APPLIED MICROBIAL AND CELL PHYSIOLOGY

Reductive dehalogenation mediated initiation of aerobicdegradation of 2-chloro-4-nitrophenol (2C4NP)by Burkholderia sp. strain SJ98

Janmejay Pandey & Hermann J. Heipieper &

Archana Chauhan & Pankaj Kumar Arora &

Dhan Prakash & M. Takeo & Rakesh K. Jain

Received: 18 January 2011 /Revised: 24 March 2011 /Accepted: 25 March 2011 /Published online: 28 May 2011# Springer-Verlag 2011

Abstract Burkholderia sp. strain SJ98 (DSM 23195) waspreviously isolated and characterized for degradation andco-metabolic transformation of a number nitroaromaticcompounds. In the present study, we evaluated its metabolicactivity on chlorinated nitroaromatic compounds (CNACs).Results obtained during this study revealed that strain SJ98can degrade 2-chloro-4-nitrophenol (2C4NP) and utilize itas sole source of carbon, nitrogen, and energy underaerobic conditions. The cells of strain SJ98 removed2C4NP from the growth medium with sequential releaseof nearly stoichiometric amounts of chloride and nitrite inculture supernatant. Under aerobic degradation conditions,2C4NP was transformed into the first intermediate that wasidentified as p-nitrophenol by high-performance liquid

chromatography, LCMS-TOF, and GC-MS analyses. Thistransformation clearly establishes that the degradation of2C4NP by strain SJ98 is initiated by “reductive dehaloge-nation”; an initiation mechanism that has not beenpreviously reported for microbial degradation of CNACunder aerobic conditions.

Keywords Chlorinated nitroaromatic compounds .

Burkholderia . Aerobic degradation

Introduction

Chloronitroaromatic compounds (CNACs) such as chloro-nitrophenols are widely used as important building blocks forthe chemical synthesis of dyes, drugs, explosives, andpesticides resulting in severe contamination of several envi-ronmental niches (Shen et al. 2008; Steinwandter 1987;Yoshida 1993; Yurawecz and Puma 1983). Studies carriedout for testing the toxicity of CNACs have suggested them tobe weakly mutagenic; however, they may cause diseases anddisorders such as methemoglobinemia, skin cancer, growthdefects, and immune suppression in humans and animals(Linch 1974; Shimizu et al. 1983). Based on the concerns fortheir potential toxicity, the European Economic Communityand the United States Environment Protection Agency haveenlisted a number of CNACs as priority pollutants (EuropeanEconomic Community 1982), thereby requiring immediateattention for regulating their production, waste management,and decontamination of the ecological niches polluted withthese compounds (Katsivela et al. 1999).

Microbial degradation could be used as an economic andenvironment friendly method to decontaminate sites con-taminated with CNACs; however, presently there is only

Electronic supplementary material The online version of this article(doi:10.1007/s00253-011-3254-y) contains supplementary material,which is available to authorized users.

J. Pandey :A. Chauhan : P. K. Arora :D. Prakash : R. K. Jain (*)Institute of Microbial Technology (CSIR),Sector 39-A,Chandigarh 160036, Indiae-mail: rkj@imtech.res.in

R. K. Jaine-mail: rakesh110@yahoo.com

H. J. HeipieperDepartment of Environmental Biotechnology,Helmholtz Centre for Environmental Research—UFZ,Permoserstrasse 15,04318 Leipzig, Germany

M. TakeoDivision of Material Engineering,Graduate Institute of Engineering, Himeji Institute of Technology,2167 Shosha, Himeji,Hyogo 671-2201, Japan

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very limited information available for the microbialdegradation of CNACs. Till date, there are only a veryfew microorganisms belonging to genera Ralstonia, Coma-monas, and Rhodococcus that have been characterized withthe ability to degrade and to utilize them as the sole sourceof carbon and energy (Ghosh et al. 2010; Katsivela et al.1999; Lenke and Knackmuss 1996; Schenzle et al. 1999).Alternatively, some studies have reported CNACs degrada-tion by mixed bacterial consortia. In one such study,degradation of 4-chloro-2-nitrophenol (4C2NP) was carriedout by mixed culture of Enterobacter cloacae and Alcali-genes sp. TK-2 (Beunink and Rehm 1990). In anotherstudy, a 4C2NP assimilatory bacterium was constructed bybringing together the nitrite removal activity and thechlorocatechol degradation ability of Pseudomonas sp.strain N31 and Ralstonia eutropha JMP134, respectively(Bruhn et al. 1988). Despite these attempts, informationavailable on the degradation of CNACs by single bacterialisolate(s) is extremely scarce. Furthermore, the mechanism,chemical reactions, and metabolic pathways involved inthe microbial degradation of CNACs are still not wellcharacterized.

