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Keywords:

  • biodegradation;
  • γ-hexachlorocylohexane;
  • metabolites;
  • pathway;
  • Xanthomonas

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aim:  To isolate γ-hexachlorocyclohexane (HCH)-degrading bacteria from contaminated soil and characterize the metabolites formed and the genes involved in the degradation pathway.

Methods and Results:  A bacterial strain Xanthomonas sp. ICH12, capable of biodegrading γ- HCH was isolated from HCH-contaminated soil. DNA-colony hybridization method was employed to detect bacterial populations containing specific gene sequences of the γ-HCH degradation pathway. linA (dehydrodehalogenase), linB (hydrolytic dehalogenase) and linC (dehydrogenase) from a Sphingomonas paucimobilis UT26, reportedly possessing γ-HCH degradation activity, were used as gene probes against isolated colonies. The isolate was found to grow and utilize γ-HCH as the sole carbon and energy source. The 16S ribosomal RNA gene sequence of the isolate resulted in its identification as a Xanthomonas species, and we designated it as strain ICH12. During the degradation of γ-HCH by ICH12, formation of two intermediates, γ-2,3,4,5,6-pentachlorocyclohexene (γ-PCCH), and 2,5-dichlorobenzoquinone (2,5-DCBQ), were identified by gas chromatography-mass spectrometric (GC-MS) analysis. While γ-PCCH was reported previously, 2,5-dichlorohydroquinone was a novel metabolite from HCH degradation.

Conclusions:  A Xanthomonas sp. for γ-HCH degradation from a contaminated soil was isolated. γ-HCH was utilized as sole source of carbon and energy, and the degradation proceeds by successive dechlorination. Two degradation products γ-PCCH and 2,5-DCBQ were characterized, and the latter metabolite was not known in contrasts with the previous studies. The present work, for the first time, demonstrates the potential of a Xanthomonas species to degrade a recalcitrant and widespread pollutant like γ-HCH.

Significance and Impact of the Study:  This study demonstrates the isolation and characterization of a novel HCH-degrading bacterium. Further results provide an insight into the novel degradation pathway which may exist in diverse HCH-degrading bacteria in contaminated soils leading to bioremediation of γ-HCH.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

γ-HCH, lindane is a chlorinated pesticide, which is used extensively to protect crops from vector borne diseases. γ-HCH is made by chlorinating benzene (ATSDR 2003) and results in the formation of four major stereo isomers in the proportions of α-HCH (70%), β-HCH (8%), γ-HCH (13%) and δ-HCH (9%). Only the gamma isomer amongst them exhibits insecticidal property. Large-scale production and use of this compound, however, leads to global contamination (Meharg et al. 1999; Li 1999; Li et al. 2003) and deterioration of environmental quality owing to its long period of persistence (Simonich and Hites 1995; Blais et al. 1998; Breivik et al. 1999). In this context, bioremediation, involving the use of natural microbes or engineered variants to detoxify and degrade environmental contaminants, has received attention as an effective biotechnological approach to clean up this pollutant (Pieper and Reineke 2000; Watanbe 2001; Furukawa 2003).

To date, there are several reports regarding the biodegradation of γ-HCH (Ohisa et al. 1980; Bachmann et al. 1988; Senoo and Wada 1989; Sahu et al. 1990; Boyle et al. 1999), and also about its other isomers (Middeldorp et al. 1996; Böltner et al. 2005; Mohn et al. 2006). Two fungi, Trametes hirsutus and Phanerochaetechrysosporium, which can degrade γ-HCH into metabolites tetrachlorocyclohexane and tetrachlorocyclohexanol, are also reported (Singh and Kuhad 1999). Genes encoding enzymes involved in γ-HCH biodegradation have been identified and cloned from different strains of Sphingobium japonicum (formerly Sphingomonas paucimobilis UT26, Nagata et al. 1999), Sphingobium indicum (formerly S. paucimobilis B90, Kumari et al. 2002), Sphingobium francense (formerly Rhodanobactor lindaniclasticus, Thomas et al. 1996) and a Microbacterium sp. (Manickam et al. 2006). In S. japonicum, the genes involved in degradation were completely cloned and sequenced (Nagata et al. 1999). In other strains, only initial genes in the pathway have been described. Diversity and distribution of several micro-organisms, able to utilize different HCH isomers, have also been recently described in detail (Phillips et al. 2005; Lal et al. 2006).

