Diversity of 16S rRNA and dioxygenase genes detected in coal-tar-contaminated site undergoing active bioremediation

Authors


Sunil Khanna, Department of Biotechnology and Bioinformatics, NIIT University, Neemrana, Rajasthan, India. E-mail: Khanna85@yahoo.com

Abstract

Aims:  In order to develop effective bioremediation strategies for polyaromatic hydrocarbons (PAHs) degradation, the composition and metabolic potential of microbial communities need to be better understood, especially in highly PAH contaminated sites in which little information on the cultivation-independent communities is available.

Methods and Results:  Coal-tar-contaminated soil was collected, which consisted of 122·5 mg g−1 total extractable PAH compounds. Biodegradation studies with this soil indicated the presence of microbial community that is capable of degrading the model PAH compounds viz naphthalene, phenanthrene and pyrene at 50 ppm each. PCR clone libraries were established from the DNA of the coal-tar-contaminated soil, targeting the 16S rRNA to characterize (i) the microbial communities, (ii) partial gene fragment encoding the Rieske iron sulfur center (α-subunit) common to all PAH dioxygenase enzymes and (iii) β-subunit of dioxygenase. Phylotypes related to Proteobacteria (Alpha-, Epsilon- and Gammaproteobacteria), Acidobacteria, Actinobacteria, Firmicutes, Gemmatimonadetes and Deinococci were detected in 16S rRNA derived clone libraries. Many of the gene fragment sequences of α-subunit and β-subunit of dioxygenase obtained from the respective clone libraries fell into clades that are distinct from the reference dioxygenase gene sequences. Presence of consensus sequence of the Rieske type [2Fe-2S] cluster binding site suggested that these gene fragments encode for α-subunit of dioxygenase gene.

Conclusions:  Sequencing of the cloned libraries representing α-subunit gene fragments (Rf1) and β-subunit of dioxygenase showed the presence of hitherto unidentified dioxygenase in coal-tar-contaminated soil.

Significance and Impact of the Study:  The combination of the Rieske primers and bacterial community profiling represents a powerful tool for both assessing bioremediation potential and the exploration of novel dioxygenase genes in a contaminated environment.

Introduction

High-molecular-weight (four or more benzene rings) polyaromatic hydrocarbons (HMW PAH) are recalcitrant pollutants and are ubiquitous in nature. These are produced during fossil fuel combustion, waste incineration, or as by-products of industrial processes, such as coal gasification and petroleum refining (Juhasz et al. 1996). Dioxygenases involved in the initial oxidation of polyaromatic hydrocarbons (PAHs) are multicomponent enzymes consisting of an electron transport chain and a terminal dioxygenase composed of large (α) and small (β) subunits (Kim et al. 2006).

The enzyme catalyzing dioxygenation of polyaromatic hydrocarbons belongs to family of aromatic-ring hydroxylating dioxygenases that introduce two atoms of oxygen into aromatic hydrocarbons to form a cis-dihydrodiol. The α-subunit of the terminal dioxygenase is thought to be critical for substrate recognition and is more highly conserved than other components of the dioxygenase complex (Kauppi et al. 1998). Hurtubise et al. (1998) reported that it is not only the α-subunit but also β-subunit that exert some influence on the enzyme–substrate interaction in conjugation with the α-subunit and thus also critical for enzyme activity. Based on the substrate range of these enzymes, nahAc gene (naphthalene dioxygenase α-subunit) appeared to be specific for the degradation of low-molecular-weight (LMW) PAH (two to three ring PAHs) (Zhou et al. 2006), whilst the nidA (naphthalene induced pyrene dioxygenase; α-subunit) gene degraded both LMW and HMW PAHs (Kim et al. 2006). Recent studies showed that α-subunit of dioxygenases involved in the degradation of LMW and HMW PAHs show only partial homology (Kimura et al. 1997) suggesting therefore the existence of novel dioxygenase genes to utilize a variety of aromatic compounds.

