Introduction

Aerobic biodegradation of polychlorinated biphenyls (PCBs) and their analog, biphenyl (BP), has been the subject of a large body of research for more than three decades. A fundamental goal has been to identify bacteria and genes involved in the PCB degradation process to optimize bioremediation processes. Numerous phylogenetically diverse bacteria have been isolated that perform BP and/or PCB degradation (Abraham et al., 2002). Genes encoding the BP (upper) degradation pathway and benzoate (lower) pathways (Pieper, 2005) in isolates have been extensively characterized, and some genes have been directly detected in the environment using molecular tools (Erb and Wagner-Dobler, 1993; Ringelberg et al., 2001; Baldwin et al., 2003). However, until recently, methodological limitations precluded researchers from ascertaining which bacteria or genes are truly active in BP/PCB degradation in the soil community.

The recent development of stable isotope probing (SIP) methods enables researchers to directly identify microorganisms involved in the degradation and/or assimilation of carbon from a specific compound without the limitations of cultivation (Friedrich, 2006; Whiteley et al., 2006). In DNA-based SIP, microbes within a complex community that grow using a ¹³C-labeled substrate incorporate ¹³C into their DNA, permitting it to be separated from the DNA of inactive organisms by isopycnic centrifugation. Subsequent analyses of ¹³C-DNA reveal the identity of organisms that derived ¹³C from the substrate, implicating them in the biodegradation of the compound or its metabolites. Previous SIP studies have revealed organisms that derive carbon from a variety of aromatic pollutants in soil, including phenol and polycyclic aromatic hydrocarbons (Madsen, 2006) and soil-derived PCB-degraders in a biofilm growing on PCB droplets (Tillmann et al., 2005). Because DNA-SIP provides entire genomes of organisms that derive ¹³C from the substrate, it also affords opportunities to investigate their functional gene contents, and degradative genes detected may be implicated in the assimilatory process (Friedrich, 2006).

A previous cultivation-based study at a PCB-contaminated site in the Czech Republic indicated that Rhodococcus was the predominant genus of PCB-degrading bacteria (Leigh et al., 2006). Culturable BP utilizers were present throughout the site, including the root zones of trees, and occurred in higher numbers in the root zone of Austrian pine (Pinus nigra L.) and goat willow (Salix caprea) than other tree species examined or nonrooted soil (Leigh et al., 2006). This finding was consistent with the hypothesis that certain plants biostimulate PCB-degrading bacteria by releasing plant aromatic compounds that function as growth substrates (Donnely et al., 1994; Singer et al., 2004), a hypothesis developed out of the fact that pathways for microbial aromatic degradation are thought to have evolved in response to plant aromatics and are widespread in root-associated soil (Harwood and Parales, 1996). It remains unknown whether the culturable PCB degraders identified accurately reflect active PCB (BP) degraders in the root zone, and which degradative genes or pathways the active organisms may be employing.

We have integrated DNA-SIP with comprehensive functional gene analyses for exploring the PCB-contaminated root zone of pine to identify bacteria that derive carbon from BP and to identify aromatic degradative genes they possess. Functional gene analyses of active BP utilizers were performed by hybridizing ¹³C-DNA to the GeoChip, a functional gene array containing 6465 probes targeting genes involved in degradation of a wide variety of aromatic compounds, including 388 probes targeting BP degradation pathways, 167 probes targeting benzoate degradation pathways and over 2400 probes targeting degradative pathways for monoaromatic compounds and plant aromatic compounds. Initial aromatic ring hydroxylating dioxygenases (ARHDs), which catalyze the critical first step in biodegradation of aromatic compounds, were further examined in ¹³C-DNA by polymerase chain reaction (PCR) and sequence analyses using a specially designed primer set. Culturable BP utilizers were also isolated and compared with SIP results to examine the extent of culture bias.

Materials and methods

Soil sample

Soil was collected from the root zone (20–40 cm depth) of a 24-year-old Austrian pine (Pinus nigra) tree growing naturally in a PCB-contaminated site (soil PCB concentration 15 mg/kg) in the Czech Republic. The date of sampling was November 2000, when culturable populations of PCB degraders were at their seasonal maximum as described previously (Leigh et al., 2001, 2006). The sample was stored at 4°C in a plastic bag for 3 years between collection and initiation of this experiment. Fresh samples were unavailable due to capping of the site; however, cultivation of BP-utilizers following protocols applied previously to newly collected soil (Leigh et al., 2006) yielded the same consortium.

Stable isotope probing microcosms

Microcosms containing soil provided with either uniformly ¹³C-labeled BP (99 atom % ¹³C; ISOTEC, Miamisburg, OH, USA) or unlabeled BP (Sigma, St Louis, MO, USA) were established to permit destructive harvesting at each time point (1, 4 and 14 days). Three replicate microcosms were established for each substrate and time point. BP was first added to empty, sterile, 160 ml serum bottles by pipetting 100 l of a 10 mg/ml acetone solution onto the inner wall and then allowing acetone to completely dry for 1–2 h in a laminar flow hood leaving behind BP crystals. A 140 g soil sample was restored to field moisture conditions (Leigh et al., 2006) by moistening with 14 ml sterile distilled water followed by incubation at 25°C for 48 h. Aliquots of homogenized soil (5 g) were then loosely placed in serum bottles containing BP. Microcosms were sealed with Teflon stoppers (Westco, Exton, PA, USA) and aluminum crimp caps to contain the headspace for later ¹³CO₂ sampling, and were incubated standing at 25°C in the dark. After 1, 4 and 14 days incubation, a sample of the headspace of one microcosm each containing ¹³C-BP and unlabeled BP was collected to determine the abundance of ¹³C in CO₂ (described below) and soil was frozen immediately at -80°C.