Bacterial populations capable of degrading HCH isomers have been isolated using culture-dependent enrichment procedures. Usually this process takes inordinate lengths of time, and also a majority of slow-growing soil bacteria remain undetected (Lloyd-Jones et al. 1999). Molecular approaches, such as polymerase chain reaction (PCR) (Purohit et al. 2003), colony hybridization (Sayler et al. 1985), DNA extraction (Martin-Laurent et al. 2001), RNA amplification (Stapleton et al. 1998) denaturing gradient gel electrophoresis (DGGE) (Mohn et al. 2006) and DNA microarray (Rhee et al. 2004; Neufeld et al. 2006) are now increasingly being used to detect organisms possessing catabolic genes involved in target xenobiotic degradation.

Several recent developments in molecular techniques also provide rapid, sensitive and accurate methods to isolate and analyse bacteria harbouring catabolic genes in the environment (Widada et al. 2002), and help to assess toxicological risks and bioremediation strategies (Power et al. 1998). We followed colony hybridization techniques to isolate cultivable bacteria possessing a metabolic pathway for the degradation of HCH. In this study, we report the isolation of a bacterium which is capable of degrading γ-HCH into a novel metabolite 2,5-dichlorobenzoquinone (2,5-DCBQ), via γ-2,3,4,5,6-pentachlorocyclohexene (γ-PCCH).

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Chemicals

Analytical grade α-, β-, γ- and δ-HCH were obtained from Riedel-deHaën, Germany. Mercuric thiocyanate, 2-phenoxy ethanol and ferric ammonium sulfate were purchased from Sigma Chemical Co, St. Louis, MO, USA. Authentic compound 2,5-dichloro-1,4-benzoquinone was procured from Aldrich Chemical Company Inc, Milwaukee, USA. Nonradioactive labelling and detection kit and nylon membrane were purchased from Roche Diagnostics GmbH, Mannheim, Germany. Reagents for PCR were purchased from Finnzyme OY, 02201 Espoo, Finland, and DNA size markers were from New England Biolabs, Beverly, MA, USA. Oligonucleotide primers were custom synthesized from MWG-Biotech AG, Ebersberg, Germany. Other chemicals for media preparations and buffers were of analytical grade reagents from Qualigens chemicals and Hi-media, Mumbai, India.

Synthesis of γ-PCCH

γ-PCCH was chemically synthesized using γ-HCH, according to the method described by Tranterik et al. (2001). Briefly, 50 mg of γ-HCH was added to 5-ml acetonitrile in a 20-ml volumetric flask. To this, 2·5-ml of 0·1-N NaOH was added to make the reaction alkaline in nature. The mixture was heated to 40°C for 20 min. To stop (neutralize) the reaction, 0·5 ml of 12-N HCl was added. The product formed was extracted twice with 5-ml hexane, and the solvent was evaporated at room temperature. The crystals of γ-PCCH were dissolved in an appropriate volume of hexane for the gas chromatography and mass spectrometry analysis.

Isolation of bacteria

Sediment samples, wastewater and rhizosphere soil were collected from an industrial site (India Pesticide Ltd, Chinhat industrial area, Lucknow, India), which is engaged in the manufacture of γ-HCH for more than 15 years. The samples were brought in sterile capped Schott bottles (Schott, Mainz, Germany), and processed within 2 days. Subsamples were stored at 4°C until their use. A portion (10-g each) of the collected samples from all of them was mixed together for enrichment culture. The culture medium used for isolation and growth on γ-HCH was prepared by using medium containing (per litre) KH2PO4, 170 mg; Na2HPO4, 980 mg; (NH4)2 SO4, 100 mg; MgSO4, 4·87 mg; FeSO4, 0·05 mg; CaCO3, 0·20 mg; ZnSO4, 0·08 mg; CuSO4.5H2O, 0·016 mg; H3BO3, 0·006 mg; in 500-ml Erlenmeyer flasks, and a final concentration of 100-μg ml−1γ-HCH was added. A total of 30 g of this soil–slurry mixture was added to 50 ml of sterile mineral medium with the above composition, and stirred for 5 h. The flask was kept static for 1 h, and 1-ml suspension from this enrichment culture was serial diluted to 103–107-fold with liquid mineral medium and 200 μl of each dilution was spread onto Luria Bertani (LB) agar plates to verify the presence of different bacteria. The plates were inverted and incubated (Innova 4230 incubator-shaker, New Brunswick scientific, NJ, USA) at 30°C, and monitored for 1 week.