Various Mycobacterium sp. have been shown to degrade HMW PAH compound and its multicomponent dioxygenase enzyme system has been studied in detail (Cheung and Kinkle 2001; Miller et al. 2004, 2007; Kim et al. 2005; Natalie et al. 2005; DeBruyn et al. 2007). There is considerable enzymatic, physiological and phylogenetic diversity amongst the presently known aromatic degrading bacteria (Yeates et al. 2000). The yet-to-be cultured forms are also likely to represent a significant resource of novel terminal dioxygenases present in the environment (Yeates et al. 2000). Therefore, the terminal dioxygenase represents a useful molecular target to investigate the presence of dioxygenase genes of uncultured aromatic hydrocarbons degraders.

Primers for the α-subunit of dioxygenase genes (nahAc, phnAc, nidA) and the conserved segments of ring-hydroxylating dioxygenases were designed and used for the detection of novel dioxygenase genes in PAH-degrading bacterial isolates and environmental samples (Hedlund et al. 1999; Lloyd-Jones et al. 1999; Wilson et al. 1999; Marlowe et al. 2002; Stach and Burns 2002; Brezna et al. 2003; Zhou et al. 2006); however these primers were not able to detect the PAH-degrading gene from some species that indicate towards the yet-to-be explored diversity of dioxygenase genes (Meyer et al. 1999).

The objective of this study was to elucidate the composition of microbial communities based on bacterial small subunit (SSU) rDNA gene in the soil of a coal-tar-mixing plant (Char coal Hot Mix plant; HM) and also the diversity of the dioxygenase genes, which could improve our understanding behind the genetics of PAH degradation.

Methods

Sample collection

Soil sample was collected from coal-tar-mixing plant (Hot mix plant, HM), Punjab, India. Soil was tightly packed with coal tar and had 7·1–10·8% moisture content with alkaline pH. To analyze the total PAH residues in the soil, 25 ml of dichloromethane (DCM) was added to 10 g soil in 250 ml flask and kept at 150 rev min−1 for 16 h. The solvent was decanted in an evaporatory dish and the same process was repeated twice. DCM extracts were pooled and evaporated under gentle air flow at room temperature. Samples were re-suspended in acetone and analyzed by Gas chromatography (Autosystem XL; Perkin Elmer) fitted with flame ionization detector (FID) and capillary column (5% phenyl polymethylsilicone, PE-1) with nitrogen as carrier gas at a flow rate of 0·75 ml min−1. Split injection was made with nitrogen as make up gas at flow rate of 30 ml min−1. Injector temperature was 300°C and the detector temperature was 350°C. The oven temperature was programmed to rise from 100°C (2 min) to 280°C at 8°C min−1 and held for 5 min at 280°C.

PAH degradation studies

To determine the bioremediation potential of the soil, degradation studies were initiated in 150-ml flasks using 0·5 g of PAH-impacted soil and 20 ml of minimal medium (Kumar et al. 2008) spiked individually with 50 mg l−1 of naphthalene (N) or phenanthrene (PH) or pyrene (PY). Flasks were shaken at 150 rev min−1 for up to 15 days at 37°C. At each time point, samples were removed and used for DNA and PAH extraction and analysis.

PAH extraction and analysis

DCM (20 ml) was added to the enrichment cultures and PAHs were extracted overnight at 150 rev min−1. Following extraction, the solvent layers were removed and dried at room temperature. Extracts (1 ml) were analyzed on a Shimadzu SPD-M20A liquid chromatograph with diode array detector set at 254 nm using a C-18 column (250 × 4·6 mm, pore size of 4 μm) (EPA Method 610 1984). The liquid chromatograph was programmed as follows: isocratic elution for 5 min using acetonitrile/water (4 : 6), then linear gradient elution to 100% acetonitrile over 30 min at 1·5 ml min−1. flow rate. The concentration of each PAH was calculated by comparison against individual PAH standard curves.