Structurally, CNACs are chemical analogues of nitro-aromatic compounds (NACs) and differ from later only bythe occurrence of one or more chlorine side groups.However, the presence of Cl− as additional electronwithdrawing group results in re-enforcement of electroni-cally destabilized aromatic ring (Cozza and Woods 1992;Wu et al. 2006). Such electronically deficient aromaticcompounds are extremely resistant towards oxidativeenzymes, which are usually involved in the degradation ofcompounds with aromatic core (Spain 1995; Symons andBruce 2006). This effect renders CNACs extremelyrecalcitrant to the electrophilic attack of oxidative enzymes.Some microorganisms can overcome this limitation bymaking use of specialized enzymes that catalyze reductionof aromatic ring by nucleophilic addition of H− in thepresence of hydride donor, e.g., NADPH (Fetzner 1998;van der Meer 1997; van den Tweel et al. 1987), andsubsequently, transformed product could be degraded viause of oxidative transformation reaction. In light of thisinformation, it could be proposed that the reactionscontributing for the “upper pathway” in general and“degradation initiation” in particular attain a pivotal rolein the microbial degradation of CNACs. In order to developfurther understanding of the degradation of CNACs, it isimportant to isolate and characterize CNACs degradingmicroorganisms.

In the present report, we describe the characterization ofmetabolic activity of Burkholderia sp. strain SJ98 on modelCNACs, i.e., chloro-nitrophenol isomers viz., 2-chloro-4-nitrophenol (2C4NP), 4-chloro-2-nitrophenol (4C2NP), 4-chloro-3-nitrophenol (4C3NP), and 3-chloro-4-nitrophenol

(3C4NP). The results obtained during the study establishedthat strain SJ98 could degrade 2C4NP and utilize it as solesource of carbon, nitrogen, and energy under aerobicconditions. Further studies were performed for the identi-fication of degradation intermediates and the characteriza-tion of some of the important reactions of the metabolicpathway for 2C4NP degradation by strain SJ98. This is oneof the few studies wherein a single microbial isolate hasbeen characterized for the degradation of a CNAC alongwith the characterization of the critical upper part of themetabolic pathway for degradation.

Materials and methods

Bacterial strain, culture conditions and measurementof growth

Burkholderia sp. SJ98 was previously isolated by “chemo-tactic enrichment culture technique” (Samanta et al. 2000) andcharacterized for degradation/co-metabolic transformation ofa number of NACs (Pandey et al. 2002; Samanta et al. 2000).Strain SJ98 has been deposited in DSMZ under thedeposition number 23195. During the present study, strainSJ98 was grown in minimal medium (MM) supplementedwith 100–500 μM of test CNACs. Minimal media wasprepared as described earlier (Ghosh et al. 2010). Nitrogen-free MM was prepared by eliminating NH4Cl from prepara-tion. Individual CNACs were added to MM from filter-sterilized stocks prepared in distilled deionized water. Nutrientbroth (NB) and nutrient agar were used as rich media forbacterial growth and culture maintenance. All incubationswere carried out at 30 °C under aerobic conditions withagitation (180 rpm) for liquid cultures. Bacterial growth wasmonitored with spectrophotometric measurement of opticaldensity of the culture at 600 nm using Perkin-Elmer UV–visspectrophotometer Lambda EZ 201.

Growth and resting cell studies

To determine whether strain SJ98 utilizes 2C4NP, 4C2NP,4C3NP, and 3C4NP as the sole source of carbon and energy,growth studies were carried out using 50 ml MM supple-mented with 100–500 μMof the above compounds as the solesource of carbon and energy. The ability to utilize the abovecompounds was determined by monitoring the growth ofstrain SJ98 spectrophotometrically as described above. Sub-sequent growth studies were performed with 2C4NP only todetermine the growth kinetics, the quantitation of substratedegradation, and the identification of degradation intermedi-ates as it was found to be the only test CNAC that wascompletely degraded and utilized as the sole source of carbonand energy. During growth studies, parallel experiments were

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performed using nitrogen-free MM supplemented with300 μM of 2C4NP as the sole source of nitrogen to determineif 2C4NP could also be used as sole source of nitrogen.

Resting cell studies were performed with minormodification of the method described earlier (Ghosh etal. 2010). Briefly, NB grown cells of strain SJ98 (250 ml)were induced at mid- log phase (OD600 ~ 0.5), harvested,washed with 100 ml MM, and finally, resuspended in20 ml of MM. Filter sterilized 2C4NP stock solution wasadded to obtain final concentration of 300 μM. Samples(0.5 ml aliquots) were collected at different time points,centrifuged, and filtered to obtain cell-free culture super-natants. These supernatants were later subjected tochromatographic analyses for the identification and thequantitation of degradation intermediates. Quantitativeanalysis was done with high-performance liquid chroma-tography (HPLC) and GC using a standard curve preparedwith authentic standards. Experiments were performed intriplicate with appropriate abiotic controls.