Colony hybridization with digoxygenin-labelled probes

The cultures obtained from the earlier section (isolation of bacteria) were then spotted in a regular numbered array on LB agar plates. Overall, 53 visibly distinguishable bacterial colony-based growth pattern and pigmentation were taken for colony hybridization. After overnight incubation at 30°C, these cultures were lifted onto nylon membranes, positively charged (Roche Diagnostics GmbH, Mannheim, Germany), and processed as described in the following. Colonies were precooled for 30 min at 4°C. The nylon membrane was placed on culture surfaces, gently spread with a glass rod and kept for at least 5 min. The membranes were placed in a plastic petri dish, culture side up, on a 3 M filter paper soaked in 3 ml of denaturing solution (0·5-mol l−1 NaOH, 1·5-mol l−1 NaCl) for 15 min two times, and then returned to the Whatman 3 M filterpaper, soaked in 3 ml of neutralizing solution (1·0-mol l−1 tris [pH 7·5], 1·5-mol l−1 NaCl) and neutralized for 15 min twice. The membrane discs were treated with proteinase-K, by keeping them on a piece of aluminum foil with 0·5 ml of 2-mg/ml proteinase-K, and after even distribution of the solution, the membrane was incubated for 1 h at 30°C. A filter paper, fully wetted with dH2O, was used to remove excess cell debris. DNA was fixed to the membrane by baking them at 80°C for 3 h. Prehybridization was done using the standard hybridization buffer, and hybridization with a digoxygenin (Dig)-labelled linA, linB and linC genes were performed at 65°C, as described by the manufacturer (Boehringer GmbH, Mannheim, Germany). Washing and detection of the DNA–DNA hybrids was performed with a Dig nucleic acid detection kit (Boehringer), and the positive colony was detected using colorimetric reagents nitroblue tetrazolium (NBT) and 5-bromo-4chloro-3-indolyl phosphate (BCIP), as described by the manufacturer.

Determination of 16S rRNA gene sequence, and identification of bacterium

The chromosomal DNA of strain ICH12 was isolated according to the procedure described by Rainey et al. (1996). The 16S rRNA gene was amplified with the primers 27f (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492r (5′-TACGG(C/T)TACCTTGTTAC GACTT-3′) universal primers. The amplified DNA was gel purified (Qiaquick gel extraction kit, Qiagen, Germany), and sequenced with four forward and three reverse primers, namely 27f (5′-AGAGTTTGATCCTGGCTCAG-3′), 357f (5′-CTCCTAC GGGAGGCAGCAG-′), 704f (5′-TAGCGGTGAAATGCGTAGA-3′), 1114f (5′-GCAA CGAGCGCAACC-3′), 685r (5′-TCTACGCATTTCACCGCTAC-3′), 1110r (5′-GGGT TGCGCTCGTTG-3′) and 1492r (5′-TACGG(C/T)TACCTTGTTACGACTT-3′), respectively (Escherichia coli numbering system). Sequence was determined by the dideoxy chain-termination method (Sanger et al. 1977) using the Big-Dye terminator kit using ABI 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). A sequence similarity search was carried out against known sequences available in the GenBank using BLASTn (Altschul et al. 1997).