Extraction of DNA from soil

DNA was directly isolated from the coal-tar-contaminated soil by the method of Zhou et al. (1996). Soil sample was mixed with 13·5 ml of DNA extraction buffer [100 mmol l−1 Tris–HCl (pH 8·0), 100 mmol l−1 sodium EDTA (pH 8·0), 100 mmol l−1 sodium phosphate (pH 8·0), 1·5 mol l−1 NaCl, 1% CTAB (w/v)] and 100 μl of proteinase K (10 mg ml−1) by horizontal shaking at 200 rev min−1. After shaking for 30 min at 37°C, 1·5 ml of 20% (w/v) SDS was added, and the samples were incubated in a water bath set at 65°C for 2 h with gentle end-over-end inversions every 15–20 min. Supernatant collected after centrifugation at 6000 g for 10 min at 25°C was mixed with an equal volume of chloroform : isoamyl alcohol (24 : 1, v/v) and the aqueous phase recovered by centrifugation was precipitated with 0·6 volume of isopropanol at 25°C for 1 h. After centrifugation at 16 000 g for 20 min at 25°C, the pellet was washed with cold 70% ethanol, and re-suspended in sterile deionized water, in a final volume of 500 μl and purified by eluting the crude extract through minicolumn (Qiagen, India).

PCR library construction

PCR library for 16S rRNA gene was constructed directly from the soil DNA using the bacterial primers (8F and 1406R) as described by Coolen et al. (2005).

8F: 5′-AGA GTT TGA TCC TGG CTC AG-3′

1406R: 5′-ACG-GGC GGT GTG TRC-3′

Similarly, PCR library for α subunit and β subunit of the dioxygenase was constructed with the following primers based on the Rieske iron-sulfur motif (Rf) sequences:

Rf1: 5′-AGG GAT CCC CAN CCR TGR TAN SWR CA-3′ (Kahng and Oh 2005)

Rr1: 5′-TGTTCCCGAACTTGTCCTTC-3′ (Zhou et al. 2006) and

Rf2-F: 5′-ATGGTNGCNACNGTNGARCA-3′

Rf2-R: 5′-CAAGCTTTTAGATCCAGAATGACAGGTT-3′ (Kim et al. 2006)

Primers Rf1 and Rr1 were used to amplify the α subunit, whereas Rf2-F and Rf2-R were used to amplify the β subunit.

PCR reactions (25 μl) included 0·5 μl of deoxynucleoside triphosphate (10 mmol l−1), 2·0 μl of MgCl2 (25 mmol l−1) and 1 μl of each primer stocks (10 mmol l−1). One unit of Taq polymerase (Life Technologies, India) and one-tenth volume of 10× buffer were added to each reaction. The thermal cycling conditions consisted of annealing temperature of 52°C 1 min. Each cycle included a 1 min denaturation step at 95°C and an extension step of 1 min at 72°C. Amplicons were cloned with TA instant cloning kit in pTZ57R/T vector (Fermentas, India) according to the manufacturer’s instructions.

Screening of the clones

To speed up the cloning processes, M13/pUC primers specific to the polylinker of the vector pTZ57R/T were used. The cloned inserts were directly amplified from transformant cells using these primers. The size of the PCR amplified product was expected to be 200 bp if no fragment is inserted into the vector. A tiny amount of cells picked up by touching the colony with a toothpick, from a Luria Broth (LB) agar plate containing ampicillin (50 μg ml−1), was added to a standard PCR mix containing 1×Taq polymerase buffer, 1 units Taq Polymerase (Life Technologies), 200 μmol l−1 dNTPs and 100 pmol of each primer in a 25-μl reaction. The reaction conditions consisted of denaturation at 94°C for 3 min, 35 cycles of 94°C for 1 min, 52°C for 1 min and 72°C for 1 min, plus one additional cycle with a final 5 min of chain elongation. The amplified PCR products were analyzed for inserts by gel electrophoresis on agarose gels using standard protocols (Sambrook et al. 1987). Gels were stained with ethidium bromide and photographed using quantity one software (Bio-Rad, India). A 100 bp ladder (MBI-Fermentas) was included on every gel.

RFLP analysis

The amplified PCR products were purified by Qiagen PCR purification system (Qiagen, USA) according to the manufacturer’s instructions. To detect unique SSU rDNA clones, RFLP analysis was carried out. One-fifth of the 25-μl-PCR-amplified products was digested with 1 U restriction enzymes HhaI, MspI and RsaI for 16S rRNA and Eco31I, AatII and EcoII for dioxygenase gene fragments (Fermentas), respectively, at 37°C overnight. The resulting RFLP products were separated by gel electrophoresis in 2% agarose (Sigma, USA) in 1× TBE at 4°C with 7 V cm−1 for 2 h. The gel was stained with 0·5 μg ethidium bromide per ml and visualized by UV excitation. The RFLP patterns were compared to score the presence of operational taxonomic units (OTUs) based on different restriction patterns (data not shown). Clones with similar restriction pattern were placed in the same group and defined as members of clone family.