Concentration and carbon isotopic composition of CO₂

Headspace gas was collected at microcosm initiation (ambient lab air), 1, 4 and 14 day time points in volumes of 1, 1, 0.5 and 0.1 ml, respectively, using an N₂-purged syringe and was stored in 12 ml serum bottles containing ultra-high purity N₂ (Linde gas, Independence, OH, USA) at room temperature. Carbon dioxide in the stored bottles was analyzed for ¹³C within 7 days on a Trace Gas system interfaced to an IsoPrime mass spectrometer (GV Instruments, Manchester, UK). Typical volumes sampled from the 12 ml serum bottles ranged from 200 to 1000 l. Before analysis, gas tight syringes were purged with ultra-high purity N₂ and a volume equal to the expected sample volume was added to the 12 ml serum bottle to maintain atmospheric pressure in the bottle. An aliquot of the sample was injected using a gas tight syringe into a direct injection port of the Trace Gas. The Trace Gas system automatically concentrates CO₂ cryogenically, removes water by a magnesium perchlorate trap and Nafion drier, and introduces a purified peak of CO₂ to the mass spectrometer following purification on a Poraplot Q gas chromatographic column with He as the carrier gas. The concentration of CO₂ was determined from the area under the mass 44 trace with corrections applied for dilution from added N₂. The carbon isotopic composition of CO₂ is expressed in atom percent.

Cultivation of BP-utilizing bacteria

BP-utilizing bacteria were cultivated by spreading organisms suspended from 0.5 g soil onto basal mineral agar prepared with Difco Noble agar (Becton, Dickinson and Co., Sparks, MD, USA) with BP vapor provided as the sole carbon source (Leigh et al., 2006). Colonies were subjected to a BP clearing zone assay using methods described previously, except that the clearing zone assay was performed with BP rather than 4-bromo-BP (Leigh et al., 2006), and colonies producing clearing zones were enumerated. Eighty-four BP-utilizing colonies were randomly selected from plates of soil at time zero for isolation and further screening. Colonies were isolated on one-eighth strength plate count agar (Leigh et al., 2006), screened again for BP clearing, and then BP utilization further verified by growth in liquid culture with BP crystals as sole carbon source in basal mineral medium.

DNA was extracted from BP-utilizing isolates using the Qiagen DNeasy Tissue kit (Qiagen, Valencia, CA, USA), and 16S rRNA genes were PCR-amplified using universal eubacterial primers 27F and 1392R (Johnson, 1994). PCR products were purified with the Qiaquick PCR purification kit (Qiagen, Valencia, CA, USA) and were submitted to Macrogen Inc. (Seoul, Korea) for single extension sequencing using primer 27F, yielding sequences of approximately 850 bp in length. Sequences were phylogenetically identified using RDPII Classifier and Sequence Match (Cole et al., 2005) and were deposited in GenBank (accession no. EF507168–EF507182).

Soil DNA extraction

DNA was extracted from 4 g aliquots of SIP microcosm soil using a freeze-grinding method modified from Zhou et al. (1996). Soil was pretreated for 10 min with 0.5 ml 50 mg/ml lysozyme at 25°C. Following extraction, DNA was precipitated with 30% polyethylene glycol 6000 (Fluka, Buchs, Switzerland) in 1.6 M NaCl (Griffiths et al., 2000) to minimize contamination with humic materials. Pellets were washed with 70% ethanol, resuspended in water, brought to 0.3 M NaCl and were added to spin columns (Bio-Rad, Hercules, CA, USA) containing 1 ml Sephacel (Sigma, St Louis, MO, USA) pre-equilibrated with 4 ml 0.3 M NaCl in TE (10 mM Tris–Cl, pH 8.0), washed with 4 ml 0.3 M TE in NaCl in TE, and eluted with 4 ml 0.5 M CaCl in TE. DNA was then precipitated with isopropanol, pellets washed with 70% ethanol, then resuspended in water (Zhou et al., 1996).

¹³C-DNA separation

Equilibrium (isopycnic) density gradient centrifugation and fractionation were adapted for DNA-SIP from methods for RNA-SIP by Manefield et al. (2002) using cesium trifluoroacetate (CsTFA; Amersham Biosciences, Piscataway, NJ, USA) solution without the addition of formamide and with a starting buoyant density (BD) of 1.60 g/ml. Samples of 1.5 g DNA were combined with CsTFA solution and then subjected to ultracentrifugation using the same tubes, rotor dimensions and conditions described previously (Manefield et al., 2002). Gradients were fractionated into 100 l fractions as described previously (Manefield et al., 2002). BD of fractions was determined gravimetrically by weighing aliquots of each fraction from blank gradients run in parallel containing water in place of DNA. Sample DNA was precipitated from fractions with isopropanol for 2 h at 25°C, and then pellets were washed twice with isopropanol and resuspended in Qiagen EB elution buffer (Qiagen, Valencia, CA, USA).