Degradation experiments

To verify that the colony hybridization-positive bacterial isolate could utilize and grow γ-HCH as sole carbon and energy source, the cells were cultured in mineral salts medium containing 0·34-mmol l−1 (100 μg ml−1) γ-HCH with additional 0·01% of yeast extract in triplicate for 8 days. Uninoculated flasks containing medium and γ-HCH served as controls. All the treatment flasks and controls were incubated at 30°C on a rotary shaker at 200 rev min−1 (Innova 4230 incubator-shaker, New Brunswick Scientific, NJ, USA). To identify metabolites formed from γ-HCH, strain ICH1 as grown in 500-ml mineral medium containing 0·34-mmol l−1 (100 μg ml−1) γ-HCH having 0·01% yeast extract was incubated at 30°C at 200 rev min−1.

Analytical methods

Characterization of metabolites formed from γ-HCH degradation The inorganic free chloride released into the liquid medium during the degradation of γ-HCH isomers was estimated by the colorimetric method (Bergmann and Sainik 1957). Individual flasks were withdrawn at 0, 24, 48 and 72 h of incubation and for gas chromatography-mass spectrometry (GC-MS) analysis of the residual γ-HCH, and the degradation products formed were extracted twice with acetone, hexane and finally once with ethyl acetate. The solvents were removed under vacuum by rotary evaporation (Büchii Rotavapor R-200, Flawit, Switzerland), and the residue was redissolved in ethyl acetate prior to analysis. Aliquots of 2–5 μl were injected directly to GC-MS autosystem XL GC, interfaced to a Turbomass mass spectrometer (Perkin-Elmer, Norwalk, CT, USA); for GC, using PE-5MS, 20 m × 0·18-mm ID, 0·18-μm film thickness capillary column and stationary phase consisting of 5% phenyl methyl siloxane. The injector temperature was 250°C, carrier gas was helium at a flow rate of 1 ml min−1. Mass spectrum conditions had the ionization energy 70 eV, ion source temperature 300°C, with single ion monitor mode. The spectra were compared with respective mass spectra of authentic compounds, and also with the mass profile of the same compound available in the National Institute of Standard Technology (NIST) library, USA.

Labelling of dehalogenase genes

The linA, linB and linC genes, cloned previously (Nagata et al. 1999) from S. paucimobilis UT26 was obtained in plasmids pIMA2, pYNA4 and pBFR41, respectively as a gift (Prof. Yuji Nagata, University of Tohuku, Sendai, Japan). The plasmids were then transformed into E. coli DH5α (Sambrook and Russell 2001). Transformants harbouring plasmids were selected on LB agar containing 100 μg of ampicillin per millilitre. Plasmids were extracted by alkaline lysis method (Sambrook and Russell 2001) and digested with respective restriction enzymes. The insert fragments were gel purified by using GenEluteTM plasmid miniprep kit (Sigma, St. Louis, MO, USA). The eluted DNA fragments were labelled with alkali-labile DIG-dUTP, using a DIG DNA labelling and detection kit (Boehringer Mannheim, Mannheim, Germany). Probes were stored at −20°C until their use.

PCR and sequencing

The oligonucleotide primers were designed based on three reported initial (linA, linB and linC) reductive dehalogenase genes for γ-HCH degradation. The primers were fwlinA 5′-GGCCGCGATTCAGGACCTCTACT-3′, revlinA 5′-CGGCCAGCG GGGTGAAATAGT-3′, for a dehydrochlorinase, linBF 5′-ATGAGCCTCGGCGCA-3′, linBR 5′-TCGCCGGACAAACGC-3′ for a hydrolytic dehalogenase, and fwlinC 5′- TGAGCGGCAAGACGATAAT-3′, revlinC 5′-CAGCGGCGGATGCGGTGTTGA- 3′ for a dehydrogenase from a S. japonicum (Nagata et al. 1999). These three pairs of primers were used to amplify homologous DNA fragments from strain ICH12. The PCR reaction mixture (50 μl) contained 50 pmol of each primer, 100 ng of chromosomal DNA as template, 100-μmol l−1 deoxynucleoside triphosphates, 1X PCR buffer, 0·7 U of DNA polymerase (Finnzyme OY, 02201 Espoo, Finland). The PCR was performed with 30 cycles of denaturing (95°C, 30 s) annealing (55°C, 30 s) and polymerization (72°C, 30 s), with 3 min of denaturation during the first cycle and an additional 10 min of polymerization during the last cycle. The nucleotide sequences of linA and linB genes was determined by the dideoxy chain-termination method (Sanger et al. 1977) using the Big-Dye terminator kit on ABI 310 Genetic Analyzer (Applied Biosystems).