DNA sequencing and phylogenetic analysis

Unique clones of 16S rRNA and α-subunit and β-subunit of the dioxygenase were sequenced by Chromous Biotech, India. Sequences were screened for the presence of vector sequence using VecScreen software (http://www.ncbi.nlm.nih.gov/VecScreen/VecScreen.html) and then Blast searched through the NCBI GenBank database. Similarly, sequences of 16S rRNA were aligned manually to the SSU rDNA sequences of the species, which showed high similarity scores aligned SSU rDNA sequence database, RDP (Ribosomal Database Project) (Maidak et al. 1997). Phylogenetic tree was constructed by neighbour-joining analysis using Molecular Evolutionary Genetics Analysis (mega) software with 500 bootstrap replicates (Kumar et al. 2004).

Statistical analysis

Accumulation curves were plotted for each clone library. The data were rarefied using EstimateS (ver. 8; R. K. Colwell, http://purl.oclc.org). The nonparametric richness estimates, Species richness (S), Chao1 (Chao and Lee, 1992), Simpson’s index, Shannon–Weaver diversity indices (H′) were calculated for each clone library (Pielou 1977; Colwell et al. 2004) and Evenness, calculated from the Shannon–Weaver diversity function by use of the equation H/Hmax, where Hmax = log2S. Per cent coverage was calculated using a standard equation (Begon et al. 1990).

Nucleotide sequence accession numbers

The nucleotide sequences of 16S rRNA, α-subunit and β-subunit of dioxygenase reported in this study were deposited in the GenBank database with the following accession numbers EU755062 to EU755117, EU755135 to EU755162 and EU755118 to EU755134, respectively.

Results

The coal-tar-contaminated soil contained 1225 mg of total extractable PAH residue per 10 g of soil. Liquid chromatography analysis indicated the presence of seven priority pollutant PAHs in the coal-tar plant soil, namely benzo[g,h,i]perylene, dibenzo[a,h]anthracene, indeno[1,2,3-c,d]pyrene, pyrene, acenaphthylene, benzo[k]fluoranthene and benzo[b]fluoranthene.

Enrichment cultures were monitored over time to examine the biodegradation potential of soil bacterial communities enriched in the presence of naphthalene, phenanthrene and pyrene as representative of the two-ring-, three-ring- and four-ring-PAH compounds. Naphthalene degradation by the enrichment cultures was >95% in 15 days (Fig. 1). On the other hand, the phenanthrene enrichment cultures showed a 24-h lag time before the onset of phenanthrene degradation after which 28% of phenanthrene was degraded after 7 days, which increased to 79% after 15 days (Fig. 1). Initiation of pyrene degradation by the enrichment cultures occurred after a 24-h lag and 53% of pyrene degradation occurred after 15 days (Fig. 1). As the coal-tar-contaminated soil was undergoing active biodegradation of PAHs, it was interesting to study the diversity of bacterial community as well as functional diversity prevailing in this site.

Figure 1.

 Biodegradation of polyaromatic hydrocarbon (PAHs) in coal-tar-contaminated soil spiked with naphthalene (N), phenanthrene (PH) and pyrene (PY) 50 mg l−1 each. Resid,ual PAH analysis was carried out by liquid chromatography and extraction efficiency of all the three compounds was >90%. The val,ues given here are the average of three experiments. (inline image N; (inline image) PH and (inline image) PY.

Bacterial community structure was determined by establishing a clone library of 1·4 kb 16S rRNA gene using DNA isolated from soil samples of coal-tar-mixing plant. Amongst the 102 clones used for RFLP analysis, 52 clones represented unique operational units, which were then sequenced. Distribution curve of the sequenced 16S rRNA clones from coal-tar-contaminated soil were most closely related to the members of Proteobacteria (classes Alpha-, Epsilon, and Gammaproteobacteria), Actinobacteria, Anaerolineae, Firmicutes, and Acidobacteria lineages. Actinobacteria (31%) and Gammaproteobacteria (30%) were the most predominant followed by Acidobacteria (12%) and Alphaproteobacteria (9%). Firmicutes (4%) and Epsilonbacteria (1%) were amongst the minor groups detected in the coal-tar-contaminated soil.