To locate fractions containing ¹³C-DNA, the distribution of DNA in gradient fractions ranging in BD from 1.561 to 1.684 g/ml was determined by real-time PCR using universal eubacterial primers 1132F (Wilmotte et al., 1993) and 1108R (Amman et al., 1995) targeting 16S rRNA genes. Quantitative PCR (Q-PCR) was performed in triplicate 15 l reactions containing SYBR green master mix (Applied Biosystems, Foster City, CA, USA), 4.5 pmol each primer and 1 l template, using a thermocycler program of 40 cycles of 95°C for 15 s and 60°C for 1 min on an ABI PRISM 7900HT sequence detection system (Applied Biosystems, Foster City, CA, USA). Gene copy number was determined in relation to a standard curve constructed with Pseudomonas stutzeri JM300 genomic DNA which contains 213 000 16S rRNA copies per ng of DNA (Ginard et al., 1997).

After the range of fractions containing ¹³C-labeled DNA or unlabeled DNA were identified, they were combined to constitute compiled 'heavy' and 'light' fractions from each sample for downstream molecular analyses. To control for background contamination of DNA throughout the density gradient, the equivalent heavy fractions from a control unlabeled sample (total community DNA at time 0) were also compiled, subjected to the same analyses, and genes detected in the background subtracted from those found in ¹³C-DNA.

Community profiling

Terminal restriction-fragment length polymorphism (T-RFLP) analyses were performed on total community DNA, density gradient fractions representing unlabeled (light) DNA, ¹³C-labeled (heavy) DNA and the background control. DNA samples were PCR-amplified using primers 27F and 1392R targeting eubacterial 16S rRNA genes (Johnson, 1994). Primer 27F was labeled with 6-carboxyfluorescien (6-FAM) on the 5' end. PCR was performed by initially amplifying 1 ng template with 5 pmol primer in a 25 l reaction using a thermocycler program of 95°C for 9 min, then 25 cycles of 95°C for 1 min, 59°C for 1 min, 72°C for 1 min 40 s and final extension at 72°C for 10 min, followed by a reconditioning step in which 5 l aliquots of initial PCR product were transferred to new reactions and amplified for three cycles under the same PCR conditions except in 50 l volumes with 10 pmol primers (Thompson et al., 2002). PCR products were purified using the Qiaquick PCR purification kit (Qiagen, Valencia, CA, USA), and then 40 ng was digested with HhaI endonuclease (New England Biolabs, Beverly, MA, USA) following the manufacturer's instructions in reaction volumes of 15 l for 4 h at 37°C. Digests were precipitated and resuspended in 10 l as described previously (Gallagher et al., 2005), and then the entire volume was loaded on an ABI PRISM 3100 genetic analyzer (Applied Biosystems, Foster City, CA, USA) and run using capillary injection time of 10 s. T-RFLPs were analyzed using GeneScan and Genotyper software systems (Applied Biosystems, Foster City, CA, USA).

16S rRNA gene sequence analyses

Cloning and sequence analyses were performed on 16S rRNA gene amplicons from total community DNA from time 0, ¹³C-DNA from each time point and the background control. PCR was performed using the same conditions as for T-RFLPs, but without the reconditioning step or the 6-FAM label on the 27F primer. For each sample, 12 replicate 25 l PCR reactions were created to generate abundant PCR products and to span variations in template and amplification bias, and then were compiled, purified and concentrated using the Qiaquick PCR cleanup kit (Qiagen, Valencia, CA, USA). Products were gel-purified using the Qiaquick gel purification kit (Qiagen, Valencia, CA, USA). Immediately before cloning, 3' poly-A overhangs were added by incubating 25 l reactions containing 20 l purified amplicon, 25 mol dATP, 0.5 U taq polymerase and 75 mol MgCl₂ in 1 taq buffer at 72°C for 10 min. Cloning was performed using the TOPO-TA cloning kit for sequencing (Invitrogen, Carlsbad, CA, USA), and clones were provided to Macrogen Inc. (Seoul, Korea) for plasmid purification and sequencing by single extension with primer 27F. Resulting sequences of approximately 650 bp in length were trimmed for a base score of >20 (error probability <0.1). Sequences from ¹³C-DNA that shared 99% sequence identity to any sequence detected in the background control library (heavy fractions from nonincubated soil) were identified using FastGroupII, a web-based program designed for dereplicating clone libraries (Yu et al., 2006). In this way, we ensured that any DNA contamination in density gradients was not misinterpreted as ¹³C-DNA. Phylogenetic analyses were performed using RDPII Classifier and Sequence Match (Cole et al., 2005) and were deposited in GenBank (accession no. EF506948–EF507159).