Chloride release, nitrite release, and ammonia release

In order to obtain preliminary information about the nature(oxidative vs. reductive) of 2C4NP degradation in strainSJ98, samples collected from the growth studies and restingcell studies were subjected to Cl−, NO2

−, and NH3 releaseassays. Chloride release was detected with chromogenicmethod as described earlier (Manickam et al. 2006).Similarly, nitrite release was detected with spectrophoto-metric method described earlier (Ghosh et al. 2010).Ammonia release was tested with the “Ammonia AssayKit” available from Sigma-Aldrich (GmbH, Steinheim,Germany). The amount of chloride and nitrite released inthe growth medium was determined by comparing with thestandard calibration curves generated with known quantitiesof NaCl and NaNO3, respectively. The amounts ofammonia were determined by comparison with the stand-ards provided along with the “Ammonia Assay Kit.”

HPLC and LCMS-TOF analysis

The cell-free aqueous samples (collected as culture super-natants) were directly analyzed with HPLC. A WatersHPLC system, model Waters-600 (Milford, Massachusetts,USA), equipped with a photodiode array detector systemover a wavelength range of 190–395 nm, was used for thequantitative analysis of 2C4NP degradation intermediates.Fifteen microliters of culture supernatant was injected fromeach sample, and metabolites were separated on a C18

reverse-phase silica column using 1% glacial acetic acid inmethanol and 1% glacial acetic acid in HPLC grade waterat a ratio of 80:20 as the mobile phase at constant flow rateof 1.0 ml min−1.

The qualitative analysis of the metabolites was per-formed with LCMS-TOF mass spectrometer (AgilentTechnologies, CA, USA) with an electrospray ionizationsource. The mass spectrometer was connected to the HPLCstream after the DAD detector. Nitrogen was used at a flowrate of l min−1 as a drying gas. The capillary temperaturewas 350 °C, and the spray voltage was 3 kV. The scanningwas done in positive ion mode, and the fragmenter and theskimmer were set at 120 and 60 V, respectively, for scans inthe range 22 of 50–450 m/z.

TLC, GC, and GC-MS analysis

The remaining aqueous samples were subjected to doublephase organic solvent extraction using the proceduredescribed earlier (Ghosh et al. 2010). Briefly, cell-freeaqueous culture supernatant was thoroughly mixed with anequal volume of ethyl acetate (under neutral pH) byagitation followed by the separation of aqueous and organiclayers. The organic layer was collected, whereas, theremaining aqueous layer was acidified to pH 2.5 with5 N–HCl and then mixed thoroughly with two volumes ofethyl acetate. The organic and aqueous layers wereseparated again. Subsequently, organic layer from acidicextraction was pooled with organic layer collected fromneutral phase extraction. The pooled organic layers wereevaporated to dryness with rotary evaporator; B-490heating bath (BUCHI-Postfach, Flawil, Switzerland) andlater dissolved in 100 μl of ethyl acetate or methanol.

For TLC analysis, 10 μl of extracted samples wasspotted on a 20×20-cm silica gel 60 F254 TLC sheets(Merck, Darmstadt, Germany) and the separation ofmetabolites was achieved with mobile phase havingtoluene, ethyl acetate, and acetic acid in the ratio of60:35:5. The Rf values for different spots (in standardsand samples) were calculated according to the standardmethod as described earlier (Pandey et al. 2003). Thesamples were also resolved on a preparative TLC usingpreparative TLC plates (20×20-cm silica gel 60 F254-coated25-mm-thick plates) from Merck (Darmstadt, Germany).The sample spots along with silica were cut out from thesheet and subjected to the organic extractions as describedabove.

Extracted samples were also analyzed with GC usingAutoSystem-XL Gas chromatograph (Perkin-Elmer Inc.Waltham, MA, USA) equipped with flame-ionizationdetector. Temperatures for injector, oven, and detector werekept constant at 280 °C, 200 °C, and 250 °C, respectively.The identities of sample analytes were predicted on thebasis of the comparison with the retention time of standardcompounds. The qualitative analysis of metabolites wascarried out with GC-MS analysis performed with a GCMS-QP2010S (Shimadzu Scientific Instruments, MD, USA).

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Metabolite peaks within the molecular weight range of40–700 were analyzed for mass fragmentation andlibrary match. Identity of the peaks and their respectivemass fragments was ascertained by comparison ofcompound database NIST62. LIB, available with theGC-MS instrument.