Members of clone family HM-9 and HM-83 showed highest similarity with Nocardioides sp. (98%), which is a PAH degrader. Members of clone family HM-162 and HM-8 were closely related to Streptomyces sp. (97%) (Fig. 2), whilst members of clone family HM-22 and HM-13 showed resemblance to Arthrobacter sp. (98%). Clone family HM-127 and HM-2 was similar to uncultured Acidobacteria, whereas members of clone family HM-84 showed maximum similarity to uncultured Actinobacterim (Fig. 2). Clone family HM-130 was clustered with uncultured Rhodobacteraceae (Fig. 2). Clone family HM-10 was clustered with low G+C Gram-positive bacteria (96%), whereas members of clone family HM-15 showed 97% similarity with uncultured Gamma proteobacteria (Fig. 2). Members of clone families HM-122, 49 and 161 clustered with uncultured Chloroflexi bacteria. Most of the clone families (HM-3, 144, 145 and 24) resembled the uncultured groups (Fig. 2). None of the clone family showed resemblance to either Betaproteobacteria or Deltaproteobacteria.

Figure 2.

 Phylogenetic tree of 16S rRNA clone sequences as determined by neighbour-joining methods from coal tar contaminated soil (HM), selected cultured isolates, and environmental clone reference sequences. Reference sequences from GenBank include the accession number. The scale bar represents substitutions per nucleotide. U bacterium, uncultured bacterium.

To assess the presence and diversity of dioxygenase in coal-tar-contaminated soil, partial subunits of dioxygenase gene representing Rieske center of the α-subunit (Rf1; c. 700 bp) and β-subunit (500 bp) were amplified. α-subunit clone library consisted of 29 unique OTU. Homology searches with sequences from GenBank confirmed that sequences deduced from the PCR products of the clones had similarity with other dioxygenase members of various bacterial genera. Analysis of the relationship of the α-subunit clone families to one another divided them into several subfamilies (Fig. 3). α-subunit library was dominated by one family Rf-1-78, which represented 11% of the clones, followed by Rf-1-106 family, which comprised 8% of the total clones. Members of clone family Rf-1-57 were most closely related to Rf1 segment of nidA3 of Mycobacterium vanbaalenii PYR-1 (Fig. 3), whilst the members of clone family Rf-1-18 was clustered with PhdA of Nocardioides sp. strain KP7 and Rf-1-3 family showed similarity with the α subunit that was identified through metagenomic approach. It was also observed that 38% of the α-subunit clone library sequences did not exhibit significant similarity to any dioxygenase sequences currently in GenBank and 33% have only low matches (<50%) to uncharacterized dioxygenase sequences. Analysis of the bacterial ring hydroxylating dioxygenase α subunit showed a signature as C-x-H-X17–CX, which is well conserved amongst Rieske nonheme iron dioxygenases. Figure 4 presents an amino acid sequence alignment for the conserved residues of α subunit (Rf1) clone families from coal tar contaminated soil with the three other α subunits recently identified amongst the hydrocarbons degrading bacteria. Clone families Rf-1-4, Rf-1-78, Rf-1-11, Rf-1-36 clearly indicated the presence of signature {CR(S)HRG} of dioxygenase gene, whereas members of clone family Rf-1-1, Rf-1-2, Rf-1-14 and Rf-1-57 possessed the critical amino acids, i.e. C-X-H (Fig. 4). Although these gene families share common sequence features with other terminal dioxygenases, the overall identity of these sequences with those of other dioxygenases was moderate.

Figure 3.

 Phylogenetic distribution of the Rieske gene fragment families from the coal-tar-contaminated soil (HM). Dendrogram was constructed from a ClustalW alignment of β-subunit gene sequences by neighbour- joining analysis using Mega 4.0. Reference sequences from GenBank include the accession number. The scale bar represents substitutions per nucleotide. U bacterium, uncultured bacterium.

Figure 4.

 Multiple alignment of deduced amino acid sequences from gene sequences of α-subunit of dioxygenase from the coal-tar-contaminated site with known dioxygenases.