Aromatic ring hydroxylating dioxygenase gene sequence analyses

ARHDs were PCR-amplified in total community DNA, ¹³C-DNA and the background control using a primer set previously developed to universally target ARHDs catalyzing the initial step in polyaromatic hydrocarbon (including BP) degradation (Bellicanta and Pellizari, 2004). The specificity of primers ARHD1F and ARHD1R for BP dioxygenase genes was tested in silico using the Fungene bphA1 Stephan Gantner database (http://flyingcloud.cme.msu.edu/fungene/). The primers ARHD1F (5'-TTYRYNTGYANNTAYCAYGGNTGGG) and ARHD1R (5'-CCCANCCRTGRTANNTRCANRYRAA) yield PCR products ranging in size from 308 to 329 bp in length.

PCR reactions were performed with 30 pmol of each primer in a total volume of 25 l, using a thermocycler program of 97°C for 3 min, 40 cycles of 94°C for 1 min, 50°C for 1 min and 72°C for 1 min, with final extension for 5 min at 72°C. PCR products from ¹³C-DNA were gel-purified and cloned as described above for 16S rRNA gene amplicons. Plasmids were extracted and subjected to single-extension sequencing using the primer ARHD1F by the Research Technology Support Facility (Michigan State University, East Lansing, MI, USA). For BP-utilizing isolates, ARHDs were PCR-amplified from genomic DNA extracts and sequenced using the same methods as for ¹³C-DNA. ARHD sequences were subjected to GenBank BLASTn searches to identify nearest matching sequences in the database. For ARHD phylogenetic tree reconstruction, partial gene sequences were translated to their amino-acid sequences and alignment was made using the CLUSTALW program, version 1.82 (Thompson et al., 1994) of the European Bioinformatics Institute (http://www.ebi.ac.uk/clustalw/#). Alignment was visually inspected and manually corrected when necessary. Alignment was exported and the parsimony algorithm was run in PAUP^* for bootstrap values with 1000 replicates (Swofford, 2000). ARHD sequences obtained from clones (accession no. EF507160–EF507167) and isolates (accession no. EF507183–EF507184) were deposited in GenBank.

GeoChip

A functional gene array, or GeoChip, containing over 24 000 probes targeting numerous functional genes important to microbial ecological processes (He et al., 2007), was used to explore the functional genes present in SIP samples. The GeoChip contains 6465 probes targeting genes involved in degradation of a wide variety of aromatic compounds, including 388 probes targeting BP, 167 targeting benzoate degradation pathways and over 2400 targeting conserved monoaromatic degradative pathways (i.e. -ketoadipate, homogentisate) and catabolic genes for plant aromatic compounds (i.e. vanillin, cymene). The GeoChip was constructed with three different 50-mer probes targeting each gene of interest designed with the highest specificity to individual organisms and sequences possible (He et al., 2007).

Three replicate hybridizations were performed for each of the following samples: total community DNA, ¹³C-DNA (14 days incubation) and the background control (heavy fractions from nonincubated soil). Whole community genome amplification (WCGA) (Wu et al., 2006) was used to amplify approximately 1 ng aliquots of each DNA sample to produce >3 g DNA for each array hybridization. WCGA was performed using a TempliPhi Amplification kit (Amersham Biosciences, Piscataway, NJ, USA). Reaction mixtures consisted of approximately 1 ng DNA plus 10 l sample buffer, 10 l reaction buffer, 1 l enzyme mix, 1 l single-stranded binding protein (260 ng/l) and 1 l spermidine (1 mM). Reactions were incubated at 30°C for 4 h and then inactivated by heating to 65°C for 10 min. Approximately 4 l of WCGA was analyzed by 1% agarose gel electrophoresis to confirm successful amplification.

WCGA-amplified DNA was Cy5-labeled using a Bioprime Array CGH Genomic Labeling System (Invitrogen, Carlsbad, CA, USA). Aliquots of WCGA products (3 g) were dried completely, combined with 20 l random octamer (3 mg/ml) and 3 l 1 mM spermidine in a 40 l reaction, heated to 99.9°C for 5 min and then chilled on ice. A 20 l aliquot of master mix was added, containing 2.5 l of dNTP mix (2.5 mM dTTP, 5 mM dATP, 5 mM dCTP and 5 mM dGTP), 1 l Cy5 dUTP (Amersham Biosciences, Piscataway, NJ, USA), 0.7 l recA (490 ng/ul), 2 l exo-Klenow and 13.8 l nuclease-free H₂O. Labeling reactions were incubated at 37°C for 6 h, and then were purified using a Qiaquick PCR purification kit. Dye incorporation was evaluated using a NanoDrop (NanoDrop Technologies, Wilmington, DE, USA), producing typical base to dye ratios of 85–100. Cy5-labeled DNA was dried completely and stored at -20°C until hybridization.