The quantitation of degradation intermediates formed atdifferent time points during the degradation of 2C4NP bystrain SJ98 was carried out with quantitative HPLC and/orquantitative GC analysis. Procedures used for quantitationwere identical to those described above. Standard curvesgenerated with known quantities of respective standards ofidentified degradation intermediates were used as thereference for the quantities.

Ring cleavage inhibition studies

The ring cleavage inhibition study was performed with ironchelator viz., 2,2′-dipyridyl, which is a known inhibitor of anumber of heme-dependent aromatic ring hydroxylatingenzymes and has been used to selectively inhibit ringcleavage during microbial degradation of aromatic com-pound (Chapman and Hooper 1968; Ferreira et al. 2008;Kadiyala and Spain 1998). One milliliter of NB grown seedculture of strain SJ98 was inoculated in 100 ml MMsupplemented with 300 μM 2C4NP and 10 mM glucose.2,2′-Dipyridyl was added to the medium to obtain aworking concentration of 1 mM. Culture supernatant(10 ml each) was collected at different time points duringthe incubation and extracted with double phase solventextraction. The extracted samples were analyzed withHPLC and GC analysis as described above.

Enzyme assays with cell-free lysate

For cell-free lysate preparation, 2C4NP-induced cells ofstrain SJ98 were harvested, washed twice with ice-cold 1×PBS, and subjected to ultra-sonication using Vibra-CellVCX 130 (Sonics & Materials, Inc., Newtown, CT, USA).The sonicated samples were centrifuged at 4 °C for 5 min at15,000 rpm, and the cell-free supernatant was carefullyseparated.

The cell-free lysate prepared from 2C4NP-inducedcells was tested for some of the catabolic enzymes(benzenetriol dioxygenase, chloro-nitrophenol reductase,p-nitrophenol (PNP)-2-monooxygenase, and 4-NC-4-monooxygenase) putatively involved in degradation (asindicated by the identification of degradation intermedi-ates). Benzenetriol dioxygenase (BtD) activity was mea-sured by spectrophotometric method using Lambda EZ201 UV–Visible spectrophotometer (Perkin-Elmer Inc,Massachusetts, USA). The reaction mixture consisted of50 mM sodium phosphate buffer (pH 7.0), 10 mM Fe

(NH4)2(SO4)2·6H2O, and ~5 mg of cell-free lysatecontaining soluble protein, in the total reaction volumeof 1 ml. Reactions were initiated by the addition of 50 μMof benzenetriol (BT) to the reaction mixture, and enzymeactivity was monitored with wavelength scan over a rangeof 220–360 nm at a regular interval of 1 min. Chloro-nitrophenol reductase activity was also measured spectro-photometrically; the reaction mixture consisted of 50 mMTris–HCl (pH 8.3), 0.2 mM NADPH, 5 mg of cell-freelysate, and 200 μM of 2C4NP. NADPH was used in thereaction as reduced co-substrate. Reaction was initiated bythe addition of 2C4NP as reaction substrate. Positiveactivity was monitored with decrease in UV–vis absor-bance at 311 nM (Amax of 2C4NP) at a regular interval of1 min. The reaction samples from chloro-nitrophenolreductase enzyme assay were also subjected to theestimation of chloride release at 0, 1, 5, and 10 min ofincubation using the method described above.

PNP-2-monooxygenase and 4-NC-4-monooxygenaseactivities were determined by measuring nitrite releasedfrom the substrate (PNP or 4-NC) upon incubation withcell-free lysate. The standard reaction mixture contained50 mM Tris–Cl (pH 8.0), 0.2 mM NADH, 0.08 mMFAD, 1 mM MgSO4, 5 mg of cell-free lysate, and 300 μMPNP or 4-NC in a total reaction volume of 1 ml. Reactionswere initiated by the addition of PNP or 4-NC in thereaction mix. The samples (100 μl reaction mixture) werecollected after 30 min and subjected to quantitation of thenitrite release in reaction mixture according to the methoddescribed above. The activity of other enzymes, e.g.,catechol-1-2-dioxygenase, 2-amino-5-chlorophenol 1,6-dioxygenase, etc., was assayed using quantitative HPLCand/or UV–vis spectroscopic methods described earlier(Haigler et al. 1994; Nishino and Spain 1993; Schlomannet al. 1990; Spain and Nishino 1987).

For the quantitation of enzyme activities, enzyme “units”were determined by using varying amounts of cell-freelysate (50 μg–5 mg) in activity assays described above. Theunit of enzyme was defined as the amount of proteinrequired to convert 1 μmol of substrate per minute. Valuesobtained from enzyme unit determination were converted inSI unit of enzyme activity, i.e., “katal” according to thefollowing relation (1 U=16.67 nanokatals) (NC-IUB 1979).The specific activities of different enzymes were deter-mined; the specific activity was defined as micromoles ofsubstrate transformed per minute per milligram of protein.Protein concentrations in cell extracts were determined byBradford method using standard procedure.