Seventy-six clones representing β-subunit of dioxygenase were retrieved from coal-tar-contaminated soil, which showed 17 unique sequences. β subunit clone families were dominated by one family HM-30, which represented 25% of the clones sequenced. Two other clone families, HM-106 and HM-25 represented 28% of the clone library. Members of clone family HM-10 was most closely related to the β subunit (ndoB) of naphthalene dioxygenase from Novosphingobium aromaticivorans (Fig. 5), whilst members of clone family HM-27 was closely related to β subunit of dioxygenase from Acinetobacter baumannii. Clone families HM-31 and HM-14 showed closeness to uncharacterized β subunit of dioxygenase. About 14% of the clone library sequences did not exhibit significant similarity to any dioxygenase sequences currently in GenBank, whilst another 21% had low similarity with β subunit of dioxygenase.

Figure 5.

 Phylogenetic distribution of the β-subunit gene fragment families in the Hot Mix Plant soil. Dendrogram was constructed from a ClustalW alignment of β-subunit gene sequences by neighbour-joining analysis using Mega 4.0. Reference sequences from GenBank include the accession number. The scale bar represents substitutions per nucleotide. U bacterium, uncultured bacterium.

The Shannon–Weaver diversity index, Chao1 and Simpson’s reciprocal index nonparametric species richness estimators were calculated for each clone library. Species richness and Shannon–Weaver diversity index indicates that 16S rRNA clone library was the most diverse, followed by α-subunit library and β-subunit library (Table 1). Simpson’s reciprocal index also indicated the same trend. The Chao1 estimate of species richness indicates a difference in the estimated species richness of the α-subunit and β-subunit libraries.

Table 1.   Statistical analyses of 16S rRNA, α- and β-subunit of dioxygenase gene clone libraries using ecological and molecular estimates of phylotype diversity
 16S rRNAα subunitβ subunit
  1. C, library coverage; S, species richness; H’, the Shannon–Weaver diversity index; Chao1, the Chao1 nonparametric richness estimate; 1/D, Simpson’s reciprocal index; E, evenness of the population.

C44·5572·8977·63
S562917
H’3·923·182·39
Chao176·3630·1421·16
1/D31·257·633·58
E0·970·940·84

Rarefaction curves were plotted for each clone library using EstimateS (ver. 8.0). The rarefaction results for 16S rRNA clone library indicates that only a portion of the richness in the bacterial communities was surveyed as the curve did not show asymptote (Fig. 6). This is an indication that further sequencing of 16S rRNA clone library may yield new bacterial families as the percentage coverage for the 16S rRNA library was only 45% (Table 1). However, species accumulation curves of α-subunit and β subunit clone libraries achieved an asymptote (Fig. 6) that indicated saturation of sampling that is, the slope neared a value of zero. The percent coverage of α-subunit and β subunit clone libraries are 77·77% and 77·63%, respectively (Table 1) was higher than that of 16S rRNA library.

Figure 6.

 Rarefaction curves determined for the various phylotypes of: 16S rRNA gene, α-subunit subunit of dioxygenase and β subunit of dioxygenase clones from coal-tar-contaminated soil. Rarefaction analysis was performed using software EstimateS. (inline image) 16S rRNA, (inline image) α-subunit of dioxygenase and (inline image) β-subunit of dioxygenase.

Discussion

The ability of bacteria to utilize individual PAHs as carbon and energy sources has been extensively documented over the last few decades. Whilst these studies have been essential for establishing metabolic pathways for the biodegradation of individual PAHs, there is increasing interest in understanding how organic pollutants affect the overall structure of microbial communities. In order to develop effective strategies for the bioremediation of PAH contaminants, a better understanding of the composition and metabolic potential of microbial communities in highly contaminated soils using cultivation-independent methods is required.

Many studies have used molecular tools such as PCR in order to detect the existence of novel dioxygenase units based on Rieske Center (78-bp) of α-subunit during biodegradation of PAHs (Chadhain et al. 2006) or from the already reported bacterial systems (Kahng and Oh 2005; Zhou et al. 2006). However, the available information concerning PAH dioxygenase gene diversity from uncultured bacteria of coal-tar-contaminated environment is still sparse.

