 l 50 mM NaHPO4 buffer, NaCl 300 mM, 10% Glycerol and stored at −20°C in 20% glycerol.
Reconstitution of hydroquinone dioxygenase (HqD) with PnpE1 and PnpE2
In order to reconstitute the HqD activity, the purified recombinant proteins PnpE1 and PnpE2 were mixed in different molar ratios ranging from 3:1 to 1:3 in 1 ml of 50 mM phosphate buffer @ pH 7.0. The reconstituted protein was incubated at 30°C for 1 h and then complex protein was purified with gel filtration chromatography using Sephacryl-200 column (GE, Healthcare, USA) on an AKTA chromatography/protein purification workstation (GE Healthcare, USA). The reconstituted HqD complex(s) were subsequently analyzed for HqD activity using a spectrophotometric activity assay. The reconstituted HqD complex showing positive HqD activity (described later) was determined by comparing the elution volume in the gel filtration chromatography and its comparison with standard proteins with known molecular weights.
HqD activity assay
The purified complexes from gel filtration chromatography described above were analyzed for HqD activity; positive HqD activity was determined spectrophotometrically by monitoring transformation of HQ used as the reaction substrate into 4-HMS (Absmax at 320 nm; ε320 = 11.0 mM -1 cm-1) as reported previously (Spain and Gibson 1991). The reaction mixture typically contained 50 mM NaHPO4 @ pH 7.5 and 100 μM FeSO4 and various concentrations of reconstituted HqD. Reactions were started by adding 100 μM hydroquinone prepared in dimethyl-formamide. One unit of HqD enzyme activity was defined as the amount of enzyme required for formation of 1 μmol HMS per minute. Protein concentration was measured routinely using Bradford reagent (Sigma, Germany). The gel filtration analysis carried out to determine the molecular weight of the reconstituted HqD and its purification to homogeneity was performed on a Sephacryl-200 column using Akta-prime (GE-Amersham, USA).
The HqD enzyme activity products were subjected to organic solvent extraction using equal volume of ethyl acetate thrice in neutral, acidic and alkaline pH and evaporated to dryness using a Rotavapour (Buchi, Switzerland). Extracted samples were re-suspended in 100 μl of ethyl acetate and subjected to gas chromatographic (GC) analysis using AutoSystem XL Gas chromatograph (Perkin Elmer, USA) equipped with the flame ionization detector. The thermal profile used for GC analysis was as follows: constant temperature for injector, oven and detector at 250°C, 200°C and 280°C respectively. Subsequent qualitative analysis of the reaction product was carried out with Gas chromatography–mass spectrometry (GC-MS) using Shimadzu QP2010 (Shimadzu Scientific Instruments., USA). The identity of the reaction product was ascertained by mass fragmentation pattern and its comparison with the compound database NIST62- LIB available on the GC-MS instrument.
Induciblity of pnpE1 and pnpE2 with RT- PCR analysis
Reverse transcription-Polymerase Chain Reaction (RT-PCR) analysis was carried out using a QIAGEN One-Step RT-PCR kit. Strain SJ98 was grown at 30°C overnight in MM with 20 mM sodium succinate. The culture was induced with 0.3 mM PNP once reached an OD600 of ~0.5 and was further incubated for 10–12 h at 30°C. Reverse transcriptase PCR (RT-PCR) analyses were performed to amplify internal fragments of pnpE1 and pnpE2 using primer pairs listed as pnpE1-RT_F, pnpE1-RT_R and pnpE2-RT_F, pnpE2-RT_R respectively (Table 2) (232 bp and 182 bp respectively) and total cellular RNA isolated as described earlier. Amplicons obtained from RT-PCR were analyzed with 1.2% agarose gel electrophoresis. Total cellular RNA isolated from cells of strain SJ98 grown on MM with 20 mM sodium succinate without PNP was used as a negative control.
Nucleotide sequence accession number
The nucleotide and amino acid sequence data reported in this paper have been deposited to the GenBank sequence database with accession numbers JN968480 and JN968481.