Arrays were preybridized at 50°C in 50% formamide, 5 sodium chloride and sodium citrate (SSC) buffer, 0.1% sodium dodecyl sulfate (SDS) and 0.1 mg/ml bovine serum albumin for 30–60 min, followed by three washings with ddH₂O, one wash in isopropanol and drying by centrifugation. For hybridization, Cy5-labeled DNA was suspended in 50% formamide, 5 SSC, 0.1% SDS, 0.1 g/l herring sperm DNA, 1.6 l recA (490 ng/l) and water for a final volume of 40 l. Hybridization was conducted at 50°C for approximately 18 h. After hybridization, arrays were washed twice in 1 SSC with 0.1% SDS at 50°C for 5 min, twice in 0.1 SSC and 0.1% SDS at 25°C and five times in 0.1 SSC and then arrays were dried by centrifugation.

Arrays were scanned using a GenePix 4000B scanner and analyzed with GenePix software (Molecular Devices, Sunnyvale, CA, USA), and then data were further analyzed using a Perl script (He et al., 2007). Briefly, spots with >2 of signal-to-noise ratio (SNR), defined as the mean fluorescence minus mean background fluorescence divided by the standard deviation (s.d.) of background fluorescence, were filtered for further analysis. SNR values for each spot were normalized by the sum of all spots' mean signals (excluding positive control spots) on the same slide. Data from the three replicate slides for each sample were combined and outliers removed (>2 s.d.). Gene detection was considered positive when a positive hybridization signal was obtained from 33% of spots targeting the gene on three replicate arrays combined.

Results

¹³C-BP incorporation

Excess ¹³CO₂ was detected in microcosm headspaces following 1 day incubation, and increased in abundance throughout the 14 days experiment (Table 1), whereas no change in ¹³CO₂ was detected in control microcosms containing unlabeled BP or no BP. On the basis of previous reports that cells growing on BP mineralize 60–80% of BP to CO₂ (Bailey et al., 1983), the amount of ¹³C-BP utilized by the microbial community in microcosms containing 5 g soil was calculated to be 0.9–1.2 g within 1 day and then reached 59–79 g by 14 days.

Table 1 - Mineralization of ¹³C-BP as determined by ¹³CO₂ evolution.

Full table

Q-PCR targeting 16S rRNA genes in density gradient fractions indicated that significant quantities of ¹³C-DNA were present on days 4 and 14, whereas a lesser but detectable amount of labeled DNA was detected at day 1 (Figure 1). Unlabeled DNA formed a peak at BD of 1.599 g/ml, whereas ¹³C-DNA occupied fractions ranging in BD from approximately 1.617 to 1.671 g/ml. Duplicate density gradients run for each same sample were nearly identical in DNA quantity and distribution. Heavy fractions (BD of 1.632–1.671 g/ml) and light fractions (BD of 1.591–1.602 g/ml) were compiled from the two replicate gradients for each sample for further analyses. In parallel with the ¹³C-BP-incubated samples, equivalent heavy fractions from background control gradients were also analyzed.

Figure 1.

Detection of ¹³C-DNA in density gradient fractions derived from DNA at (a) the initiation of the experiment (t=0) and following incubation times of (b) 1 day; (c) 4 days; and (d) 14 days using Q-PCR targeting 16S rRNA genes. (a) (solid symbols) and (b) (open symbols) denote replicate density gradients run for each sample.

Full figure and legend (47K)

Community and phylogenetic analyses

T-RFLP analyses of the background control fractions revealed the presence of five major and several minor T-RFs contaminating the heavy fractions of density gradients, and these peaks were thus omitted in subsequent analyses of T-RFLPs of ¹³C-DNA samples (Figure 2). As incubation time increased, ¹³C-DNA samples produced larger numbers of T-RFs (Figure 3). Some T-RFs associated with ¹³C-labeled populations were also detected in the total community profile, whereas others were only found in ¹³C-DNA samples. The number of T-RFs shared among ¹³C-DNA and the total community was similar between 1 and 4 days, but then increased between 4 and 14 days incubation (Figure 3).

Figure 2.

T-RFLP profiles of 16S rRNA gene amplicons digested with HhaI. Labeled peaks correspond to predicted T-RF size from sequenced clones (A) Variovorax, Polaromonas, Anaerolinea; (B) Pseudonocardia, Kribbella; (C) Nocardiodes, Actinoplanes, Streptomyces; (D) Streptomyces; (E) Anaerolinea; (F) Nocardiodes; (G) unclassified Actinobacteria.

Full figure and legend (84K)

Figure 3.

Number of T-RFs detected in ¹³C-DNA following SIP incubation of 1, 4 and 14 days (after subtraction of background peaks) and in total community DNA from time 0.

Full figure and legend (17K)

Clone libraries of 16S rRNA gene amplicons were constructed from total community DNA at time 0, ¹³C-DNA from 1, 4 and 14 days, and from the heavy background control. Subtraction of sequences detected in the background that shared 99% sequence identity from ¹³C-DNA libraries resulted in the removal of 5, 5 and 3 sequences from the libraries from 1, 4 and 14 days SIP incubation, resulting in valid libraries of 50, 77 and 86 sequences, respectively. Rarefaction curves indicated that full community coverage was not achieved in any library, and Chao1 richness estimates were 785, 1000 and 1123 ribotypes for 1, 4 and 14 days valid ¹³C-DNA libraries. Seventy-five different genera were found to derive carbon from BP, with the majority of ¹³C-labeled organisms being Actinomycetes and Proteobacteria (Table 2). The most abundant sequences detected in ¹³C-DNA libraries were members of the genera Pseudonocardia, Nocardiodes, Kribbella and Sphingomonas, whereas many other organisms were detected in lower abundance. Fourteen percent of sequences were not classified by RDPII classifier at the 80% confidence threshold and may be novel organisms (Table 2).