Primers used in the present study have been successfully used to amplify the dioxygenase gene from the cultureable micro-organisms (Kim et al. 2006; Zhou et al. 2006); however, they were not used to amplify the dioxygenase gene from the environmental sample directly. These primers successfully yielded 29 clone families for α subunit and 17 clone families for β subunit of dioxygenase gene from coal-tar-contaminated soil. Deduced amino acid sequences of the members of α subunit contain the consensus pattern of bacterial ring-hydroxylating dioxygenase α-subunits: C-x-HR-[GAR]-x(7,8)-[GEKVI]-[NERAQ]-x(4,5)-C-x-[FY]-H (PROSITE: PS00570), (Hulo et al. 2006). This consensus includes the cysteines and histidines (Cys81, and His83) in Pseudomonas putida NCIB9816-4, (Kauppi et al. 1998) that are involved in the coordination of the iron ions in the Rieske [2Fe-2S] center of many dioxygenases (Ferraro et al. 2005). However, we also amplified gene fragments that showed little or no homology to the known dioxygenase genes (38% for α subunit and 14% for β subunit). Previous studies also reported the presence of dioxygenase genes, which showed low or no homology to that available in the database (Taylor et al. 2002; Kahng and Oh 2005; Taylor and Janssen, 2005; Gomes et al. 2007; Lozada et al. 2008). Presence of diagnostic conserved cysteine and histidine residues leads us to believe that the amplified fragments are derived from previously unreported genes.

It is believed that β subunit of dioxygenase plays a critical role in combination of α subunit (Hurtubise et al. 1998). Structural features of both subunits may influence the dimension and shape of the catalytic site to determine which PAHs can be oxygenated, as well as the orientations of the adjacent reactive carbons towards the active site. Hurtubise et al. (1998) demonstrated that both α- and β-subunits of the aryl-hydroxylating dioxygenases influence the enzyme–substrate interaction. Thus, we also examined if any heterogeneity of β subunit existed in the coal-tar-contaminated soil. Interestingly, we were able to detect putative class of β subunit of dioxygenase. Rarefaction curves as well as diversity indices indicated a low diversity of β subunit compared to α-subunit clones, which was also indicated by the high average sequence identity of the β subunit clones relative to the average sequence identity of known β subunit sequences.

Phylogenetic composition showed the presence of ten different groups of bacteria in the coal-tar-contaminated soil, which was dominated by Actinobacteria and Gammaproteobacteria. Chadhain et al. (2006) showed the dominance of Gammaproteobacteria during the enrichment on naphthalene, phenanthrene and pyrene; however, members of Actinobacteria were missing in all the three enrichment systems. We were unable to detect any genera of Betaproteobacteria and Deltaproteobacteria; however, we detected very low number of clones belonged to the Gemmatimonadetes and Deinococci lineages. In contrast, Bakermans and Madsen (2002) showed the presence of Betaproteobacteria in coal-tar-waste-contaminated aquifer and reported the dominance of facultative aerobic and anaerobic bacteria. Most of the clones showed similarity to the sequences from uncultured bacteria indicating the presence of diverse class of yet-to-be uncultured bacteria which may play a role in the degradation of PAH compounds.

Our study with the 16S rRNA clone families revealed the presence of genera that showed relatedness to Arthrobacter sp., uncultured Gammaproteobacteria sp. Nocardioides sp., uncultured Rhodobacteraceae bacterium and uncultured low G+C Gram-positive bacterium. The role of these bacteria in the PAH-biodegradation in the environment has been well established and their dioxygenase genes have been recently identified (Larkin et al. 1999; Saito et al. 2000; Zhou et al. 2006). Thus, there seems to be a correlation between the presence of dioxygenase genes and the presence of certain genera in the coal-tar-contaminated soil. We used traditional laboratory biodegradation studies combined with culture independent molecular ecological techniques to explore the presence of diverse microbial community and functional gene profiles in a PAH-contaminated site undergoing bioremediation. Overall, our study suggests that a wide diversity of dioxygenase genes can be present in contaminated environments, which results in the succession of different dioxygenase gene populations for the degradation of different PAHs. The combination of the Rieske primers and bacterial community profiling represents a powerful tool for both assessing bioremediation potential in and for the exploration of novel dioxygenase genes in a contaminated environment.

Acknowledgement

This research work was supported by the grant from Department of Biotechnology (DBT), New Delhi, India.

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