Nucleotide sequence accession number

The 16S rRNA gene sequence of the ICH12 strain generated in this study was submitted to the GenBank database with the accession number AY864860. The linA and linB functional gene sequences obtained from strain ICH12 have been submitted to the GenBank, and have now received accession number DQ910544-DQ910545.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Colony hybridization and isolation of the bacteria

Fifty-three different colonies with distinct morphologies could be identified after samples from HCH pesticide contaminated soil and sediment were plated on LB agar and incubated. These were present in dilution numbers ranging from 10−4 to 10−5 per millilitre, and the total cell numbers that appeared per plate were 335 and 121 in the respective dilutions. Labelled and specific gene probes for dehalogenases, such as dehydrochlorinase (linA), hydrolytic dehalogenase (linB) and dehydrogenase (linC) were used to screen against a colony blot of the 53 isolates. After colony hybridization, using the DIG-nucleic acid detection kit, the images were obtained as shown in Fig. 1. Of the three genes tested, only the probes corresponding to linA and linB resulted in hybridization to a colony placed at position number 12 in both the blots (Fig. 1a,b) and the linC gene probe did not cross-react with any of the bacterial colonies (Fig. 1c). The signal intensity of the positive colony was strong enough to differentiate from the background. However, we have also observed the background interference in the blot showing some nonspecific bacterial flora. These nonspecific colonies on LB agar plates showed growth with some mucilaginous secretions (Fig. 1) when compared with the rest of the colonies. The hybridization-positive isolate was selected for further studies to test its ability to degrade γ-HCH.

image

Figure 1.  DNA-colony hybridization blots showing the bacteria cross-hybridizing with linA and linB genes for the degradation of γ-hexachlorocyclohexane (γ-HCH). Fifty-three visibly distinguishable colonies obtained from a HCH-contaminated soil were grown on Luria-Bertani (LB) agar in triplicate. Gene probes for dehydrochlorinase (linA) (a), hydrolytic dehalogenase (linB) (b) and dehydrogenase (linC) (c) were hybridized.

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Identification of the bacterium

The strain designated as ICH12 formed smooth colonies on solid medium that turned bright yellow. The bacterium stained gram negative, rod shaped, oxidase and catalase positive. A 1490-bp DNA fragment corresponding to the 16S rRNA gene was amplified by universal primers described previously (Lane 1991). The amplified 16S rRNA sequence had the strong match of 97% to a 16S rRNA sequence in GenBank for Xanthomonas sp. isolated from a lake in Korea (accession no. AY689031) and 95% to the sequence of Xanthomonas axonopodis (accession no. AJ244722) species characterized from Brazil. The strain isolated in this study was consequently denoted as Xanthomonas sp. strain ICH12.

Biodegradation of γ-HCH

The strain ICH12 was found to utilize γ-HCH isomer as the sole carbon and energy source. It was determined that during the growth, >95% of free chloride from γ-HCH was released into the medium, and perhaps the rest 5% is still on the compound or unavailable for the estimation. Degradation of 0·34 mmol l−1 (100 μg ml−1) γ-HCH was observed in 8 days (Fig. 1). Cultures grown on γ-HCH reached an OD600 of about 0·2 in 6 days from the initial inoculum of a singly colony from plate culture. When 0·01% of yeast extract was provided to the medium, the growth of the culture was increased to 0·27 from the initial inoculum of 0·1 OD600 but did not influence the degradation of γ-HCH in any significant manner. Disappearance of the input HCH, increase in cell mass and free chloride ions released in stoichiometric amounts confirmed that the bacterium possesses degradative capability for this isomer. Other major isomers, such as α-, β- and δ-HCH did not support the growth, and correspondingly no degradation products were observed. We presume therefore, this strain does not possess the ability to degrade the other isomers of HCH. However, several other species with the capability to degrade all the isomers of HCH have also been reported (Table 1).