Isolation of pnp gene cluster from strain SJ98
In order to isolate and clone genes responsible for PNP degradation by Burkholderia sp. strain SJ98, a cosmid genomic library was constructed. In an earlier study the use of a partial btd gene fragment (~540 bp) as a probe to screen plasmid based genomic library has been described (Chauhan et al. 2010). The cosmid library was also screened using the same probe and a total of 8 positive cosmid clones (Additional file 1: Figure S1A and Additional file 1: Figure S1B). These positive clones were also verified by the amplification of btd specific DNA amplicon using colony PCR assay (Additional file 1: Figure S1C). Restriction digestion analysis was carried out to determine the length of the insert of the positive clones and found insert length in the range of 38 to 41 Kb (Additional file 1: Figure S1D). One of the positive clones designated as pSJC88 with insert size of ~41 Kb was selected for sequencing and further characterization.
Sequence annotation of pSJC88
Amino acid sequence comparison of the ORFs identified in pnp gene cluster of Burkholderia sp. strain SJ98
Hydroxyquinol dioxygenase, Pseudomonas sp. WBC-3
Maleylacetate reductase, P. fluorescens
Hydroquinone dioxygenase Large subunit, Pseudomonas sp. WBC-3
Hydroquinone dioxygenase Small subunit, Pseudomonas sp. WBC-3
4-hydroxymuconic semialdehyde dehydrogenase, Pseudomonas fluorescens
Heterologous expression of pnp E1 and pnp E2
Reconstitution of active HqD complex
Catalytic properties, substrate specificity and stability of HqD from strain SJ98
Catalytic properties of HqD were routinely analyzed with spectrophotometric assay that monitors ring cleavage using HQ as reaction substrate and resulting in formation of 4 − HMS (as described above). HqD activity was observed only in presence of Fe(II) and maximum activity was measured at 100 μM FeSO4. HqD from strain SJ98 shows a broad pH optima ranging from pH 7.0 – 8.0 with maximum activity at pH 7.2 (Figure 3Bi). Optimal temperature for HqD activity was observed in a range of 25 – 30°C (Figure 3Bii). Kinetic analyses showed a Vmax of 9.85 ± 0.4152 μmole min-1 μg-1 and Km of 65.32 ± 10.49 μM (at pH 7.0 and 25°C) as determined from the Michaelis-Menten plot for kinetic analyses (Figure 3Biii). Reconstituted HqD catalyzed ring cleavage in a substrate specific manner with only HQ as the reaction substrate. Neither substitute hydroquinones (e.g. Chlorohydroquinone) nor other biphenolic compounds (e.g. catechol, resorcinol, chlorocatechol and nitrocatechol) were used as the reaction substrate by this enzyme (data not shown). In presence of ferrous ion chelator e.g. 2,2’-dipyridyl, the activity of HqD from strain SJ98 was completely abolished. The inactivation of enzyme activity appeared irreversible since it could not be restored by extended incubation at lower temperature with or without Fe(II). Rapid and complete inactivation of reconstituted HqD also occurred upon incubation with 100 μM hydrogen peroxide. Incubation at 40°C for 30 min and 45°C for 15 min also resulted in complete inactivation of the HqD activity.
Induction of pnpE1 and pnpE2 in strain SJ98 during growth on PNP
It is important to understand whether pnpE1 and pnpE2 are selectively induced during growth of strain SJ98 on PNP. DNA-free total cellular RNA isolated from cells of strain SJ98, grown with and without PNP. These isolates were analyzed using RT-PCR, targeting transcripts corresponding to the above genes. In this analysis, the primer pairs were designed to amplify internal fragments corresponding to pnpE1 and pnpE2 transcript. RT-PCR products of expected size could be successfully amplified using RNA purified from SJ98 culture grown on PNP but not from cultures grown without PNP (Additional file 1: Figure S3). The RT- PCR products were also not observed when chemical analogues of HQ e.g. 2-chlorohydroquinone, 2-methylhydroquinone or intermediates involved in the ‘BT’ cleavage pathway e.g. 4-NC and BT were used as inducer during cell growth of strain SJ98 (data not shown). These results provide strong indication for involvement of HqD is involved in PNP degradation process.