Table 2 - Phylogenetic identification of organisms based on 16S rRNA gene sequences using RDP Classifier.

Full table

Twenty-two of the isolates were capable of growth on BP as a sole carbon source on agar plates, clearing BP sprayed over colonies and growth on BP as sole carbon source in liquid medium. Sequence analyses of 16S rRNA genes revealed that the majority (73%) of isolates were members of the genus Rhodococcus, whereas Arthobacter, Pseudomonas, Staphylococcus and Stenotrophomonas were also isolated (Table 2). All BP-utilizing isolates produced 16S rRNA gene sequences that differed from those obtained from ¹³C-DNA, with the exception of one sequence that was obtained from two Arthobacter isolates and two Arthrobacter clones. Plate counts of BP utilizers showed no significant change over time. Mean numbers with s.d.s in parentheses detected at time 0, 1, 4 and 14 days were 1.42 10⁵ (6.48 10⁴), 2.18 10⁵ (3.37 10⁴), 1.47 10⁵ (7.88 10⁴) and 1.44 10⁵ (1.55 10⁵) organisms per gram of fresh soil, respectively.

T-RFs were predicted by in silico digestion of sequences obtained from clones and were matched with major peaks in T-RFLPs (Figure 2). Sequences that were detected with highest frequency in ¹³C-DNA clone libraries were associated with many of the major T-RFs. The dominant peak in ¹³C-DNA was 356 bp in size, and matched T-RFs predicted from Pseudonocardia clones, with a secondary overlapping peak attributed to Kribbella (357–358 bp). The Pseudonocardia peak was present at low intensity at 1 day, and then increased in height to become the dominant peak at 4 and 14 days. Some Pseudonocardia clones also produced predicted T-RFs below the 50 bp cutoff for accurate T-RF measurement. Nocardiodes sequences produced T-RFs of several different sizes, including two T-RFs that matched detectable peaks (Figure 2). Sphingomonas sequences produced T-RFs of both 80 bp and below 50 bp.

Functional gene analyses

PCR amplification using ARHD primers produced amplicons of appropriate size (308–329 bp) visible on agarose gels from ¹³C-DNA fractions after 4 and 14 days incubation. No PCR products were visible from total community DNA, ¹³C-DNA following 1 day incubation and background control samples. When DNA from BP-utilizing isolates was amplified, only Rhodococcus spp. produced amplicons.

Sequences obtained from isolates and cloned amplicons of ¹³C-DNA (14 days) were subjected to BLASTn searches, and along with nearest matches and reference sequences were translated and aligned to produce a phylogenetic tree (Figure 4). The most abundant ARHD sequence obtained from ¹³C-DNA (25 out of 39 clones sequenced) is represented by Clone A04 in Figure 4. This sequence shared 100% amino-acid sequence similarity to Rhodococcus isolates C28 and C22, respectively, within the 77 amino-acid residues compared, whereas the nucleotide sequence similarity was 99 and 100%, respectively. The sequence represented by Clone B03 was detected only once in the library, and produced nearest GenBank matches of ARHDs targeting BP, isopropylbenzene, benzene and toluene associated with Rhodococcus spp., however, did not match any Genbank sequence exactly. When the 56 amino-acid residues available for Clone B03 were compared to the nearest GenBank matches that shared the same clade on the phylogenetic tree (Figure 4), amino-acid sequence similarities of 98% were obtained in all cases.

Figure 4.

Phylogenetic tree based on shared amino acids of translated sequences of ADHRs genes. Sequences generated in this study for isolates (square) and PCR amplification from ¹³C-DNA (circles) are marked. The scale is the expected number of substitutions per position. Numbers at the nodes are shown only for percentage bootstrap values above 50% of 1000 resamplings using the parsimony method. The NahAC sequence from Pseudomonas stutzeri was treated as the outgroup.

Full figure and legend (46K)

Whereas clones A04 and B03 nearly matched known ARHDs in GenBank along their full (300 bp) nucleotide sequence length, 12 sequences obtained from ¹³C-DNA matched only short (30 bp) segments of other initial aromatic dioxygenases in GenBank. ARHDs were the only gene type represented in the nearest GenBank matches with E values below 1 for these sequences. In our phylogenetic tree (Figure 4), the sequences clustered distantly from the Gram-positive or Gram-negative groups of ARHDs. Sequences were analyzed using a Rieske protein Hidden Markov model (HMM) to determine if the Rieske center was present in the sequences obtained. Because one ARHD primer is within the Rieske center, only approximately one-third of the Rieske region (5' end) could be evaluated. This region in the sequences obtained matched closely to the Rieske HMM model constructed by Pfam database (http://www.sanger.ac.uk/cgi-bin/Pfam/getacc?PF00355).