Chemicals

All the CNACs isomers and putative degradation inter-mediates were obtained from Sigma-Aldrich (GmbH,

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Steinheim, Germany). Ferric ammonium sulfate, Fe(NH4)·(SO4)2, and mercuric thiocyanate, Hg(SCN)2, wereprocured from Fluka Chemicals (Buchs, Switzerland). N-(1-naphthyl)-ethylenediamine dihydrochloride, sulphanilicacid, and nitric acid were obtained from the local vendors.The solvents were obtained from Merck Limited (Darm-stadt, Germany). All the other chemicals and media usedduring the study were of the highest purity grade.

Results

Degradation of 2C4NP by strain SJ98

Initial screening carried out for the growth of strain SJ98on 2C4NP, 4C2NP, 4C3NP, and 3C4NP established thatstrain SJ98 could degrade 2C4NP and utilize it as thesole source of carbon and energy, whereas other com-pounds are only co-metabolized in the presence ofalternative carbon source (data not shown). The con-sumption of 2C4NP was accompanied by concomitantincrease in cell growth that reached a maximum growthequivalent to OD600 of 0.39±0.016 (Fig. 1a). The similargrowth of strain SJ98 was also observed with nitrogen-free MM supplemented with 2C4NP as the only source ofnitrogen (Fig. 1a). This observation indicates that strainSJ98 can also utilize 2C4NP as the sole source ofnitrogen. In comparison, no growth of strain SJ98 wasobserved in cultures established in MM without 2C4NP orany other carbon source. Subsequent studies demonstratedthat strain SJ98 could degrade 300 μM 2C4NP after 40 hof incubation (Fig. 1a). Pre-induction carried out bygrowing cells in NB in the presence of 300 μM 2C4NPinduces the degradation activity, and the use of pre-induced cells as inoculum resulted in the improvedkinetics of degradation as observed by the consumptionof 300 μM substrate in 20 h compared to 38–40 h withnon-induced cells (Fig. 1a).

Identification of degradation intermediates

Colorimetric analyses of chloride, nitrite,and ammonia release

Culture supernatant collected at different time pointsduring growth studies demonstrated nearly stoichiometricrelease of nitrite (278±12.42 μM) at complete degrada-tion of 2C4NP (Fig. 1b). However, culture supernatantcollected from nitrogen free MM showed accumulation ofsignificantly lesser amounts (32±8.91 μM) of nitrite in themedium (Fig. 1b). This observation further substantiatedthe notion that strain SJ98 utilizes 2C4NP as the solesource of nitrogen. These samples were also found to have

stoichiometric amounts of chloride (285±9.64 and 288±11.72 μM) for MM and nitrogen MM, respectively;(Fig. 1b). Ammonia was not detected in samples collectedfrom either growth studies or resting cell studies. Based onthis observation, it could be proposed that the degradationof 2C4NP in strain SJ98 proceeds via oxidative mecha-nism for removal of the nitro group. Another noticeablefinding was the time course of nitrite and chloride release.As represented in Fig. 1b, chloride release preceded therelease of nitrite in culture medium during degradation of2C4NP by strain SJ98.

Chromatographic analyses of degradation intermediates

Results obtained with chromatographic studies alsocorroborated with the observations from chloride, nitrite,and ammonia release assays. HLPC-MS analysis showedthe appearance of metabolites with retention times; Rt=1.92 and 3.64 min with gradual decrease of parentsubstrate (2C4NP) with Rt of 2.12 min (Fig. 2). Theretention times of metabolites matched precisely to thoseobtained with standard PNP, 4-NC, and 2C4NP. The massfragmentation analysis of the above metabolite peaksascertained their identity as PNP and 4-NC as revealedby the occurrence of characteristic protonated pseudo-molecular ions with (m/z + H+) values of 140.03 and156.03, respectively (Fig. 2). The other peaks observedbetween Rt of 6 and 10 min did not match withcompounds listed in metabolite database, including puta-tive ortho or meta ring cleavage of 4-NC.

TLC and GC analyses also confirmed the presence ofPNP and 4-NC as 2C4NP degradation intermediate.Noticeably, TLC and GC analyses showed the presence ofan additional metabolite that was later identified as BT withGC-MS analysis. As shown in Table 1, the retention timesof metabolites were in accordance with those of theauthentic standards PNP, 4-NC, and BT. The massfragmentation analysis on metabolite peaks showed molec-ular ions with m/z values of 173, 139, 126, and 155 (Fig. S1and Table 1) that are in agreement with the molecularmasses of 2C4NP, PNP, BT, and 4-NC, respectively. Theseresults together ascertained PNP, 4-NC, and BT as themajor characterized degradation intermediates of 2C4NPdegradation by strain SJ98.