Genetic characterization of gene(s) and regulatory elements involved in degradation of xenobiotic compounds is extremely important for successful development of bioremediation technologies as well as for basic understanding of biochemical and molecular mechanisms of degradation process. Hitherto, a number of studies have reported cloning and characterization of PNP degradation gene clusters. During present study, we cloned, sequenced and characterized a PNP degrading DNA fragment from Burkholderia sp. strain SJ98. The sequence analyses and annotation of this fragment indicated that the organization of pnp gene cluster in strain SJ98 is very similar to that of the nph gene cluster of Arthrobacter sp. strain JS443 (Perry and Zylstra 2007). However, the only striking difference is the presence of two orfs within pnp cluster of strain SJ98 that share strong sequence similarity with genes encode for cognate subunits of HqD. During earlier studies, we detected HQ as a transient metabolite in PNP degradation by strain SJ98; however, in absence of genetic information, HQ could not be ascertained as a true degradation intermediate. Instead, it was suggested that strain SJ98 may non- specifically transform PNP into HQ as a dead end product which does not contribute to the active catabolism of PNP. Similar observation has also been reported with PNP degradation by strain JS443 (Jain et al. 1994; Perry and Zylstra 2007). Results obtained during the present study and previous study with strain SJ98 clearly establish presence of a functional genes encoding for enzymes involved in both the branches of PNP degradation (viz., HqD and BtD) within the PNP degradation gene cluster of strain SJ98. Characterization of HqD carried out during present study established that this enzyme catalyzes Fe(II) dependent transformation of HQ to 4-HMS. This observation indicates that HqD from strain SJ98 belongs to ‘Ferrous ion dependent type II hydroquinone dioxygenase. Also, the active reconstituted HqD complex as predicted on the basis of the molecular weights of complex, PnpE1 and PnpE2 was found to be a heterotetrameric complex consisting of 2 molecules of both subunits. These observations are in agreement with earlier reports of HqD from other microorganisms. Ferrous ion dependent, heterotetrameric HqD complex has been previously reported to catalyze ring cleavage of HQ formed during degradation of hydroxyacetophenone (Moonen et al. 2008) and PNP (Shen et al. 2010; Zhang et al. 2009). The apparent molecular weight of reconstituted HqD from strain SJ98 (~ 120 kDa) is also within the range of molecular weights (112 kDa – 120 kDa) of other reported HqDs (Shen et al. 2010; Spain and Gibson 1991; Moonen et al. 2008; Kolvenbach et al. 2011). Noticeably, HqD from strain SJ98 also showed stringent substrate specificity in the activity assay and is selectively induced during growth of strain SJ98 on PNP; these observations clearly indicate it to be involved in transformation of HQ produced during PNP catabolism by strain SJ98. Conspicuously, stringent substrate specificity has not been observed with HqDs characterized from Sphingomonas sp. strain TTNP-3, Pseudomonas fluorescens strain ABC, Cupriavidus necator JMP134 and Pseudomonas sp. strain NyZ402 (Moonen et al. 2008; Kolvenbach et al. 2011; Yin and Zhou 2010; Wei et al. 2010b). The reason and molecular mechanism for such different behavior is unclear and is still under investigation.
Based on the results presented above, we propose orf pnp E1 and pnp E2 as genes encoding for functional cognate subunits of HqD that catalyze transformation reaction involved in HQ branch. Previously, orfs pnpC and pnpD were characterized as genes encoding for BtD and MaR that are involved in BT branch of PNP degradation pathway. Based on these results we show a graphical representation of the branching organization of pnp gene cluster from strain SJ98 (Figures 1A and B). Results obtained from RT-PCR analyses targeting mRNA transcripts corresponding to pnpE1 and pnpE2 in strain SJ98 cells grown with PNP demonstrated PNP specific induction of these genes. Previously, Chauhan et al. (2010) detected BtD and MaR specific transcripts in PNP induced cells of strain SJ98 indicating their inducible nature. Together these results present conclusive evidence for branching of PNP degradation pathway in any PNP degrading isolate. Similar observation was reported by Zhang et al. in Pseaudomonas sp. stain 1–7 where activity of four PNP degradation genes (pdcDEFG) expressed in heterologous system in vitro, supports the existence of HQ and BT mediated degradation pathways (Zhang et al. 2012).