GeoChip analyses, after subtraction of all genes also detected in background, positively detected 30 genes associated with organic contaminant degradation in the ¹³C-DNA (Table 3). The majority (90%) of organic contaminant degradation genes detected were related to aromatic degradation. Four BP degradation genes were detected in ¹³C-DNA; bpdB and bpdF from Rhodococcus sp. M5 and bphD and bphB from Bacillus sp. JF8. Genes encoding components of oxygenase enzymes catalyzing benzoate, dibenzofuran, phenol, phenylpropionate and protocatechuate degradation were also detected, as were several genes associated with the -ketoadipate pathway.

Table 3 - Organic contaminant degradation genes detected in ¹³C-DNA following 14 days incubation with ¹³C-BP and subtraction of background contamination.

Full table

Discussion

SIP revealed a wealth of bacteria in the root zone of pine that derived carbon from BP, either directly through BP degradation or indirectly via growth on metabolic intermediates released from BP utilizers or their detritus (Table 2). Pseudonocardia, Nocardiodes, Kribella, Sphingomonas, Variovorax and Polaromonas were the predominant genera in the pine root zone that derived carbon from BP, as evidenced by early (1–4 days), sustained and frequent detection in 16S rRNA clone libraries constructed from ¹³C-DNA over the incubation period. Because clone libraries did not achieve full coverage based on rarefaction curves, T-RFLP peak heights are more reliable indicators of relative abundance than clone frequency although as in all PCR-based analyses amplification bias cannot be ruled out. On the basis of both abundance in clone libraries and associated T-RFLP peak height, Pseudonocardia was the most abundant utilizer of BP-derived carbon detected in this study.

PCB degradation capabilities have previously been reported for Sphingomonas spp. (Pieper, 2005), whereas the other dominant genera that derived carbon from BP were not known to degrade PCBs or BP although they were known to degrade other aromatics. Pseudonocardia spp. are known to degrade the monoaromatics benzoate, quinate and hydroxybenzoate (Grund and Kutzner, 1998; Hammann and Kutzner, 1998), toluene (Juteau et al., 1999) and tetrachlorobenzene (Kampfer and Kroppenstedt, 2004). Nocardiodes spp. are capable of degrading phenanthrene (Saito et al., 2000) and monoaromatics 2,4-dichlorophenoxyacetate (2,4-D) (Ferraroni et al., 2005) and nitrophenols (Ebert et al., 1999). A previous SIP experiment identified Variovorax as a naphthalene degrader (Padmanabhan et al., 2003), and it is also known to degrade plant aromatics vanillylamine and capsaicin (Flagan and Leadbetter, 2006) and other aromatics including linuron (Dejonghe et al., 2003) and 2,4-D (Kamagata et al., 1997). Polaromonas has been reported to degrade naphthalene (Jeon et al., 2004). The numerous other organisms identified as likely BP utilizers by virtue of acquiring ¹³C within 1 day were not previously known to degrade BP (Table 2).

A pronounced disparity was observed between BP utilizers identified using SIP and the cultivation method. One Arthobacter strain was the only organism identified as a BP utilizer using both methods. Rhodococcus spp. represented 73% of BP-utilizing isolates, however, were not detected in any 16S rRNA gene clone libraries produced by SIP. This discrepancy may be due to the slow growth rate of rhodococci, which required up to 21 days to form colonies on agar plates, whereas the SIP incubation only extended for 14 days. This is supported by the apparent lack of growth of culturable BP utilizers in soil over the course of the study as determined by plate counts. The possibility of poor PCR priming of rhodococci was also considered; however, when primers used to generate 16S rRNA gene clone libraries (27F and 1392R) were compared to sequences of closely related rhodococci obtained from the RDP database, no sequence mismatches were found. If the cloning process was biased against rhodococci, then large T-RFs attributable to rhodococci would be expected on T-RFLP profiles; however, this was not the case. T-RFs predicted from 16S rRNA gene sequences of Rhodococcus isolates overlapped with very small peaks also assigned to the close relatives, Nocardiodes and Kribella, which were present in ¹³C-DNA clone libraries.

Our findings differ from the only previously published SIP study aimed at identifying PCB-degrading bacteria (Tillmann et al., 2005). In the previous study, Burkholderia were found to be the only species that degraded ¹³C-labeled 2,2'-dichlorobiphenyl in a biofilm that was formed from a soil inoculum but was growing on PCB droplets. Culture-based studies of the biofilm were in agreement with the SIP results. The difference in taxa and species richness identified in our work are likely a result of differences in dominant PCB degraders in the soils studied as well as the distinct difference in community growth conditions between the two studies.

The increase in the number of ¹³C-associated T-RFs observed over the course of the incubation period (Figure 3) suggests that carbon flowed through BP utilizers into other organisms in the soil community. Secondary carbon flow is most evident at the 14 days time point, when an increased number of T-RFs detected in the total community became ¹³C-labeled (Figure 3), indicating that abundant soil heterotrophs began scavenging ¹³C. Clone libraries from ¹³C-DNA (Table 2) revealed eight different genera detected only after 14 days incubation, including Lysobacter, which was previously identified using SIP as a micropredator of ¹³C-labeled Escherichia coli cells added to soil (Lueders et al., 2006). Slower growing BP utilizers may also be expected to appear late in the incubation period. Unfortunately, after significant secondary carbon flow is underway, primary substrate degraders cannot be distinguished from secondary feeders with confidence.