Inhibition studies and time course analysesof intermediate formation

The samples collected from inhibition studies showedaccumulation of a metabolite with GC retention time of4.21 min (Fig. S2), which corresponded to that of standardBT. This result indicated BT as the terminal aromaticintermediate of 2C4NP degradation by strain SJ98. It is

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perceivable that both BT and 4-NC are used as the substratefor cleavage substrate reaction during 2C4NP degradationby SJ98; wherein BT is acted upon by a heme-dependentdioxygenase, and 4-NC is acted upon by a non-heme

dioxygenase. However, 3-nitro-cis,cis-muconic acid and 5-nitro-2-hydroxymuconate (reaction products expected fromortho and meta ring cleavage of 4-NC, respectively) werenever detected. Therefore, it could be proposed that the ring

Fig. 1 a Growth characterization of Burkholderia sp. strain SJ98 on2-chloro-4-nitrophenol and time course of 2C4NP degradation by non-induced and pre-induced cells of strain SJ98. b Estimation of chloride(Cl−) and nitrite (NO2

2−) released in the culture medium during

growth of strain SJ98 on 2C4NP as the sole source of carbon,nitrogen, and energy. Experiments were carried out in triplicates andresults are presented as average mean values. Error bars showstandard errors

Fig. 2 HPLC and LCMS-TOF analysis of sample collected during growth of strain SJ98 on 2C4NP. a HPLC chromatograph showing retentiontime of metabolites and b mass fragmentation pattern of metabolites corresponded with those of standards PNP and 4NC

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cleavage using 4-NC either constitute the minor pathway oris not a part of degradation. Further molecular character-ization of genes involved in degradation process would becarried out in subsequent studies to ascertain which reactionconstitutes the major ring cleavage reactions in thisdegradation process. The time course analysis carried outfor determining the amounts of intermediates formed duringthe degradation of 2C4NP by strain SJ98 showed theappearance of PNP as the first intermediate (first observedat 8 h after incubation) followed by sequential appearanceof 4-NC and BT (first observed at 12 h and 16 h afterincubation respectively; Fig. 3).

Enzyme activity in cell-free lysate

Cell-free lysate of 2C4NP grown cells of strain SJ98demonstrated positive activity for BtD and converted BT(Amax of 282 nm) into a metabolite with UV–vis Amax of243 nm (Fig. S3). This absorbance is characteristic ofstandard maleylacetate (MA). Activity of BtD was found tobe Fe3+ dependent and it was inhibited by presence of ironchelator(s) vis., 2,2′ dipyridyl and o-phenanthroline in thereaction mixture. Also, when NADH was included in thereaction mixture, the absorbance peak at 243 nM alsodisappeared, indicating potential activity of maleylacetatereductase. These results indicated that BT and MA serves asreaction substrate and product respectively for heme-dependent ring cleavage dioxygenase viz., Benzenetrioldioxygenase in degradation of 2C4NP by strain SJ98. Wedid not attempt to identify further downstream intermedi-ates, since MA is almost always degraded via β-ketoadipateand formation of acetyl-CoA and succinyl-CoA as subse-quent intermediates in gram positive as well as gramnegative microorganism.

Cell lysates prepared from 2C4NP-induced cells of strainSJ98 also showed positive activity for “chloro-nitrophenolreductase” when 2C4NP was used as the reaction substrate.Positive activity was monitored with time-dependent decreasein absorbance at 311 nm corresponding to Amax of 2C4NP(Fig. S3). Cell lysate prepared from cells of strain SJ98grown on NB did not show positive activity indicating thatthe enzyme is induced during growth and degradation of2C4NP (Fig. S3). The specific activity for this reaction wasfound to be 0.310 μmol min−1 mg−1 protein. Noticeably,similar high values for specific activities (0.264 and0.248 μmol min−1 mg−1 protein) were also observed when

Fig. 3 Quantitative HPLC/GC analysis showing time course forconcentration of intermediates generated during degradation of2C4NP by induced cells of strain SJ98. Experiments were performedin triplicate; results are presented as arithmetic means values obtainedand error bars show standard deviations

Table 1 Identification of intermediates of 2C4NP degradation by strain SJ98 carried out by comparison of Rf values, retention times in GC andHPLC analysis, and mass fragmentation pattern obtained from the mass spectroscopy analysis