Few earlier studies on genetic characterization of PNP degrading Pseudomonads viz., Pseudomonas sp. strain WBC and Pseudomonas putida strain DLL-E4 also reported the presence of both BtD and HqD within the same genetic locus (Shen et al. 2010; Wei et al. 2010a; Zhang et al. 2009). However, the PNP degradation in these strains proceeds only via HQ ring cleavage pathway and utilizes HqD whereas the BT cleavage enzyme BtD is only selectively utilized in 4-NC degradation (Kitagawa et al. 2004). Similar involvement for both HQ and BT branches of degradation has been reported in the degradation of 4-chlorophenol (4-CP) by A. chlorophenolicus A6 (Nordin et al. 2005). Nordin et al. (2005) reported involvement of two pathways but the terminal aromatic ring cleavage intermediate is the hydroxyquinol (Nordin et al. 2005). One branch of degradation of 4-CP by A. Chlorophenolicus A6 is proceeds via the hydroquinone and then finally into the hydroxyquinol whereas the other branch of this pathway ocuurs via the conversion of 4-CP to chlorohydroxyquinol and finally into the hydroquinone which subsequently transformed into hydroxyquinol (Nordin et al. 2005; Unell et al. 2007; Unell et al. 2009).
The annotated pnp gene cluster of Strain SJ98 also indicates presence of other genes required for the respective pathways. Further, studies for heterologous expression, purification, and biochemical characterization of other genes are currently underway to rigorously demonstrate involvement of both the pathways. Results obtained with biochemical characterization of purified HqD during the present study clearly demonstrate that HqD from strain SJ98 is a Fe(II) requiring, type II, heterotetrameric HqD and it represents the first enzyme of this type characterized from a member of the genus Burkholderia. It is actively involved in catabolic degradation of PNP by strain SJ98. Furthermore it gets selectively induced during growth of strain SJ98 on PNP. Data gathered in this study and also supported by our previous findings present conclusive evidence for co-occurrence of both ‘HQ and BT’ ring cleavage branches of PNP degradation pathway in strain SJ98. We propose Burkholderia sp. strain SJ98 as an important model system for further studies on evolution of PNP degradation pathways.
This study was carried out in part with financial support from Council for Scientific and Industrial Research (CSIR)-India, and Indo-Australian collaboration in Biotechnology (IACB). SV, JP, AG and FK acknowledge the research fellowship granted by CSIR India. We are thankful to Ms. Archana Chauhan for suggestion during course of the study and to Mr. Dhan Prakash for his technical help in carrying out some of the experiments. This is the IMTECH communication number 085/2011.
