Polycyclic aromatic hydrocarbons (PAH) are widespread environmental pollutants of considerable risk to human health. The aerobic degradation of PAH via oxygenase reactions has been studied for several decades. In contrast, it was not until very recent that the first key enzyme involved in anaerobic PAH degradation, the dearomatizing 2-naphthoyl-CoA reductase, was isolated and characterized. In this work, a PCR-based functional assay was developed to detect microorganisms that have the ability to anaerobically degrade naphthalene, as a model for larger PAH. The degenerative oligonucleotide probes introduced here amplified a highly conserved region of the gene encoding 2-naphthoyl-CoA reductase (Ncr) in numerous sulfate-reducing pure cultures and environmental enrichments. The assay provides the first molecular tool for monitoring the anaerobic degradation of a model PAH.
Polycyclic aromatic hydrocarbons (PAH) are widespread environmental pollutants of considerable risk to human health. The limited water solubility and strong adsorption of PAH to sediment matrices contribute to slow rates of natural attenuation and long-term environmental persistence, especially under anoxic conditions. Recent investigations in the field of anaerobic PAH degradation have focused on naphthalene as a model system to understand the enzymatic reactions governing the biodegradation process (Meckenstock & Mouttaki, 2011).
The observation that microorganisms can mineralize PAH under anaerobic conditions is a relatively recent development. Enrichment cultures have been reported that degrade these compounds under nitrate-, iron-, sulfate-reducing, and methanogenic conditions (e.g. Sharak Genthner et al., 1997; Zhang & Young, 1997; Annweiler et al., 2002; Chang et al., 2002; Trably et al., 2003; Christensen et al., 2004; Meckenstock et al., 2004; Pravecek et al., 2005; Yuan & Chang, 2007; Kleemann & Meckenstock, 2011). However, to date, there are only three pure cultures that possess the ability the degrade naphthalene under anoxic conditions, the marine sulfate-reducing strains NaphS2, NaphS3, and NaphS6 (Galushko et al., 1999; Musat et al., 2009), and one highly enriched sulfate-reducing culture, N47 (Meckenstock et al., 2000), that was isolated from a contaminated freshwater aquifer located near Stuttgart (DE).
The initial reactions of anaerobic naphthalene degradation were elucidated using the N47 culture and comprise the following steps: (1) the carboxylation of naphthalene to 2-naphthoic acid (Mouttaki et al., 2012); (2) the ATP-dependent activation to 2-naphthoyl-CoA (not yet demonstrated, in vitro); (3) the ATP-independent reductive dearomatization of the nonsubstituted ring of 2-naphthoyl-CoA to 5,6,7,8-tetrahydronaphthoyl-CoA catalyzed by 2-naphthoyl-CoA reductase (Ncr); and (4) the ATP-dependent reduction in the substituted ring to a hexahydronaphthoyl-CoA compound (Fig. 1, Eberlein et al., 2013a). Ncr is the only enzyme involved in anaerobic PAH degradation that has been isolated and characterized to date (Eberlein et al., 2013b). The 150 kDa dimeric enzyme catalyses the reduction in 2-naphthoyl-CoA to 5,6,7,8-tetrahydro-2-naphthoyl-CoA (Eberlein et al., 2013b), a characteristic reaction for anaerobic naphthalene, 1- or 2-carboxy-, and 2-methylnaphthalene ring reduction.
To date, no assay targeting a characteristic gene involved in anaerobic PAH degradation is available. The purpose of this work was to identify a conserved segment of the gene encoding 2-naphthoyl-CoA reductase, ncr, to design degenerate oligonucleotide probes that specifically detect the ability of microorganisms to anaerobically degrade naphthalene. The potential to efficiently detect Ncr-containing microorganisms should facilitate further study of anaerobic PAH degradation in a culture-independent manner.
Materials and methods
The enrichment culture N47 (Meckenstock et al., 2000) and the pure culture NaphS2 (Galushko et al., 1999) were grown at 30 °C in anoxic minimal salts medium as described therein. The cells were routinely subcultured monthly by transferring 10% inoculum (v/v) into 500 mL of fresh, sterile, anoxic medium in 1 L Schott® bottles containing 0.5 M 2-naphtholic acid as the carbon source under sulfate-reducing conditions.
A total of four environmental cultures used for this study originate from Eastern Germany (DE), Soběslav (CZ), and Michle (CZ). They were cultivated in defined mineral salts medium as described for N47 (Meckenstock et al., 2000), using 2 mL of naphthalene dissolved in 2,2,4,4,6,8,8-heptamethylnonane as the carbon source instead of 2-naphthoic acid. The two cultures from Eastern Germany originate from groundwater (BR3) and sediment (MP8) and were enriched using material from a fuel storage depot located close to the river Elbe. The site has been used for storage since the 1920s, with major organic contamination consisting of ethylbenzene, xylene, and benzene in the range of 1300, 2500, and 15 000 μg L−1 groundwater−1, respectively. Sulfate-reducing and methanogenic conditions prevail in most contaminated areas of the Eastern German site. The Soběslav culture (Sob) was enriched using groundwater taken from the property of an operational creosote-based wood preservation facility in southern Bohemia, near the town of Soběslav (CZ). The site has been used for wood treatment since the mid-19th century and is heavily contaminated with PAH and heavy metals, with concentrations ranging from 640 mg kg−1 sediment−1 total PAH to 1000 mg kg−1 sediment−1 total chromium (Cr). The last enrichment culture (MIC) was developed using groundwater from a former gas works facility near the river Botič, north of Prague (CZ). Since the early 19th century, the site has been used for gas production through coke-making and later hydrocarbon cracking. These activities have contributed to the site being contaminated by PAH and BTEX, with typical PAH concentrations of 280 mg kg−1 sediment−1 and c. 4600 μg L−1 BTEX in the groundwater. The biogeochemistry of the Czech sites is characterized by sulfate- and iron-reducing conditions.