Table 1.   Growth and degradation of hexachlorocyclohexane isomers by various bacteria studied recently
No.Bacterial speciesDegradationReference
αβγδ
  1. *Indicates that these bacteria have been reclassified as Sphinogobium japonicum, Sphinogobium indicum and Sphinogobium francense (Pal et al. 2005).

  2. −, +Degradation activity present or absent.

1Pseudomonas vesicularis P59+Huntjens et al. (1996)
2*Sphingomonas paucimobilis UT26++++Nagata et al. (1999)
3*Rhodanobacter lindaniclasticus+Nalin et al. (1999)
4*S. pauciomobilis B90++++Kumari et al. (2002)
5Pandoraea sp.++Okeke et al. (2002)
6Pseudomonas aeruginosa ITRC5++++Kumar et al. (2005)
7Sphingomons strains++++Böltner et al. (2005)
8Microbacterium sp. ITRC1++++Manickam et al. (2006)
9Sphingomons strains+++Mohn et al. (2006)
10Xanthomonas sp. ICH12+This study

Characterization of metabolites produced by γ-HCH degradation

Gas chromatograms and mass spectra of the metabolic intermediate formed during the degradation of γ-HCH are shown in Fig. 2. The chromatogram, after 48 hs of culture incubation, showed the appearance of a metabolite at 16·79 min (a1) with γ-HCH at 23·04 min of retention time. Cultures extracted after 72 h of incubation possessed another metabolite with 12·55 min (b1) retention time concomitantly with the disappearance of small amounts of γ-HCH and the first metabolite. The peaks corresponding to a1 reduced significantly at 72 h, implying that this is a transient intermediate.

image

Figure 2.  Degradation of γ-hexachlorocyclohexane (γ-HCH) by the strain ICH12 (UI, uninoculated, I, inoculated). (bsl00001) γ-HCH degradation, UI; (□) γ-HCH degradation, I; (bsl00066) growth, UI; (bsl00084) growth I; (•) chloride release, UI; (○) chloride release, I. The initial concentration of the γ-HCH in the liquid mineral medium was 0·34 mmol l−1, and strain ICH12 colony from LB-agar plate was inoculated and the culture was incubated in a shaker at 30°C. The values are mean of three experiments and the standard deviation was <10%.

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The mass fragmentation patterns of both the metabolic intermediates obtained were compared with the NIST mass spectral database for chemicals. The mass fragmentation patterns of a1 gave major fragments at m/z of 181 and 145. The same fragmentation pattern was also observed in the spectrum of authentic compound γ-PCCH (Fig. 3a,a1). This is a known reaction of dehydrodechlorination in the γ-HCH degradation pathway of S. paucimobilis strain UT26.

image

Figure 3.  Characterization of the metabolites formed by strain ICH12 during its growth on γ-hexachlorocyclohexane (γ-HCH). (a) mass spectrum of the metabolite (M1) by strain ICH12; (a1) the authentic γ-2,3,4,5,6-pentachlorocyclohexene (γ-PCCH). (b) Mass spectrum of the metabolite (M2) by strain ICH12; (b1) the authentic 2,5-dichlorobenzoquinone (2,5-DCBQ).

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The mass spectrum of metabolite b1 with the retention time of 12·55 min showed a strong molecular peak at m/z 176 and distinct peaks at m/z 60 and 88. The NIST library search resulted in its identification as 2,5-DCBQ. An authentic 2,5-DCBQ was purchased (Aldrich Chemical Company Inc, Milwaukee, USA), and its mass spectrum was measured (Fig. 3b,b1). The fragmentation pattern of the degradation product b1 and the authentic 2,5-DCBQ had same signature profiles. These results suggest that the pathway may divulge via novel intermediates in this strain, after the initial dechlorination step. Similarly, in a Sphingobium chlorophenolicum, the formation of tetrachlorbenzoquinone was observed as a dechlorination product from pentachlorophenol (PCP) degradation (Dai et al. 2003).