Metaboliteandstandards

Rf valuesin TLCanalysis

Rt (min)in GCanalysis

Rt (min)in HPLCanalysis

m/z values of observed fragment ions Identificationof metabolite

(i) 0.71 2.68 2.12 2C4NP

(ii) 0.69 2.43 1.92 139* [M+], 109 [M+-H-NO], 81 [109-C2H4], 53 [base peak] PNP

(iii) 0.34 4.14 3.64 155* [M+], 107 [M+-NO2− −], 98 [M+-(C2H4)2], 79 [98-H-OH], 53 [base peak] 4-NC

(iv) 0.29 5.98 126* [M+], 97 [M+-H-C2H4], 80 [97-OH], 53 [base peak] BT

2C4NP 0.72 2.66 2.16

PNP 0.68 2.44 1.98

4-NC 0.34 4.18 3.58

BT 0.29 5.99 4.21

CHQ 0.42 3.27 2.96

HQ 0.39 3.74 3.17

4-ACP 0.51 2.93 2.38

(i), (ii), (iii), and (iv) were major identified metabolites in the samples collected from resting cell studies. Authentic standards for possibleintermediates were used for comparison

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4C2NP and 4C3NP were used as the reaction substrates(Table 2). However, 3C4NP showed relatively low specificactivity. The positive activity of chloro-nitrophenol reductasewas also monitored by Cl− release in the reaction mixture,detected by colorimetric method describe earlier. Resultsfrom this analysis showed that amount of measurable Cl− inreaction mixture correlated with conversion of reactionsubstrate, i.e., chloro-nitrophenol (as observed by decreaseat UV–vis absorbance at 311 nm). The specific activitiesdetermined with Cl− release were 0.296, 0.271, and0.266 μmol min−1 mg−1 with the use of 2C4NP, 4C2NP,and 4C3NP as reaction substrate, respectively. These valuesare comparable to those obtained with spectrophotometricenzyme activity assay.

Cell lysate also catalyzed the release of stoichiometricamounts of nitrite from PNP and 4-NC. The enzymaticactivities for PNPmonooxygenase and 4-NCmonooxygenasewere found to be dependent on the presence of NADH andFAD as the reaction cofactors and did not occur in thepresence of FMN. The amount of measurable nitrite releasedwith PNP and 4-NC was stoichiometrically related to theamount of substrate used in the activity assay. The specificactivity values of PNP monooxygenase, 4-NC monooxyge-nase, and other tested enzymes are enlisted in Table 2.

The positive activities of chloro-nitrophenol reductase,PNP monooxygenase, 4-NC monooxygenase, and benzene-triol dioxygenase in cell lysate prepared from 2C4NP

grown cells of strain SJ98 were in accordance withidentification of degradation pathway intermediates withchromatographic and mass spectroscopic methods. Togetherthese results ascertain the identity of PNP, 4-NC, BT, and MAas the major characterized intermediates.

Metabolic pathway for degradation of 2C4NPby strain SJ98

In light of the observations and results presented above, wepropose that strain SJ98 degrades 2C4NP and uses it as asole source of carbon, nitrogen, and energy. The kineticstudies for degradation clearly indicated that the catabolicpathway is of inducible nature. The identification ofdegradation intermediates establish that the catabolicpathway for degradation of 2C4NP in strain SJ98 proceedsvia sequential formation of monophenolic (PNP), biphe-nolic (4-NC), and triphenolic (BT) compounds. Subse-quently, BT is acted upon by “ring cleaving enzyme” andleads to the formation of MA. The proposed pathway isshown in Fig. 4.

Discussion

CNACs constitute an important class of organic com-pounds; however, they are extremely recalcitrance towards

Table 2 Specific activities of putative catabolic enzyme in cell extracts of 2C4NP induced resting cells of strain SJ98

Reaction substrate Putative catabolic enzyme Enzyme unit(nanokatal)

Specific activity(μmol min−1 mg−1)

Comment

2C4NP ortho chloro-nitrophenol reductase 53.77 0.310 Involved in 2C4NPdegradation

4C2NP para chloro-nitrophenol reductase 63.14 0.264 Involved in 2C4NPdegradation

4C3NP para chloro-nitrophenol reductase 67.21 0.248 Involved in 2C4NPdegradation

3C4NP meta chloro-nitrophenol reductase 4,167.5 0.004 No activity

PNP para-nitrophenol monooxygenase 6.33 2.63 Involved in 2C4NPdegradation

4-NC 4-NC monooxygenase 4.50 3.71 Involved in 2C4NPdegradation

BT Benzenetriol dioxygenase 16.92 0.985 Involved in 2C4NPdegradation

2,5-Dichlorohydroquninone 2,5-Dichlorohydroquinone reductivedehalogenase

>16,670 <0.001 No activity

2-Chlorohydroquinone Chlorohydroquinone reductivedehalogenase

>16,670 <0.001 No activity

2-Chloro-4-hydroxylaminophenol

2-Chloro-4-hydroxylaminopheol mutase >16,670 <0.001 No activity

4-Amino-2-chlorophenol 4-Amino-2-chloropheol dioxygenase >16,670 <0.001 No activity