- Bhushan B, Chauhan A, Samanta SK, Jain RK: Kinetics of biodegradation of p-nitrophenol by different bacteria. Biochem Biophys Res Commun 2000, 274: 626–630. 10.1006/bbrc.2000.3193View ArticlePubMedGoogle Scholar
- Chauhan A, Chakraborti AK, Jain RK: Plasmid-encoded degradation of p-nitrophenol and 4-nitrocatechol by Arthrobacter protophormiae. Biochem Biophys Res Commun 2000, 270: 733–740. 10.1006/bbrc.2000.2500View ArticlePubMedGoogle Scholar
- Chauhan A, Pandey G, Sharma NK, Paul D, Pandey J, Jain RK: p-Nitrophenol degradation via 4-nitrocatechol in Burkholderia sp. SJ98 and cloning of some of the lower pathway genes. Environ Sci Technol 2010, 44: 3435–3441. 10.1021/es9024172View ArticlePubMedGoogle Scholar
- Eppink MH, Cammaart E, Van Wassenaar D, Middelhoven WJ, van Berkel WJ: Purification and properties of hydroquinone hydroxylase, a FAD-dependent monooxygenase involved in the catabolism of 4-hydroxybenzoate in Candida parapsilosis CBS604. Eur J Biochem 2000, 267: 6832–6840. 10.1046/j.1432-1033.2000.01783.xView ArticlePubMedGoogle Scholar
- Jain RK, Dreisbach JH, Spain JC: Biodegradation of p-nitrophenol via 1,2,4-benzenetriol by an Arthrobacter sp. Appl Environ Microbiol 1994, 60: 3030–3032.PubMed CentralPubMedGoogle Scholar
- Kadiyala V, Spain JC: A two-component monooxygenase catalyzes both the hydroxylation of p-nitrophenol and the oxidative release of nitrite from 4-nitrocatechol in Bacillus sphaericus JS905. Appl Environ Microbiol 1998, 64: 2479–2484.PubMed CentralPubMedGoogle Scholar
- Kitagawa W, Kimura N, Kamagata Y: A novel p-nitrophenol degradation gene cluster from a gram-positive bacterium, Rhodococcus opacus SAO101. J Bacteriol 2004, 186: 4894–4902. 10.1128/JB.186.15.4894-4902.2004PubMed CentralView ArticlePubMedGoogle Scholar
- Kolvenbach BA, Lenz M, Benndorf D, Rapp E, Fousek J, Vlcek C, Schäffer A, Gabriel FL, Kohler HP, Corvini PF: Purification and characterization of hydroquinone dioxygenase from Sphingomonas sp. strain TTNP3. AMB Express 2011, 1: 8. 10.1186/2191-0855-1-8PubMed CentralView ArticlePubMedGoogle Scholar
- Liu H, Zhang JJ, Wang SJ, Zhang XE, Zhou NY: Plasmid-borne catabolism of methyl parathion and p-nitrophenol in Pseudomonas sp. strain WBC-3. Biochem Biophys Res Commun 2005, 334: 1107–1114. 10.1016/j.bbrc.2005.07.006View ArticlePubMedGoogle Scholar
- Moonen MJ, Synowsky SA, van den Berg WA, Westphal AH, Heck AJ, van den Heuvel RH, Fraaije MW, van Berkel WJ: Hydroquinone dioxygenase from Pseudomonas fluorescens ACB: a novel member of the family of nonheme-iron(II)-dependent dioxygenases. J Bacteriol 2008, 190: 5199–5209. 10.1128/JB.01945-07PubMed CentralView ArticlePubMedGoogle Scholar
- Nordin K, Unell M, Jansson JK: Novel 4-chlorophenol degradation gene cluster and degradation route via hydroxyquinol in Arthrobacter chlorophenolicus A6. Appl Environ Microbiol 2005, 71: 6538–6544. 10.1128/AEM.71.11.6538-6544.2005PubMed CentralView ArticlePubMedGoogle Scholar
- Paul D, Rastogi N, Krauss U, Schlomann M, Pandey G, Pandey J, Ghosh A, Jain RK: Diversity of ‘benzenetriol dioxygenase’ involved in p-nitrophenol degradation in soil bacteria. Indian J Microbiol 2008, 48: 279–286. 10.1007/s12088-008-0038-xPubMed CentralView ArticlePubMedGoogle Scholar
- Perry LL, Zylstra GJ: Cloning of a gene cluster involved in the catabolism of p-nitrophenol by Arthrobacter sp. strain JS443 and characterization of the p-nitrophenol monooxygenase. J Bacteriol 2007, 189: 7563–7572. 10.1128/JB.01849-06PubMed CentralView ArticlePubMedGoogle Scholar