Growth of the strain on 2,5-DCBQ and cell-free enzymatic assay

Strain ICH12, when inoculated in flasks having 2,5-DCBQ as carbon source, no growth was observed till 8 days of incubation. This implies that 2,5-DCBQ did not support ICH12, and utilize it as substrate at concentrations ranging from 10 to 100 μg ml−1. When cells cultivated in mineral medium having 100-μg ml γ-HCH was washed and added to 50 μg ml−1 of 2,5-DCBQ, we observed that the compound underwent conversion as noticed by the GC analysis. However, we did not observe the formation of any new product, but noticed that during the incubation, the reaction mixture changed to dark brown in colour, indicating the formation of some oxidation product. We are presently unable to characterize the metabolic product formed using 2,5-DCBQ as susbstrate, from the whole cells, and the crude cell extract of ICH12 to assay the enzyme catalysed turnover.

PCR amplification of linA, linB and linC genes from strain ICH12

To verify the presence of the initial three catabolic genes for γ-HCH degradation in the strain ICH12, PCR amplification was performed. Primer sets linAF plus linAR and linBF plus LinBR, both the linA and linB dehalogenases primer sets amplified the expected amplicons sizing 409 and 876 bp, respectively, using ICH12 genomic DNA as a template. These two primers pairs also amplified same size of products from plasmids clones (pIMA2 and pYNA4) bearing insert gene for linA and linB to serve as positive controls. In the same conditions, the third gene belonging to a short-chain alcohol dehydrogenase (linC)-specific primers did not produce any amplicon. However, the same set of primers for linC yielded an expected product sizing of 470 bp from the positive linC plasmid (pBFR41) as template. This implies that a gene with different nucleotide sequence composition than the known linC gene may be present in strain ICH12, or alternately this gene may be absent.

The sequences obtained from strain ICH12 were compared with the GenBank (http://www.ncbi.nlm.nih.gov/blast) database. We found that the deduced amino acid sequence for linA gene was highly similar as it had 98% homology to all the reported sequences. It had differences of two amino acids, threonine and cysteine, at position numbers 110 and 111, respectively, in ICH12, where it was the amino acid alanine in all other reported linA gene (Nagata et al. 1999; Kumari et al. 2002). Likewise, for the linB gene sequences, we have observed 11 amino acid differences located at various positions when aligned with the known linB genes (Nagata et al. 1999; Kumari et al. 2002; Böltner et al. 2005).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The results presented here demonstrate that micro-organisms for the degradation of γ-HCH may exist in contaminated environment belonging to different genera. Previously, degradation of γ-HCH and genes involved in its degradation pathway were studied in detail only from Sphingomonas sp. (Thomas et al. 1996; Nagata et al. 1999; Kumari et al. 2002), with an exception of a recent report of a gram-positive Microbacterium sp. (Manickam et al. 2006). Degradation of γ-HCH by S. paucimobilis UT26 (Nagata et al. 1999) led to the production of 2,5-dichlorohydroquinone (2,5-DCHQ) as the intermediate after three steps of chloride removal by three different dehalogenase enzymes. The formation of 2,5-DCHQ was attributed to the catabolic activity of a reductive dehalogenase using 2,5-dichloro-2,5-cyclohexadiene-1,4-diol (2,5-DDOL) as a substrate by strain UT26 (Nagata et al. 1994). In this study, we observed the formation of γ-2,3,4,5,6-PCCH and 2,5-DCBQ as intermediates by the strain ICH12. Though the initial three steps of the reaction by two different dechlorinase enzymes (linA and linB) can be carried out by this strain (Fig. 4), the homologous gene for linC is found to be absent. In addition, it is possible that multiple gene clusters may be operative in this strain as reported recently for the chloroaromatic compounds, such as 4-chlorophenol and 2,4-dichlorophenol, though they are structurally unrelated chemicals to HCH isomers (Moiseeva et al. 2002; Thiel et al. 2005)

image

Figure 4.  Upper pathway for the aerobic degradation of γ-hexachlorocyclohexane (γ-HCH) in a Sphingomonas paucimobilis UT26, and a possible deviation of pathway in the Xanthomonas sp. ICH12. 1. γ-hexachlorocyclohexane (γ-HCH), 2. γ-2,3,4,5,6-pentachlorocyclohexane (γ–PCCH), 3. 1,3,4,6-tetrachloro-1,4-cyclohexadiene (1,4-TCDN), 4. 1,2,4-trichlorobenzene (1,2,4-TCB), 5. 2,4,5-trichloro-2,5-cyclohexadiene-1-ol (2,4,5-DNOL), 6. 2,5-dichlorophenol (2,5-DCP), 7. 2,5 dichloro-cyclohexadiene-1,4-diol (2,5-DDOL), 8. 2,5-dichlorohydroquinone (2,5-DCHQ), 9. 2,5-dichlorobenzoquinone (2,5-DCBQ).