Clone libraries consisting of 2-napthoyl-CoA reductase (ncr) sequences from N47, NaphS2, and enrichment cultures from Eastern Germany (BR3) and Soběslav (Sob) were created using one set of primers (Ncr1for: 5′-CGTTATWCKCCYTGCCGTG-3′, Ncr1rev: 5′-CGATAAGCCATRCADATRGG-3′) designed to amplify the corresponding protein-coding sequences from both N47 (GI 308271890) and NaphS2 (GIs 301060407 and 301062483). The libraries were developed using a TOPO-TA cloning kit (Life Technologies™) according to manufacturer's instructions. The PCR cycling times for both clone library construction and novel PCR-primer design and validation consisted of an initial denaturation step for 3 min at 95 °C, followed by 30 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 2 min, and a final elongation step at 72 °C for 10 min. Positive clones were screened using blue/white selection and grown overnight in 96-well plates at 37 °C. The DNA sequences for 12 positive clones from each of the four cultures were amplified using the T7 primer, purified, and sequenced by GATC Biotech AG (Konstanz, DE). These sequences were used to generate novel degenerate primers designed to target sulfate-reducing, naphthalene-degrading cultures. It is this second set of primers (Ncr2for: 5′-TGGACAAAYAAAMGYACVGAT-3′; Ncr2rev: 5′-GATTCCGGCTTTTTTCCAAVT-3′) that form the basis for the novel PCR-based functional assay presented in this work. The assay targeted amino acids 194 through 299 of the Ncr sequences available. The results from a total of eight anaerobic, naphthalene-degrading cultures are presented below.
Results and discussion
The 2-naphthoyl-CoA reductase (Ncr) represents a novel class of dearomatizing arylcarboxyl-CoA reductases and is currently known to be exclusively involved in anaerobic PAH degradation (Eberlein et al., 2013b; Boll et al., 2014). The enzyme shows sequence similarity to the old yellow enzyme (OYE) family of flavin-containing proteins (Williams & Bruce, 2002).
With the newly developed PCR-based assay, the presence of the ncr gene was reliably amplified in NaphS2 (Galushko et al., 1999), NaphS3 and NaphS6 (Musat et al., 2009), N47 (Meckenstock et al., 2000), and the four environmental enrichment cultures from Eastern Germany (BR3 and MP8), Soběslav (Sob), and Michle (MIC) (Fig. 2a, only results from the Sob and BR3 sites are shown). The amplified fragments from each culture have been deposited in NCBI under the accession numbers KJ161160-65. In contrast, no corresponding fragment was amplified using DNA from Azoarcus evansii and Thauera aromatica strain K172, both serving as negative controls. These two well-known monocyclic aromatic compound-degrading, denitrifying bacteria contain 1,5-dienoyl-CoA oxidase (Dco), a member of the OYE protein family involved in oxygen detoxification in some monoaromatic compound-degrading anaerobic bacteria. The gene encoding Dco is the most closely related relative to Ncr (35% amino acid sequence identity, Thiele et al., 2008; Fig. 2b) currently in the NCBI database, yet was not amplified by the assay. Therefore, the c. 320 bp DNA fragments were specifically amplified only in the presence of DNA from ncr-containing anaerobic cultures and are indicative of the ability to specifically degrade the naphthalene ring system under anoxic conditions. Sequencing confirmed that the targeted regions of ncr were successfully amplified in all cases; no unspecific amplicons were observed.
The genes from the Sob and BR3 enrichment cultures were both 98% identical to N47 along the amplified region. Two additional environmental cultures were tested, one seeded with sediment from the MP8 and one from the MIC site. The amplified sequences from these two cultures were both 99% identical to the ncr from N47. Notably, N47 and the four enrichment cultures originate from freshwater aquifers, supporting the plausibility that N47-like organisms are involved in anaerobic naphthalene degradation in each of the four systems, all under sulfate-reducing conditions. For the pure cultures that stem from marine systems, NaphS2, NaphS3, and NaphS6, the sequences from NaphS3 and NaphS6 were 99% identical to the targeted region of Ncr1/Ncr2 from NaphS2. The neighbor-joining tree in Fig. 2b shows the separation of ncr sequences from freshwater and marine systems. Along the intended regions of the amplified DNA fragment, ncr from N47 and NaphS2 are 71% identical on the nucleotide level. For reference, the amino acid similarities between Ncr from N47 and NaphS2 are 67% identical. Current ongoing work has shown that both catalyze the identical reductive dearomatization of 2-naphthoyl-CoA (Eberlein et al., 2013b).
The PCR-based microbial assay presented here provides the first molecular method for detecting a functional gene involved in anaerobic PAH degradation. It may be applied to explore the diversity of anaerobic PAH-degrading bacteria in nature and to assess and monitor biodegradation of naphthalene at contaminated sites. The assay or variations thereof may also be useful for detecting anaerobic PAH-degrading bacteria that use alternative electron acceptors or other PAH compounds.
The authors thank Florin Musat, at the MPI for Marine Microbiology (Bremen), for providing DNA samples for NaphS3 and NaphS6. We also thank Rainer U. Meckenstock (Helmholtz Centre Munich) for providing strain N47. This work was funded by the collaborative EU-FP7 network (MAGIC-PAH/FP7-KBBE-2009-245226) and the DFG (German Research Council) Scientific Priority Program SPP1319.