4-Aminophenol 4-Aminophenol dioxygenase >16,670 <0.001 No activity

a Cell extracts were added to the reaction mixture to give a final concentration of 50 μg–5 mg. The activity assays were performed in triplicates and theresults are presented as arithmetic mean of values obtained

604 Appl Microbiol Biotechnol (2011) 92:597–607

corresponding chloroaniline (Susarla et al. 1996) or (b)oxidative removal of the nitro group and the formation ofchlorocatechol as the first identified intermediate of thedegradation pathway (Ghosh et al. 2010).

The most significant salient feature of 2C4NP degrada-tion by strain SJ98 is the involvement of “reductivedehalogenation” for the conversion of 2C4NP into PNP asthe initial step for aerobic degradation of a CNAC.Reductive dehalogenation is known to occur predominantlyunder anaerobic environments and often constitutes theinitial step in anaerobic biodegradation of halogenatedaromatic compounds (Mohn and Tiedje 1992; Struijs andRogers 1989; van den Tweel et al. 1987). It has also beenreported as the initial degradation step in the aerobicdegradation of few chlorinated aliphatic compounds includ-ing hexachlorocyclohexane, trichloroethene, etc. (Mohnand Tiedje 1992); however, as indicated by a number ofreviews, reductive dechlorination is not a common initia-tion mechanism for the aerobic degradation of aromatichalogenated compounds as it has been rarely observed(Apajalahti and Salkinoja-Salonen 1987; Janssen et al.2001; Mohn and Tiedje 1992; Romanov and Hausinger

Fig. 4 Proposed pathway for degradation of 2C4NP by strain SJ98 and its comparison with earlier reported pathways for CNACs degradation

Appl Microbiol Biotechnol (2011) 92:597–607 605

microbial degradation (Symons and Bruce 2006; Wu et al.2006). Consequently, information pertaining to the micro-bial degradation of CNACs and metabolic pathwaysinvolved in the degradation of CNACs is relatively scarce.During present study, we found that Burkholderia sp. strainSJ98 degrades 2C4NP under aerobic conditions withsequential and stoichiometric release of Cl− and NO2

− release.This finding strongly indicated towards the involvement of acatabolic pathway which is different from the other pathwayspreviously reported for CNACs degradation, wherein NO2

−

group is partially reduced to give rise to hydroxylaminointermediate and subsequent degradation proceeds viaremoval of ammonia in the growth medium (Fig. 4 (i, ii,iii); Katsivela et al. 1999; Schenzle et al. 1999; Wu et al.2006). The identification of PNP, 4-NC, BT, and MA as themajor degradation intermediates also ascertained that meta-bolic pathway for 2C4NP degradation in strain SJ98 is incontrast to pathways previously reported for degradation ofother CNACs. Furthermore, this degradation pathway is alsoin contrast with other lesser common pathways for CNACdegradation and reported to initiate via (a) completereduction of the nitro group leading to the formation of

1996; van den Tweel et al. 1987). Furthermore, a reductivedehalogenation mediated initiation has never been reportedin the case of the aerobic degradation of CNACs. Lenkeand Knackmuss (1996) reported an initial reductivedechlorination for 2-chloro-4,6-dinitrophenol by Rhodococ-cus erythropolis HL24-1; however, the complete degrada-tion and identification of further metabolites remainedelusive. Apart from its ability to carry out the initial stepof degradation, the putative reductive dehalogenase in-volved in 2C4NP degradation by strain SJ98 also exhibits“relaxed substrate” specificity for ortho and para dechlo-rination. Further characterization of this putative reductivedehalogenase of strain SJ98 could be useful for basic aswell as applied aspect. Also, as observed, 2C4NP degrada-tion pathway in strain SJ98 merges in metabolic pathwaycharacterized for degradation of PNP (Chauhan et al. 2010).It would be useful to evaluate the genetics of 2C4NP andPNP degradation and to determine if 2C4NP degradation isa cognate function of PNP degradation in strain SJ98 or if itis encoded by physically distinct genetic locus.

Acknowledgments This study was supported in part by theDepartment of Biotechnology (DBT), India and the Council forScientific and Industrial Research (CSIR), India. We are thankful toMs. Mahima Pant and Mr. Vivek Mahajan for their technical helpduring this study. JP, AC, and PKA acknowledge the researchscholarship provided by CSIR, India.

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