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Earlier, the formation of various substituted quinones has been identified as intermediates during the degradation of PCP (Dai et al. 2003). In case of PCP degradation by a S. chlorophenolicum, it has been believed for more than a decade that PCP was converted to tetrachlorohydroquinone. Subsequently, it has been proved that PCP is actually converted to tetrachlorobenzoquinone (Dai et al. 2003). However, HCH and PCP have two different chemistries, and degradation mechanisms are followed via different pathways, as PCP is converted to tetrachlorobenzoquinone in a NADPH-dependent oxygenation reaction. Different batch cultures of Xanthomonas on γ-HCH grown for 8 days, extraction and analysis of samples on GC, and GC-MS, confirmed the formation of 2,5-DCBQ. To date, the formation of 2,5-DCBQ or other chlorinated benzoquinones has not been reported from HCH, suggesting that the gene sequences for enzyme (gene homologous to linC as in strain UT26) converting 2,5-DDOL to 2,5-DCBQ in this strain may be recently inserted from bacteria degrading other chlorinated compounds. Apparently, more supportive data may be obtained by genetic investigation by cloning the genes present in this bacterium for the HCH-degradation activity. Based on the formation of 2,5-DCBQ metabolite and absence of linC-like DNA sequences, we presume that a novel downstream pathway may be present in strain ICH12. To our knowledge, this is perhaps the first report of HCH degradation by a Xanthomonas sp.

Micro-organisms have the capacity to remove many contaminants from the environment by a diversity of enzymatic processes. Extensive lists of micro-organisms that carry out bioremediation reactions are reported (Anderrson and Lovley 1997;Wackett and Hershberger 2001). The isolation and characterization of pure cultures has been, and will continue to be, crucial for bioremediation itself and analysis of molecular ecology. Investigations of their biodegradative reactions also will help to understand the metabolic products formed and their fate in the environment.

Specifically in India, the industrial sites with high levels of HCH contaminations are numerous. Some of them are: (1) India Pesticide Limited (IPL), Lucknow, Uttar Pradesh; (2) Hindustan Insecticides Limited, Udyogmandal Industrial Estate, Kerala; (3) Yawalkar Pesticides Limited, Nagpur, Maharastra; and (4) Kanoria Chemicals & Industries Limited, Renookut, Uttar Pradesh; and so on. All the aforementioned industries in the past manufactured γ-HCH for pest management and health care programmes. A large-scale organized bioremediation may be possible to reduce the HCH contamination by using the strain ICH12 and also cultures reported in the literature. A number of studies have been reported (Doelman et al. 1985; Bachmann et al. 1988; Manish et al. 2005) where the degradation of HCH isomers in contaminated soil has been demonstrated. Therefore, while the pure cultures are applied for bioremediation, there is a strong need for the consortium of micro-organisms required to express their degradative activities in varying conditions in the field scale treatment of HCH-contaminated soils.

Further, it is reported that 2,5-DCBQ is used as a precursor in the preparation of several types of natural and unnatural quinones. One of the preparations 1,5-diazaanthraquinone, made from 2,5-DCBQ, has potent antitumor activity (Lopez-Alvarado et al. 2002). Future studies required to explore the use of this strain ICH12 to produce commercial quantities of 2,5-DCBQ from γ-HCH, and perhaps the gene convert 2,5-DCBQ into further metabolites may be blocked for this purpose.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This study was supported by a networked project No. COR 008, of the Council of Scientific and Industrial Research (CSIR), Government of India. We are thankful to Prof Yuji Nagata, Department of Biotechnology, University of Tohuku, Sendai, Japan, for providing plasmids harbouring lin genes. This manuscript carries the ITRC communication No. 2370.

References

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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