When the metagenome of organisms that derived carbon from BP was explored using the GeoChip functional gene array, 28 different genes associated with aromatic degradation were detected. Four genes in the BP degradation pathway associated with Rhodococcus sp. M5 (bpdB, bpdF) and Bacillus sp. JF8 (bphD, bphB) were ¹³C-enriched. However, known genes associated with other steps in BP degradative pathways were not detected in ¹³C-DNA, nor were ARHDs that target BP or other polyaromatic compounds. Probing the pine rhizosphere with an array constructed from sequences of known degradative genes likely significantly undersamples the variety of aromatic degradative genes present and involved in BP utilization.

The latter hypothesis is supported by sequences obtained from ¹³C-DNA using primers designed to target the ARHDs, all of which were novel compared to known sequences present in GenBank. Some sequences clustered among known ARHDs, whereas five of the sequences were highly distinct from any known ARHD sequences although they possessed the portion of the Rieske center explored by the primers and matched short regions (30 bp) of other known aromatic dioxygenases. The novelty of these sequences invites further inquiry to obtain full sequences and examine their substrate specificity to evaluate their potential value to bioremediation.

The most frequently detected ARHD sequences (25 of 39 clones) were most closely related to Rhodococcus aromatic dioxygenases, and shared 99–100% nucleotide sequence similarity to ARHDs amplified from Rhodococcus isolates. This suggests two possible hypotheses. First, rhodococci may have derived carbon from BP, despite the absence of Rhodococcus sequences in 16S rRNA gene clone libraries. Alternatively, Rhodococcus-like dioxygenases may be more phylogenetically widespread than previously known, and could be present in related Actinobacteria that were frequently detected with SIP, such as Pseudonocardia or Nocardiodes, or other more distant organisms as a result of lateral gene transfer.

Benzoate (chlorobenzoate) is a major intermediate produced during BP (PCB) degradation and is subject to mineralization via various monoaromatic degradative pathways, including the -ketoadipate pathway (Harwood and Parales, 1996; Pieper, 2005). The -ketoadipate pathway is a convergent pathway common among soil bacteria that serves to mineralize a variety of monoaromatic xenobiotics as well as plant aromatics by funneling them through the central intermediate -ketoadipate via either protocatechuate or catechol branches (Harwood and Parales, 1996). Organisms that derived carbon from BP in the pine root zone possessed several genes encoding enzymes of the -ketoadipate pathway, including protocatechuate 3,4-dioxygenase and four different enzymes involved in the catechol branch of the pathway (Table 3). Thus, organisms active in BP mineralization in the root zone of pine, in theory, possess the capability to derive carbon and energy from plant aromatics. This provides further support for the hypothesis that plant-released aromatics have the capacity to support the growth of bacteria involved in PCB degradation (Singer et al., 2004).

SIP successfully concentrated genes of interest associated with organisms truly involved in BP mineralization that were not detectable in the total community due to their low abundance. Sequences of novel ARHD genes, many other functional genes and 16S rRNA genes detected in ¹³C-DNA were not detected directly in the total community DNA (Table 3, Figure 3). These findings underscore the utility of SIP for facilitating the discovery of genes of value to bioremediation and biocatalysis.

Together, this novel phylogenetic and functional gene information provides an improved understanding of BP degradation and carbon flow in soil communities and sheds new light on culture bias. Knowing the identity of bacteria and genes involved in biodegradation also enables the development of molecular tools to detect, quantify and monitor populations truly active in bioremediation processes. These tools could be applied to identify effective plant species for rhizoremediation by quantifying active PCB degraders in spatial association with different plants with much greater accuracy than cultivation methods used previously (Leigh et al., 2006). Future SIP studies could also provide new insights into the mechanisms of rhizoremediation by determining whether plant aromatics function as growth substrates for pollutant-degrading bacteria in the root zone.

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Acknowledgements

This work benefited from the technical assistance of Anthony Gaca, Cleopatra Mwansa and Najibah Rehman. Thanks to Mike Manefield and Mark J Bailey for training in SIP techniques. We thank Liyou Wu for providing GeoChips, Gene Wickham for training in hybridization methods and Ye Deng and Dan Cardin for bioinformatic support. T-RF prediction and Rieske center HMM analyses were performed by Benli Chai. SIP incubation methods were developed by Joonhong Park, Debora Rodrigues provided real-time PCR protocols and Stephan Gantner contributed to soil DNA extraction method development. Field site access was provided generously by Colorlak, logistical support provided by Vítek Matj, Martina Macková and Tomá Macek and Nathan Stewart assisted with field work. This work was supported by David L Boren (NSEP) Graduate International Fellowships and an NSF Postdoctoral Fellowship to MBL, NIEHS grant 2P42ES04911 under the Superfund Basic Science Program, and The Center for Microbial Ecology.