- Samanta SK, Bhushan B, Chauhan A, Jain RK: Chemotaxis of a Ralstonia sp. SJ98 toward Different Nitroaromatic Compounds and Their Degradation. Biochem Biophys Res Commun 2000, 269: 117–123. 10.1006/bbrc.2000.2204View ArticlePubMedGoogle Scholar
- Shen W, Liu W, Zhang J, Tao J, Deng H, Cao H, Cui Z: Cloning and characterization of a gene cluster involved in the catabolism of p-nitrophenol from Pseudomonas putida DLL-E4. Bioresour Technol 2010, 101: 7516–7522. 10.1016/j.biortech.2010.04.052View ArticlePubMedGoogle Scholar
- Spain JC: Biodegradation of nitroaromatic compounds. Annu Rev Microbiol 1995, 49: 523–555. 10.1146/annurev.mi.49.100195.002515View ArticlePubMedGoogle Scholar
- Spain JC, Gibson DT: Pathway for Biodegradation of p-Nitrophenol in a Moraxella sp. Appl Environ Microbiol 1991, 57: 812–819.PubMed CentralPubMedGoogle Scholar
- Takeo M, Murakami M, Niihara S, Yamamoto K, Nishimura M, Kato D, Negoro S: Mechanism of 4-nitrophenol oxidation in Rhodococcus sp. Strain PN1: characterization of the two-component 4-nitrophenol hydroxylase and regulation of its expression. J Bacteriol 2008, 190: 7367–7374. 10.1128/JB.00742-08PubMed CentralView ArticlePubMedGoogle Scholar
- Unell M, Abraham PE, Shah M, Zhang B, Ruckert C, et al.: Impact of phenolic substrate and growth temperature on the Arthrobacter chlorophenolicus proteome. J Proteome Res 2009, 8: 1953–1964. 10.1021/pr800897cView ArticlePubMedGoogle Scholar
- Unell M, Nordin K, Jernberg C, Stenstrom J, Jansson JK: Degradation of mixtures of phenolic compounds by Arthrobacter chlorophenolicus A6. Biodegradation 2007, 19: 495–505.View ArticlePubMedGoogle Scholar
- Wei M, Zhang JJ, Liu H, Zhou NY: para-Nitrophenol 4-monooxygenase and hydroxyquinol 1,2-dioxygenase catalyze sequential transformation of 4-nitrocatechol in Pseudomonas sp. strain WBC-3. Biodegradation 2010, 21: 915–921. 10.1007/s10532-010-9351-2View ArticlePubMedGoogle Scholar
- Wei Q, Liu H, Zhang JJ, Wang SH, Xiao Y, Zhou NY: Characterization of a para-nitrophenol catabolic cluster in Pseudomonas sp. strain NyZ402 and construction of an engineered strain capable of simultaneously mineralizing both para- and ortho-nitrophenols. Biodegradation 2010, 21: 575–584. 10.1007/s10532-009-9325-4View ArticlePubMedGoogle Scholar
- Yin Y, Zhou NY: Characterization of MnpC, a hydroquinone dioxygenase likely involved in the meta-nitrophenol degradation by Cupriavidus necator JMP134. Curr Microbiol 2010, 61: 471–476. 10.1007/s00284-010-9640-3View ArticlePubMedGoogle Scholar
- Zhang JJ, Liu H, Xiao Y, Zhang XE, Zhou NY: Identification and characterization of catabolic para-nitrophenol 4-monooxygenase and para-benzoquinone reductase from Pseudomonas sp. strain WBC-3. J Bacteriol 2009, 191: 2703–2710. 10.1128/JB.01566-08PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang S, Sun W, Xu L, Zheng X, Chu X, Tian J, Wu N, Fan Y: Identification of the para-nitrophenol catabolic pathway, and characterization of three enzymes involved in the hydroquinone pathway, in Pseudomonas sp. 1–7. BMC Microbiol 2012, 12: 27. 10.1186/1471-2180-12-27PubMed CentralView ArticlePubMedGoogle Scholar
- Zheng Y, Liu D, Liu S, Xu S, Yuan Y, XIONG L: Kinetics and mechanisms of p-nitrophenol biodegradation by Pseudomonas aeruginosa HS-D38. J Environ Sci 2009, 21: 1194–1199. 10.1016/S1001-0742(08)62403-1View ArticleGoogle Scholar
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